Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.

Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of keymore » binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.« less

Structure of a Pheromone Receptor-Associated Mhc Molecule With An Open And Empty Groove

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olson, R.; Huey-Tubman, K.E.; Dulac, C.

2006-10-06

Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-bindingmore » MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.« less

Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

PubMed

Dai, Qi; Ren, Aiming; Westholm, Jakub O; Duan, Hong; Patel, Dinshaw J; Lai, Eric C

2015-01-01

Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional properties of this new family of transcription factors. © 2015 Dai et al.; Published by Cold Spring Harbor Laboratory Press.

Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family

PubMed Central

Dai, Qi; Ren, Aiming; Westholm, Jakub O.; Duan, Hong; Patel, Dinshaw J.

2015-01-01

Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain (“BEN-solo” factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional properties of this new family of transcription factors. PMID:25561495

Negatively Cooperative Binding of High Density Lipoprotein to the HDL Receptor SR-BI†

PubMed Central

Nieland, Thomas J.F.; Xu, Shangzhe; Penman, Marsha; Krieger, Monty

2011-01-01

Scavenger receptor class B, type I (SR-BI) is a high-density lipoprotein (HDL) receptor, which also binds low density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a co-receptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high affinity binding sites (one site model). We have re-investigated the ligand concentration dependence of 125I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [3H]CE from [3H]CE-HDL using an expanded range of ligand concentrations (<1 µg protein/ml, lower than previously reported). Scatchard and non-linear least squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites, or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects (‘ lattice model’). Similar results were observed for LDL. Application of the ‘infinite dilution’ dissociation rate method established that the binding of 125I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport and cell signaling. PMID:21254782

Structural and Functional Dissection of Human Cytomegalovirus US3 in Binding Major Histocompatibility Complex Class I Molecules

PubMed Central

Lee, Sungwook; Yoon, Juhan; Park, Boyoun; Jun, Youngsoo; Jin, Mirim; Sung, Ha Chin; Kim, Ik-Hwan; Kang, Seongman; Choi, Eui-Ju; Ahn, Byung Yoon; Ahn, Kwangseog

2000-01-01

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule. PMID:11070025

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for presentation of transformed self

PubMed Central

Mohammed, Fiyaz; Cobbold, Mark; Zarling, Angela L.; Salim, Mahboob; Barrett-Wilt, Gregory A.; Shabanowitz, Jeffrey; Hunt, Donald F.; Engelhard, Victor H.; Willcox, Benjamin E.

2008-01-01

Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex (MHC) class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphorylated peptides on cancer cells provides an immunological signature of “transformed self”. Here, we demonstrate that phosphorylation can radically increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide–HLA-A2 complexes. These revealed a novel peptide binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting MHC binding, or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy. PMID:18836451

A Triad of Molecular Regions Contribute to the Formation of Two Distinct MHC Class II Conformers

PubMed Central

Drake, Lisa A.; Drake, James R.

2016-01-01

MHC class II molecules present antigen-derived peptides to CD4 T cells to drive the adaptive immune response. Previous work has established that class II αβ dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4 T cells. Motif 1 (M1) paired I-Ak class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2 mAb selectively binds M1 paired I-Ak class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2 mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and β chain Glu-86. These findings provide insight into the complexity of 11-5.2 mAb recognition of the M1 paired I-Ak class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition. PMID:27148821

Recent advances in Major Histocompatibility Complex (MHC) class I antigen presentation: Plastic MHC molecules and TAPBPR-mediated quality control

PubMed Central

van Hateren, Andy; Bailey, Alistair; Elliott, Tim

2017-01-01

We have known since the late 1980s that the function of classical major histocompatibility complex (MHC) class I molecules is to bind peptides and display them at the cell surface to cytotoxic T cells. Recognition by these sentinels of the immune system can lead to the destruction of the presenting cell, thus protecting the host from pathogens and cancer. Classical MHC class I molecules (MHC I hereafter) are co-dominantly expressed, polygenic, and exceptionally polymorphic and have significant sequence diversity. Thus, in most species, there are many different MHC I allotypes expressed, each with different peptide-binding specificity, which can have a dramatic effect on disease outcome. Although MHC allotypes vary in their primary sequence, they share common tertiary and quaternary structures. Here, we review the evidence that, despite this commonality, polymorphic amino acid differences between allotypes alter the ability of MHC I molecules to change shape (that is, their conformational plasticity). We discuss how the peptide loading co-factor tapasin might modify this plasticity to augment peptide loading. Lastly, we consider recent findings concerning the functions of the non-classical MHC I molecule HLA-E as well as the tapasin-related protein TAPBPR (transporter associated with antigen presentation binding protein-related), which has been shown to act as a second quality-control stage in MHC I antigen presentation. PMID:28299193

Toxic shock syndrome toxin 1 binds to major histocompatibility complex class II molecules.

PubMed Central

Scholl, P; Diez, A; Mourad, W; Parsonnet, J; Geha, R S; Chatila, T

1989-01-01

Toxic shock syndrome toxin 1 (TSST-1) is a 22-kDa exotoxin produced by strains of Staphylococcus aureus and implicated in the pathogenesis of toxic shock syndrome. In common with other staphylococcal exotoxins, TSST-1 has diverse immunological effects. These include the induction of interleukin 2 receptor expression, interleukin 2 synthesis, proliferation of human T lymphocytes, and stimulation of interleukin 1 synthesis by human monocytes. In the present study, we demonstrate that TSST-1 binds with saturation kinetics and with a dissociation constant of 17-43 nM to a single class of binding sites on human mononuclear cells. There was a strong correlation between the number of TSST-1 binding sites and the expression of major histocompatibility complex class II molecules, and interferon-gamma induced the expression of class II molecules as well as TSST-1 binding sites on human skin-derived fibroblasts. Monoclonal antibodies to HLA-DR, but not to HLA-DP or HLA-DQ, strongly inhibited TSST-1 binding. Affinity chromatography of 125I-labeled cell membranes over TSST-1-agarose resulted in the recovery of two bands of 35 kDa and 31 kDa that comigrated, respectively, with the alpha and beta chains of HLA-DR and that could be immunoprecipitated with anti-HLA-DR monoclonal antibodies. Binding of TSST-1 was demonstrated to HLA-DR and HLA-DQ L-cell transfectants. These results indicate that major histocompatibility complex class II molecules represent the major binding site for TSST-1 on human cells. Images PMID:2542966

New horizons in mouse immunoinformatics: reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity.

PubMed

Hattotuwagama, Channa K; Guan, Pingping; Doytchinova, Irini A; Flower, Darren R

2004-11-21

Quantitative structure-activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide-protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2-D(b), H2-K(b) and H2-K(k). As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online ( http://www.jenner.ac.uk/MHCPred).

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing.

PubMed

Morozov, Giora I; Zhao, Huaying; Mage, Michael G; Boyd, Lisa F; Jiang, Jiansheng; Dolan, Michael A; Venna, Ramesh; Norcross, Michael A; McMurtrey, Curtis P; Hildebrand, William; Schuck, Peter; Natarajan, Kannan; Margulies, David H

2016-02-23

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE PAGES

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.; ...

2016-02-11

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function.

PubMed

Follin, Elna; Karlsson, Maria; Lundegaard, Claus; Nielsen, Morten; Wallin, Stefan; Paulsson, Kajsa; Westerdahl, Helena

2013-04-01

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1-α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.

Structural Definition of Duck Major Histocompatibility Complex Class I Molecules That Might Explain Efficient Cytotoxic T Lymphocyte Immunity to Influenza A Virus

PubMed Central

Wu, Yanan; Wang, Junya; Fan, Shuhua; Chen, Rong; Liu, Yanjie; Zhang, Jianhua; Yuan, Hongyu; Liang, Ruiying

2017-01-01

ABSTRACT A single dominantly expressed allele of major histocompatibility complex class I (MHC I) may be responsible for the duck's high tolerance to highly pathogenic influenza A virus (HP-IAV) compared to the chicken's lower tolerance. In this study, the crystal structures of duck MHC I (Anpl-UAA*01) and duck β2-microglobulin (β2m) with two peptides from the H5N1 strains were determined. Two remarkable features were found to distinguish the Anpl-UAA*01 complex from other known MHC I structures. A disulfide bond formed by Cys95 and Cys112 and connecting the β5 and β6 sheets at the bottom of peptide binding groove (PBG) in Anpl-UAA*01 complex, which can enhance IAV peptide binding, was identified. Moreover, the interface area between duck MHC I and β2m was found to be larger than in other species. In addition, the two IAV peptides that display distinctive conformations in the PBG, B, and F pockets act as the primary anchor sites. Thirty-one IAV peptides were used to verify the peptide binding motif of Anpl-UAA*01, and the results confirmed that the peptide binding motif is similar to that of HLA-A*0201. Based on this motif, approximately 600 peptides from the IAV strains were partially verified as the candidate epitope peptides for Anpl-UAA*01, which is a far greater number than those for chicken BF2*2101 and BF2*0401 molecules. Extensive IAV peptide binding should allow for ducks with this Anpl-UAA*01 haplotype to resist IAV infection. IMPORTANCE Ducks are natural reservoirs of influenza A virus (IAV) and are more resistant to the IAV than chickens. Both ducks and chickens express only one dominant MHC I locus providing resistance to the virus. To investigate how MHC I provides IAV resistance, crystal structures of the dominantly expressed duck MHC class I (pAnpl-UAA*01) with two IAV peptides were determined. A disulfide bond was identified in the peptide binding groove that can facilitate Anpl-UAA*01 binding to IAV peptides. Anpl-UAA*01 has a much wider recognition spectrum of IAV epitope peptides than do chickens. The IAV peptides bound by Anpl-UAA*01 display distinctive conformations that can help induce an extensive cytotoxic T lymphocyte (CTL) response. In addition, the interface area between the duck MHC I and β2m is larger than in other species. These results indicate that HP-IAV resistance in ducks is due to extensive CTL responses induced by MHC I. PMID:28490583

Structural Definition of Duck Major Histocompatibility Complex Class I Molecules That Might Explain Efficient Cytotoxic T Lymphocyte Immunity to Influenza A Virus.

PubMed

Wu, Yanan; Wang, Junya; Fan, Shuhua; Chen, Rong; Liu, Yanjie; Zhang, Jianhua; Yuan, Hongyu; Liang, Ruiying; Zhang, Nianzhi; Xia, Chun

2017-07-15

A single dominantly expressed allele of major histocompatibility complex class I (MHC I) may be responsible for the duck's high tolerance to highly pathogenic influenza A virus (HP-IAV) compared to the chicken's lower tolerance. In this study, the crystal structures of duck MHC I ( Anpl -UAA*01) and duck β2-microglobulin (β2m) with two peptides from the H5N1 strains were determined. Two remarkable features were found to distinguish the Anpl -UAA*01 complex from other known MHC I structures. A disulfide bond formed by Cys 95 and Cys 112 and connecting the β5 and β6 sheets at the bottom of peptide binding groove (PBG) in Anpl -UAA*01 complex, which can enhance IAV peptide binding, was identified. Moreover, the interface area between duck MHC I and β2m was found to be larger than in other species. In addition, the two IAV peptides that display distinctive conformations in the PBG, B, and F pockets act as the primary anchor sites. Thirty-one IAV peptides were used to verify the peptide binding motif of Anpl -UAA*01, and the results confirmed that the peptide binding motif is similar to that of HLA-A*0201. Based on this motif, approximately 600 peptides from the IAV strains were partially verified as the candidate epitope peptides for Anpl -UAA*01, which is a far greater number than those for chicken BF2*2101 and BF2*0401 molecules. Extensive IAV peptide binding should allow for ducks with this Anpl -UAA*01 haplotype to resist IAV infection. IMPORTANCE Ducks are natural reservoirs of influenza A virus (IAV) and are more resistant to the IAV than chickens. Both ducks and chickens express only one dominant MHC I locus providing resistance to the virus. To investigate how MHC I provides IAV resistance, crystal structures of the dominantly expressed duck MHC class I (p Anpl -UAA*01) with two IAV peptides were determined. A disulfide bond was identified in the peptide binding groove that can facilitate Anpl -UAA*01 binding to IAV peptides. Anpl -UAA*01 has a much wider recognition spectrum of IAV epitope peptides than do chickens. The IAV peptides bound by Anpl -UAA*01 display distinctive conformations that can help induce an extensive cytotoxic T lymphocyte (CTL) response. In addition, the interface area between the duck MHC I and β2m is larger than in other species. These results indicate that HP-IAV resistance in ducks is due to extensive CTL responses induced by MHC I. Copyright © 2017 Wu et al.

Improved pan-specific MHC class I peptide-binding predictions using a novel representation of the MHC-binding cleft environment.

PubMed

Carrasco Pro, S; Zimic, M; Nielsen, M

2014-02-01

Major histocompatibility complex (MHC) molecules play a key role in cell-mediated immune responses presenting bounded peptides for recognition by the immune system cells. Several in silico methods have been developed to predict the binding affinity of a given peptide to a specific MHC molecule. One of the current state-of-the-art methods for MHC class I is NetMHCpan, which has a core ingredient for the representation of the MHC class I molecule using a pseudo-sequence representation of the binding cleft amino acid environment. New and large MHC-peptide-binding data sets are constantly being made available, and also new structures of MHC class I molecules with a bound peptide have been published. In order to test if the NetMHCpan method can be improved by integrating this novel information, we created new pseudo-sequence definitions for the MHC-binding cleft environment from sequence and structural analyses of different MHC data sets including human leukocyte antigen (HLA), non-human primates (chimpanzee, macaque and gorilla) and other animal alleles (cattle, mouse and swine). From these constructs, we showed that by focusing on MHC sequence positions found to be polymorphic across the MHC molecules used to train the method, the NetMHCpan method achieved a significant increase in the predictive performance, in particular, of non-human MHCs. This study hence showed that an improved performance of MHC-binding methods can be achieved not only by the accumulation of more MHC-peptide-binding data but also by a refined definition of the MHC-binding environment including information from non-human species. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jia, Xiaofei; Singh, Rajendra; Homann, Stefanie

The HIV-1 protein Nef inhibits antigen presentation by class I major histocompatibility complex (MHC-I). We determined the mechanism of this activity by solving the crystal structure of a protein complex comprising Nef, the MHC-I cytoplasmic domain (MHC-I CD) and the {mu}1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-{mu}1 interface, which encompasses the cargo-recognition site of {mu}1 and the proline-rich strand of Nef. The Nef C terminus induces a previously unobserved conformational change in {mu}1, whereas the N terminus binds the Nef core tomore » position it optimally for complex formation. Positively charged patches on {mu}1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity.« less

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

PubMed Central

Hawse, William F.; Gloor, Brian E.; Ayres, Cory M.; Kho, Kevin; Nuter, Elizabeth; Baker, Brian M.

2013-01-01

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity. PMID:23836912

Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

PubMed

Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

2007-01-01

Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made are freely available online at the URL http://www.jenner.ac.uk/MHCPred.

Structure of HLA-A*0301 in complex with a peptide of proteolipid protein: insights into the role of HLA-A alleles in susceptibility to multiple sclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMahon, Róisín M.; University of Oxford, Roosevelt Drive, Oxford OX3 7BN; Friis, Lone

The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-ray crystallography to 2.7 Å resolution. The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-ray crystallography to 2.7 Å resolution. HLA-A3 is a predisposing allele for multiple sclerosis (MS), an autoimmune disease of the central nervous system. The KLIETYFSK peptide is a naturally processed epitope of proteolipid protein, a myelin protein and candidate target for immune-mediated myelin destruction in MS.more » Comparison of the structure of HLA-A3 with that of HLA-A2, an MHC class I molecule which is protective against MS, indicates that both MHC class I molecules present very similar faces for T-cell receptor recognition whilst differing in the specificity of their peptide-binding grooves. These characteristics may underlie the opposing (predisposing versus protective) associations that they exhibit both in humans and in mouse models of MS-like disease. Furthermore, subtle alterations within the peptide-binding groove of HLA-A3 and other A3-like MHC class I molecules, members of the so-called A3 superfamily, may be sufficient to alter their presentation of autoantigen peptides such as KLIETYFSK. This in turn may modulate their contribution to the associated risk of autoimmune disease.« less

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

PubMed Central

Valés-Gómez, M; Reyburn, H T; Erskine, R A; López-Botet, M; Strominger, J L

1999-01-01

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells. PMID:10428963

Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens

PubMed Central

Xing, Dongxia; Li, Sufang; Robinson, Simon N.; Yang, Hong; Steiner, David; Komanduri, Krishna V.; Shpall, Elizabeth J.

2009-01-01

In the control of T-helper type I (Th-1) polarization, dendritic cells (DCs) must interpret a complex array of stimuli, many of which are poorly understood. Here we demonstrate that Th-1 polarization is heavily influenced by DC-autonomous phenomena triggered by the loading of DCs with antigenically matched major histocompatibility complex (MHC) class I and class II determinants, that is, class I and II peptide epitopes exhibiting significant amino acid sequence overlap (such as would be physiologically present during infectious processes requiring Th-1 immunity for clearance). Data were derived from 13 independent antigenic models including whole-cell systems, single-protein systems, and 3 different pairs of overlapping class I and II binding epitopes. Once loaded with matched class I and II antigens, these “Th-1 DCs” exhibited differential cytokine secretion and surface marker expression, a distinct transcriptional signature, and acquired the ability to enhance generation of CD8+ T lymphocytes. Mechanistically, tRNA-synthetases were implicated as components of a putative sensor complex involved in the comparison of class I and II epitopes. These data provide rigorous conceptual explanations for the process of Th-1 polarization and the antigenic specificity of cognate T-cell help, enhance the understanding of Th-1 responses, and should contribute to the formulation of more effective vaccination strategies. PMID:19171878

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

PubMed

Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

2018-01-24

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Viral peptides-MHC interaction: Binding probability and distance from human peptides.

PubMed

Santoni, Daniele

2018-05-23

Identification of peptides binding to MHC class I complex can play a crucial role in retrieving potential targets able to trigger an immune response. Affinity binding of viral peptides can be estimated through effective computational methods that in the most of cases are based on machine learning approach. Achieving a better insight into peptide features that impact on the affinity binding rate is a challenging issue. In the present work we focused on 9-mer peptides of Human immunodeficiency virus type 1 and Human herpes simplex virus 1, studying their binding to MHC class I. Viral 9-mers were partitioned into different classes, where each class is characterized by how far (in terms of mutation steps) the peptides belonging to that class are from human 9-mers. Viral 9-mers were partitioned in different classes, based on the number of mutation steps they are far from human 9-mers. We showed that the overall binding probability significantly differs among classes, and it typically increases as the distance, computed in terms of number of mutation steps from the human set of 9-mers, increases. The binding probability is particularly high when considering viral 9-mers that are far from all human 9-mers more than three mutation steps. A further evidence, providing significance to those special viral peptides and suggesting a potential role they can play, comes from the analysis of their distribution along viral genomes, as it revealed they are not randomly located, but they preferentially occur in specific genes. Copyright © 2018 Elsevier B.V. All rights reserved.

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics.

PubMed

Gul, Ahmet; Erman, Burak

2018-01-16

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

NASA Astrophysics Data System (ADS)

Gul, Ahmet; Erman, Burak

2018-03-01

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.

PubMed

Hamada, K; Gleason, S L; Levi, B Z; Hirschfeld, S; Appella, E; Ozato, K

1989-11-01

Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, but not to other MHC cis-acting sequences or to mutant region II sequences, similar to the naturally occurring region II factor in mouse cells. The deduced amino acid sequence of H-2RIIBP revealed two putative zinc fingers homologous to the DNA-binding domain of steroid/thyroid hormone receptors. Although sequence similarity in other regions was minimal, H-2RIIBP has apparent modular domains characteristic of the nuclear hormone receptors. Further analyses showed that both H-2RIIBP and the natural region II factor bind to the estrogen response element (ERE) of the vitellogenin A2 gene. The ERE is composed of a palindrome, and half of this palindrome resembles the region II binding site of the MHC CRE. These results indicate that H-2RIIBP (i) is a member of the superfamily of nuclear hormone receptors and (ii) may regulate not only MHC class I genes but also genes containing the ERE and related sequences. Sequences homologous to the H-2RIIBP gene are widely conserved in the animal kingdom. H-2RIIBP mRNA is expressed in many mouse tissues, in agreement with the distribution of the natural region II factor.

Staphylococcal enterotoxins bind H-2Db molecules on macrophages

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1995-01-01

We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

Recognition of Human Histocompatibility Leukocyte Antigen (HLA)-E Complexed with HLA Class I Signal Sequence–derived Peptides by CD94/NKG2 Confers Protection from Natural Killer Cell–mediated Lysis

PubMed Central

Borrego, Francisco; Ulbrecht, Matthias; Weiss, Elisabeth H.; Coligan, John E.; Brooks, Andrew G.

1998-01-01

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical HLA class I molecule, the gene for which is transcribed in most tissues. It has recently been reported that this molecule binds peptides derived from the signal sequence of HLA class I proteins; however, no function for HLA-E has yet been described. We show that natural killer (NK) cells can recognize target cells expressing HLA-E molecules on the cell surface and this interaction results in inhibition of the lytic process. Furthermore, HLA-E recognition is mediated primarily through the CD94/NKG2-A heterodimer, as CD94-specific, but not killer cell inhibitory receptor (KIR)–specific mAbs block HLA-E–mediated protection of target cells. Cell surface HLA-E could be increased by incubation with synthetic peptides corresponding to residues 3–11 from the signal sequences of a number of HLA class I molecules; however, only peptides which contained a Met at position 2 were capable of conferring resistance to NK-mediated lysis, whereas those having Thr at position 2 had no effect. Interestingly, HLA class I molecules previously correlated with CD94/NKG2 recognition all have Met at residue 4 of the signal sequence (position 2 of the HLA-E binding peptide), whereas those which have been reported not to interact with CD94/NKG2 have Thr at this position. Thus, these data show a function for HLA-E and suggest an alternative explanation for the apparent broad reactivity of CD94/NKG2 with HLA class I molecules; that CD94/NKG2 interacts with HLA-E complexed with signal sequence peptides derived from “protective” HLA class I alleles rather than directly interacting with classical HLA class I proteins. PMID:9480992

A Peptide Filtering Relation Quantifies MHC Class I Peptide Optimization

PubMed Central

Goldstein, Leonard D.; Howarth, Mark; Cardelli, Luca; Emmott, Stephen; Elliott, Tim; Werner, Joern M.

2011-01-01

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human Immunodeficiency Virus Gag-Pol polyprotein. PMID:22022238

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2011-11-01

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mikeska, Ruth; Wacker, Roland; Arni, Raghuvir

2005-01-01

The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1. The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R{sub free} = 23.6%) and 20.9 (R{sub free} = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chainmore » has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.« less

Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using (/sup 125/I)FK33824

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rothman, R.B.; Jacobson, A.E.; Rice, K.C.

1987-11-01

Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites (25,35), consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, (/sup 3/H)oxymorphone, (/sup 3/H)D-ala2-MePhe4, Gly-ol5-enkephalin (DAGO), and (/sup 125/I)D-ala2-Me-Phe4-met(o)-ol)enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu bindingmore » sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand (/sup 125/I)FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.« less

The production and crystallization of the human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 complexed with deamidated gliadin peptides implicated in coeliac disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henderson, Kate N.; Reid, Hugh H.; Borg, Natalie A.

2007-12-01

The production and crystallization of human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 in complex with deamidated gliadin peptides is reported. Crystals of HLA-DQ2{sup PQPELPYPQ} diffracted to 3.9 Å, while the HLA-DQ8{sup EGSFQPSQE} crystals diffracted to 2.1 Å, allowing structure determination by molecular replacement. The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4{sup +} T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 α-I (PQPELPYPQ) and DQ8more » α-I (EGSFQPSQE), respectively, are reported.« less

Discrete structural features among interface residue-level classes.

PubMed

Sowmya, Gopichandran; Ranganathan, Shoba

2015-01-01

Protein-protein interaction (PPI) is essential for molecular functions in biological cells. Investigation on protein interfaces of known complexes is an important step towards deciphering the driving forces of PPIs. Each PPI complex is specific, sensitive and selective to binding. Therefore, we have estimated the relative difference in percentage of polar residues between surface and the interface for each complex in a non-redundant heterodimer dataset of 278 complexes to understand the predominant forces driving binding. Our analysis showed ~60% of protein complexes with surface polarity greater than interface polarity (designated as class A). However, a considerable number of complexes (~40%) have interface polarity greater than surface polarity, (designated as class B), with a significantly different p-value of 1.66E-45 from class A. Comprehensive analyses of protein complexes show that interface features such as interface area, interface polarity abundance, solvation free energy gain upon interface formation, binding energy and the percentage of interface charged residue abundance distinguish among class A and class B complexes, while electrostatic visualization maps also help differentiate interface classes among complexes. Class A complexes are classical with abundant non-polar interactions at the interface; however class B complexes have abundant polar interactions at the interface, similar to protein surface characteristics. Five physicochemical interface features analyzed from the protein heterodimer dataset are discriminatory among the interface residue-level classes. These novel observations find application in developing residue-level models for protein-protein binding prediction, protein-protein docking studies and interface inhibitor design as drugs.

Discrete structural features among interface residue-level classes

PubMed Central

2015-01-01

Background Protein-protein interaction (PPI) is essential for molecular functions in biological cells. Investigation on protein interfaces of known complexes is an important step towards deciphering the driving forces of PPIs. Each PPI complex is specific, sensitive and selective to binding. Therefore, we have estimated the relative difference in percentage of polar residues between surface and the interface for each complex in a non-redundant heterodimer dataset of 278 complexes to understand the predominant forces driving binding. Results Our analysis showed ~60% of protein complexes with surface polarity greater than interface polarity (designated as class A). However, a considerable number of complexes (~40%) have interface polarity greater than surface polarity, (designated as class B), with a significantly different p-value of 1.66E-45 from class A. Comprehensive analyses of protein complexes show that interface features such as interface area, interface polarity abundance, solvation free energy gain upon interface formation, binding energy and the percentage of interface charged residue abundance distinguish among class A and class B complexes, while electrostatic visualization maps also help differentiate interface classes among complexes. Conclusions Class A complexes are classical with abundant non-polar interactions at the interface; however class B complexes have abundant polar interactions at the interface, similar to protein surface characteristics. Five physicochemical interface features analyzed from the protein heterodimer dataset are discriminatory among the interface residue-level classes. These novel observations find application in developing residue-level models for protein-protein binding prediction, protein-protein docking studies and interface inhibitor design as drugs. PMID:26679043

Evolution of major histocompatibility complex class I and class II genes in the brown bear

PubMed Central

2012-01-01

Background Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia. PMID:23031405

Evolution of major histocompatibility complex class I and class II genes in the brown bear.

PubMed

Kuduk, Katarzyna; Babik, Wiesław; Bojarska, Katarzyna; Sliwińska, Ewa B; Kindberg, Jonas; Taberlet, Pierre; Swenson, Jon E; Radwan, Jacek

2012-10-02

Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South-north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia.

The CD1 family: serving lipid antigens to T cells since the Mesozoic era.

PubMed

Zajonc, Dirk M

2016-08-01

Class I-like CD1 molecules are in a family of antigen-presenting molecules that bind lipids and lipopeptides, rather than peptides for immune surveillance by T cells. Since CD1 lacks the high degree of polymorphism found in their major histocompatibility complex (MHC) class I molecules, different species express different numbers of CD1 isotypes, likely to be able to present structurally diverse classes of lipid antigens. In this review, we will present a historical overview of the structures of the different human CD1 isotypes and also discuss species-specific adaptations of the lipid-binding groove. We will discuss how single amino acid changes alter the shape and volume of the CD1 binding groove, how these minor changes can give rise to different numbers of binding pockets, and how these pockets affect the lipid repertoire that can be presented by any given CD1 protein. We will compare the structures of various lipid antigens and finally, we will discuss recognition of CD1-presented lipid antigens by antigen receptors on T cells (TCRs).

The CD1 family: serving lipid antigens to T cells since the Mesozoic era

PubMed Central

Zajonc, Dirk M.

2016-01-01

Class I-like CD1 molecules are in a family of antigen-presenting molecules that bind lipids and lipopeptides, rather than peptides for immune surveillance by T cells. Since CD1 lacks the high degree of polymorphism found in their major histocompatibility complex (MHC) class I molecules, different species express different numbers of CD1 isotypes, likely to be able to present structurally diverse classes of lipid antigens. In this review, we will present a historical overview of the structures of the different human CD1 isotypes and also discuss species-specific adaptations of the lipid-binding groove. We will discuss how single amino acid changes alter the shape and volume of the CD1 binding groove, how these minor changes can give rise to different numbers of binding pockets, and how these pockets affect the lipid repertoire that can be presented by any given CD1 protein. We will compare the structures of various lipid antigens and finally, we will discuss recognition of CD1-presented lipid antigens by antigen receptors on T cells (TCRs). PMID:27368414

Genomic localization of the human gene encoding Dr1, a negative modulator of transcription of class II and class III genes.

PubMed

Purrello, M; Di Pietro, C; Rapisarda, A; Viola, A; Corsaro, C; Motta, S; Grzeschik, K H; Sichel, G

1996-01-01

Dr1 is a nuclear protein of 19 kDa that exists in the nucleoplasm as a homotetramer. By binding to TBP (the DNA-binding subunit of TFIID, and also a subunit of SL1 and TFIIIB), the protein blocks class II and class III preinitiation complex assembly, thus repressing the activity of the corresponding promoters. Since transcription of class I genes is unaffected by Dr1. it has been proposed that the protein may coordinate the expression of class I, class II and class III genes. By somatic cell genetics and fluorescence in situ hybridization, we have localized the gene (DR1), present in the genome of higher eukaryotes as a single copy, to human chromosome region 1p21-->p13. The nucleotide sequence conservation of the coding segment of the gene, as determined by Noah's ark blot analysis, and its ubiquitous transcription suggest that Dr1 has an important biological role, which could be related to the negative control of cell proliferation.

Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers

PubMed Central

Chattopadhyay, Pratip K.; Melenhorst, J. Joseph; Ladell, Kristin; Gostick, Emma; Scheinberg, Philip; Barrett, A. John; Wooldridge, Linda; Roederer, Mario; Sewell, Andrew K.; Price, David A.

2008-01-01

The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated tetrameric peptide-MHC class I (pMHCI) complexes in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where TCR-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer-binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidity, minimize background noise and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. PMID:18836993

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

PubMed Central

Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

2015-01-01

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. PMID:25533465

Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings

PubMed Central

Ayres, Cory M.; Corcelli, Steven A.; Baker, Brian M.

2017-01-01

Structural biology of peptides presented by class I and class II MHC proteins has transformed immunology, impacting our understanding of fundamental immune mechanisms and allowing researchers to rationalize immunogenicity and design novel vaccines. However, proteins are not static structures as often inferred from crystallographic structures. Their components move and breathe individually and collectively over a range of timescales. Peptides bound within MHC peptide-binding grooves are no exception and their motions have been shown to impact recognition by T cell and other receptors in ways that influence function. Furthermore, peptides tune the motions of MHC proteins themselves, which impacts recognition of peptide/MHC complexes by other proteins. Here, we review the motional properties of peptides in MHC binding grooves and discuss how peptide properties can influence MHC motions. We briefly review theoretical concepts about protein motion and highlight key data that illustrate immunological consequences. We focus primarily on class I systems due to greater availability of data, but segue into class II systems as the concepts and consequences overlap. We suggest that characterization of the dynamic “energy landscapes” of peptide/MHC complexes and the resulting functional consequences is one of the next frontiers in structural immunology. PMID:28824655

Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings.

PubMed

Ayres, Cory M; Corcelli, Steven A; Baker, Brian M

2017-01-01

Structural biology of peptides presented by class I and class II MHC proteins has transformed immunology, impacting our understanding of fundamental immune mechanisms and allowing researchers to rationalize immunogenicity and design novel vaccines. However, proteins are not static structures as often inferred from crystallographic structures. Their components move and breathe individually and collectively over a range of timescales. Peptides bound within MHC peptide-binding grooves are no exception and their motions have been shown to impact recognition by T cell and other receptors in ways that influence function. Furthermore, peptides tune the motions of MHC proteins themselves, which impacts recognition of peptide/MHC complexes by other proteins. Here, we review the motional properties of peptides in MHC binding grooves and discuss how peptide properties can influence MHC motions. We briefly review theoretical concepts about protein motion and highlight key data that illustrate immunological consequences. We focus primarily on class I systems due to greater availability of data, but segue into class II systems as the concepts and consequences overlap. We suggest that characterization of the dynamic "energy landscapes" of peptide/MHC complexes and the resulting functional consequences is one of the next frontiers in structural immunology.

Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1.

PubMed

Heix, J; Zomerdijk, J C; Ravanpay, A; Tjian, R; Grummt, I

1997-03-04

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.

Neurobehavioral toxicology of pyrethroid insecticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crofton, K.M.

1986-01-01

Pyrethroid insecticides are classified as either Type I or Type II based upon in vivo toxic signs, and neurophysiological and biochemical data. Both axonal sodium channels and the ..gamma..-aminobutyric acid (GABA) receptor complex have been proposed as the major site of action of the Type II pyrethroids. This investigation characterized the behavior and biochemical effects of low dosages of pyrethroids in rats. Type I and II pyrethroids were tested for effects on figure-eight maze activity and the acoustic startle response (ASR). All compounds decreased figure-eight maze activity. Interactions of Type I and II pyrethroids with the three major binding sitesmore » on the GABA complex were determined in vivo. Radioligand binding experiments assessed in vitro interactions of pyrethroids with the three major GABA-complex binding sites. None of the pyrethroids competed for (/sup 3/H)-muscimol or (/sup 3/H)-flunitrazepam binding. Only Type II pyrethroids inhibited binding of (/sup 35/S)-t-butylbicyclophosphorothionate (TBPS) in cortical synaptosome preparations with K/sub i/ values of 5 to 10 ..mu..M. The (/sup 35/S)-TBPS data implicate the TBPS/picrotoxinin binding site in the mechanism of Type II pyrethroid toxicity. The results of these experiments support the classification of pyrethroids into two classes, and demonstrate the utility of the figure-eight maze and the ASR in studies to elucidate neurotoxic mechanisms. The interaction of the Type II pyrethroids is probably restricted to the TBPS/picrotoxinin binding domain on the GABA complex as shown by both the in vivo and in vitro studies.« less

Structures of native and affinity-enhanced WT1 epitopes bound to HLA-A*0201: implications for WT1-based cancer therapeutics.

PubMed

Borbulevych, Oleg Y; Do, Priscilla; Baker, Brian M

2010-09-01

Presentation of peptides by class I or class II major histocompatibility complex (MHC) molecules is required for the initiation and propagation of a T cell-mediated immune response. Peptides from the Wilms Tumor 1 transcription factor (WT1), upregulated in many hematopoetic and solid tumors, can be recognized by T cells and numerous efforts are underway to engineer WT1-based cancer vaccines. Here we determined the structures of the class I MHC molecule HLA-A*0201 bound to the native 126-134 epitope of the WT1 peptide and a recently described variant (R1Y) with improved MHC binding. The R1Y variant, a potential vaccine candidate, alters the positions of MHC charged side chains near the peptide N-terminus and significantly reduces the peptide/MHC electrostatic surface potential. These alterations indicate that the R1Y variant is an imperfect mimic of the native WT1 peptide, and suggest caution in its use as a therapeutic vaccine. Stability measurements revealed how the R1Y substitution enhances MHC binding affinity, and together with the structures suggest a strategy for engineering WT1 variants with improved MHC binding that retain the structural features of the native peptide/MHC complex. Copyright 2010 Elsevier Ltd. All rights reserved.

Structure of Ristocetin A in Complex with a Bacterial Cell-wall Mimetic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nahoum, V.; Spector, S; Loll, P

2009-01-01

Antimicrobial drug resistance is a serious public health problem and the development of new antibiotics has become an important priority. Ristocetin A is a class III glycopeptide antibiotic that is used in the diagnosis of von Willebrand disease and which has served as a lead compound for the development of new antimicrobial therapeutics. The 1.0 A resolution crystal structure of the complex between ristocetin A and a bacterial cell-wall peptide has been determined. As is observed for most other glycopeptide antibiotics, it is shown that ristocetin A forms a back-to-back dimer containing concave binding pockets that recognize the cell-wall peptide.more » A comparison of the structure of ristocetin A with those of class I glycopeptide antibiotics such as vancomycin and balhimycin identifies differences in the details of dimerization and ligand binding. The structure of the ligand-binding site reveals a likely explanation for ristocetin A's unique anticooperativity between dimerization and ligand binding.« less

Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets'.

PubMed

Wang, Yong-Qiang; Melzer, Rainer; Theissen, Günter

2010-10-01

Several lines of evidence suggest that the identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of MADS-domain proteins. These bind to specific cis-regulatory elements ('CArG-boxes') of their target genes involving DNA-loop formation, thus constituting 'floral quartets'. Gymnosperms, angiosperms' closest relatives, contain orthologues of floral homeotic genes, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. We have comprehensively studied the dimerization and DNA-binding of several classes of MADS-domain proteins from the gymnosperm Gnetum gnemon. Determination of protein-protein and protein-DNA interactions by yeast two-hybrid, in vitro pull-down and electrophoretic mobility shift assays revealed complex patterns of homo- and heterodimerization among orthologues of floral homeotic class B, class C and class E proteins and B(sister) proteins. Using DNase I footprint assays we demonstrate that both orthologues of class B with C proteins, and orthologues of class C proteins alone, but not orthologues of class B proteins alone can loop DNA in floral quartet-like complexes. This is in contrast to class B and class C proteins from angiosperms, which require other factors such as class E floral homeotic proteins to 'glue' them together in multimeric complexes. Our findings suggest that the evolutionary origin of floral quartet formation is based on the interaction of different DNA-bound homodimers, does not depend on class E proteins, and predates the origin of angiosperms. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Binding Selectivity of Methanobactin from Methylosinus trichosporium OB3b for Copper(I), Silver(I), Zinc(II), Nickel(II), Cobalt(II), Manganese(II), Lead(II), and Iron(II)

NASA Astrophysics Data System (ADS)

McCabe, Jacob W.; Vangala, Rajpal; Angel, Laurence A.

2017-12-01

Methanobactin (Mb) from Methylosinus trichosporium OB3b is a member of a class of metal binding peptides identified in methanotrophic bacteria. Mb will selectively bind and reduce Cu(II) to Cu(I), and is thought to mediate the acquisition of the copper cofactor for the enzyme methane monooxygenase. These copper chelating properties of Mb make it potentially useful as a chelating agent for treatment of diseases where copper plays a role including Wilson's disease, cancers, and neurodegenerative diseases. Utilizing traveling wave ion mobility-mass spectrometry (TWIMS), the competition for the Mb copper binding site from Ag(I), Pb(II), Co(II), Fe(II), Mn(II), Ni(II), and Zn(II) has been determined by a series of metal ion titrations, pH titrations, and metal ion displacement titrations. The TWIMS analyses allowed for the explicit identification and quantification of all the individual Mb species present during the titrations and measured their collision cross-sections and collision-induced dissociation patterns. The results showed Ag(I) and Ni(II) could irreversibly bind to Mb and not be effectively displaced by Cu(I), whereas Ag(I) could also partially displace Cu(I) from the Mb complex. At pH ≈ 6.5, the Mb binding selectivity follows the order Ag(I)≈Cu(I)>Ni(II)≈Zn(II)>Co(II)>>Mn(II)≈Pb(II)>Fe(II), and at pH 7.5 to 10.4 the order is Ag(I)>Cu(I)>Ni(II)>Co(II)>Zn(II)>Mn(II)≈Pb(II)>Fe(II). Breakdown curves of the disulfide reduced Cu(I) and Ag(I) complexes showed a correlation existed between their relative stability and their compact folded structure indicated by their CCS. Fluorescence spectroscopy, which allowed the determination of the binding constant, compared well with the TWIMS analyses, with the exception of the Ni(II) complex. [Figure not available: see fulltext.

Binding Selectivity of Methanobactin from Methylosinus trichosporium OB3b for Copper(I), Silver(I), Zinc(II), Nickel(II), Cobalt(II), Manganese(II), Lead(II), and Iron(II).

PubMed

McCabe, Jacob W; Vangala, Rajpal; Angel, Laurence A

2017-12-01

Methanobactin (Mb) from Methylosinus trichosporium OB3b is a member of a class of metal binding peptides identified in methanotrophic bacteria. Mb will selectively bind and reduce Cu(II) to Cu(I), and is thought to mediate the acquisition of the copper cofactor for the enzyme methane monooxygenase. These copper chelating properties of Mb make it potentially useful as a chelating agent for treatment of diseases where copper plays a role including Wilson's disease, cancers, and neurodegenerative diseases. Utilizing traveling wave ion mobility-mass spectrometry (TWIMS), the competition for the Mb copper binding site from Ag(I), Pb(II), Co(II), Fe(II), Mn(II), Ni(II), and Zn(II) has been determined by a series of metal ion titrations, pH titrations, and metal ion displacement titrations. The TWIMS analyses allowed for the explicit identification and quantification of all the individual Mb species present during the titrations and measured their collision cross-sections and collision-induced dissociation patterns. The results showed Ag(I) and Ni(II) could irreversibly bind to Mb and not be effectively displaced by Cu(I), whereas Ag(I) could also partially displace Cu(I) from the Mb complex. At pH ≈ 6.5, the Mb binding selectivity follows the order Ag(I)≈Cu(I)>Ni(II)≈Zn(II)>Co(II)>Mn(II)≈Pb(II)>Fe(II), and at pH 7.5 to 10.4 the order is Ag(I)>Cu(I)>Ni(II)>Co(II)>Zn(II)>Mn(II)≈Pb(II)>Fe(II). Breakdown curves of the disulfide reduced Cu(I) and Ag(I) complexes showed a correlation existed between their relative stability and their compact folded structure indicated by their CCS. Fluorescence spectroscopy, which allowed the determination of the binding constant, compared well with the TWIMS analyses, with the exception of the Ni(II) complex. Graphical abstract ᅟ.

Analysis of MHC class I folding: novel insights into intermediate forms

PubMed Central

Simone, Laura C.; Tuli, Amit; Simone, Peter D.; Wang, Xiaojian; Solheim, Joyce C.

2012-01-01

Folding around a peptide ligand is integral to the antigen presentation function of major histocompatibility complex (MHC) class I molecules. Several lines of evidence indicate that the broadly cross-reactive 34-1-2 antibody is sensitive to folding of the MHC class I peptide-binding groove. Here, we show that peptide-loading complex proteins associated with the murine MHC class I molecule Kd are found primarily in association with the 34-1-2+ form. This led us to hypothesize that the 34-1-2 antibody may recognize intermediately, as well as fully, folded MHC class I molecules. In order to further characterize the form(s) of MHC class I molecules recognized by 34-1-2, we took advantage of its cross-reactivity with Ld. Recognition of the open and folded forms of Ld by the 64-3-7 and 30-5-7 antibodies, respectively, has been extensively characterized, providing us with parameters against which to compare 34-1-2 reactivity. We found that the 34-1-2+ Ld molecules displayed characteristics indicative of incomplete folding, including increased tapasin association, endoplasmic reticulum retention, and instability at the cell surface. Moreover, we demonstrate that an Ld-specific peptide induced folding of the 34-1-2+ Ld intermediate. Altogether, these results yield novel insights into the nature of MHC class I molecules recognized by the 34-1-2 antibody. PMID:22329842

A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.

PubMed

Ross, Peter; Holmes, Jennifer C; Gojanovich, Gregory S; Hess, Paul R

2012-12-15

Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species. Copyright © 2012 Elsevier B.V. All rights reserved.

Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II.

PubMed

Saul, F A; Rovira, P; Boulot, G; Damme, E J; Peumans, W J; Truffa-Bachi, P; Bentley, G A

2000-06-15

Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes.

Nuclear binding of progesterone in hen oviduct. Binding to multiple sites in vitro.

PubMed Central

Pikler, G M; Webster, R A; Spelsberg, T C

1976-01-01

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1x10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1x10(-9)-1x10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei. PMID:182147

Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

PubMed Central

Chappell, Paul E; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antoni G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

2015-01-01

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

Narrow Groove and Restricted Anchors of MHC Class I Molecule BF2*0401 Plus Peptide Transporter Restriction can Explain Disease Susceptibility of B4 Chickens

PubMed Central

Zhang, Jianhua; Chen, Yong; Qi, Jianxun; Gao, Feng; Liu, Yanjie; Liu, Jun; Zhou, Xuyu; Kaufman, Jim; Xia, Chun; Gao, George F.

2016-01-01

The major histocompatibility complex (MHC) has genetic associations with many diseases, often due to differences in presentation of antigenic peptides by polymorphic MHC molecules to T lymphocytes of the immune system. In chickens, only a single classical class I molecule in each MHC haplotype is expressed well due to co-evolution with the polymorphic transporters associated with antigen presentation (TAPs), which means that resistance and susceptibility to infectious pathogens are particularly easy to observe. Previously, structures of chicken MHC class I molecule BF2*2101 from B21 haplotype showed an unusually large peptide-binding groove that accommodates a broad spectrum of peptides to present as epitopes to cytotoxic T lymphocytes (CTL), explaining the MHC-determined resistance of B21 chickens to Marek's disease. Here, we report the crystal structure of BF2*0401 from the B4 (also known as B13) haplotype, showing a highly positively-charged surface hitherto unobserved in other MHC molecules, as well as a remarkably narrow groove due to the allele-specific residues with bulky side chains. Together, these properties limit the number of epitope peptides that can bind this class I molecule. However, peptide-binding assays show that in vitro BF2*0401 can bind a wider variety of peptides than are found on the surface of B4 cells. Thus, a combination of the specificities of the polymorphic TAP transporter and the MHC results in a very limited set of BF2*0401 peptides with negatively charged anchors to be presented to T lymphocytes. PMID:23041567

Effect of chain length on the conformation and T cell recognition of synthetic hemagglutinin fragments

NASA Astrophysics Data System (ADS)

Tóth, Gábor K.; Holly, Sándor; Majer, Zsuzsa; Hollósi, Miklós; Rajnavölgyi, Éva; Laczkó, Ilona

2000-01-01

Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-E d major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N-terminal three residues resulted in a shift to an increased α-helical conformer population in the 317-329 H1 peptide and the breakage of the 3 10 or weakly H-bonded (nascent) α-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-E d-peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation.

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

PubMed

Murray, J S; Fois, S D S; Schountz, T; Ford, S R; Tawde, M D; Brown, J C; Siahaan, T J

2002-03-01

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.

Cloning of murine RNA polymerase I-specific TAF factors: Conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1

PubMed Central

Heix, Jutta; Zomerdijk, Joost C. B. M.; Ravanpay, Ali; Tjian, Robert; Grummt, Ingrid

1997-01-01

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP–TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein–protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP–TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription. PMID:9050847

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice.

PubMed

Harrison, L C; Honeyman, M C; Trembleau, S; Gregori, S; Gallazzi, F; Augstein, P; Brusic, V; Hammer, J; Adorini, L

1997-03-17

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity*S⃞

PubMed Central

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P.; Lewis, Timothy A.; Maglathin, Rebecca L.; McLean, Thomas H.; Bochkarev, Alexey; Plotnikov, Alexander N.; Vedadi, Masoud; Arrowsmith, Cheryl H.

2008-01-01

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators. PMID:18285338

Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity.

PubMed

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P; Lewis, Timothy A; Maglathin, Rebecca L; McLean, Thomas H; Bochkarev, Alexey; Plotnikov, Alexander N; Vedadi, Masoud; Arrowsmith, Cheryl H

2008-04-25

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy.

PubMed

Bowen, Alice M; Johnson, Eachan O D; Mercuri, Francesco; Hoskins, Nicola J; Qiao, Ruihong; McCullagh, James S O; Lovett, Janet E; Bell, Stephen G; Zhou, Weihong; Timmel, Christiane R; Wong, Luet Lok; Harmer, Jeffrey R

2018-02-21

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe 2 S 2 ] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe 2 S 2 ] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

PubMed

Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

2003-02-01

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

PubMed

Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

2017-11-28

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape. Copyright © 2017 Du et al.

HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail

PubMed Central

Roeth, Jeremiah F.; Williams, Maya; Kasper, Matthew R.; Filzen, Tracey M.; Collins, Kathleen L.

2004-01-01

To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the μ1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef–MHC-I complex is an important step required for inhibition of antigen presentation by HIV. PMID:15569716

Immunoinformatics Approach in Designing Epitope-based Vaccine Against Meningitis-inducing Bacteria (Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae Type b).

PubMed

Zahroh, Hilyatuz; Ma'rup, Ahmad; Tambunan, Usman Sumo Friend; Parikesit, Arli Aditya

2016-01-01

Meningitis infection is one of the major threats during Hajj season in Mecca. Meningitis vaccines are available, but their uses are limited in some countries due to religious reasons. Furthermore, they only give protection to certain serogroups, not to all types of meningitis-inducing bacteria. Recently, research on epitope-based vaccines has been developed intensively. Such vaccines have potential advantages over conventional vaccines in that they are safer to use and well responded to the antibody. In this study, we developed epitope-based vaccine candidates against various meningitis-inducing bacteria, including Streptococcus pneumoniae , Neisseria meningitidis , and Haemophilus influenzae type b. The epitopes were selected from their protein of polysaccharide capsule. B-cell epitopes were predicted by using BCPred, while T-cell epitope for major histocompatibility complex (MHC) class I was predicted using PAProC, TAPPred, and Immune Epitope Database. Immune Epitope Database was also used to predict T-cell epitope for MHC class II. Population coverage and molecular docking simulation were predicted against previously generated epitope vaccine candidates. The best candidates for MHC class I- and class II-restricted T-cell epitopes were MQYGDKTTF, MKEQNTLEI, ECTEGEPDY, DLSIVVPIY, YPMAMMWRNASNRAI, TLQMTLLGIVPNLNK, ETSLHHIPGISNYFI, and SLLYILEKNAEMEFD, which showed 80% population coverage. The complexes of class I T-cell epitopes-HLA-C*03:03 and class II T-cell epitopes-HLA-DRB1*11:01 showed better affinity than standards as evaluated from their Δ G binding value and the binding interaction between epitopes and HLA molecules. These peptide constructs may further be undergone in vitro and in vivo testings for the development of targeted vaccine against meningitis infection.

Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.

PubMed

Andreatta, Massimo; Lund, Ole; Nielsen, Morten

2013-01-01

Proteins recognizing short peptide fragments play a central role in cellular signaling. As a result of high-throughput technologies, peptide-binding protein specificities can be studied using large peptide libraries at dramatically lower cost and time. Interpretation of such large peptide datasets, however, is a complex task, especially when the data contain multiple receptor binding motifs, and/or the motifs are found at different locations within distinct peptides. The algorithm presented in this article, based on Gibbs sampling, identifies multiple specificities in peptide data by performing two essential tasks simultaneously: alignment and clustering of peptide data. We apply the method to de-convolute binding motifs in a panel of peptide datasets with different degrees of complexity spanning from the simplest case of pre-aligned fixed-length peptides to cases of unaligned peptide datasets of variable length. Example applications described in this article include mixtures of binders to different MHC class I and class II alleles, distinct classes of ligands for SH3 domains and sub-specificities of the HLA-A*02:01 molecule. The Gibbs clustering method is available online as a web server at http://www.cbs.dtu.dk/services/GibbsCluster.

Fine specificity of antigen binding to two class I major histocompatibility proteins (B*2705 and B*2703) differing in a single amino acid residue

NASA Astrophysics Data System (ADS)

Rognan, Didier; Krebs, Stefan; Kuonen, Oliver; Lamas, , José R.; Castro, José A. López de; Folkers, Gerd

1997-09-01

Starting from the X-ray structure of a class I majorhistocompatibility complex (MHC)-encoded protein (HLA-B*2705), a naturallypresented self-nonapeptide and two synthetic analogues were simulated in thebinding groove of two human leukocyte antigen (HLA) alleles (B*2703 andB*2705) differing in a single amino acid residue. After 200 ps moleculardynamics simulations of the solvated HLA-peptide pairs, some molecularproperties of the complexes (distances between ligand and protein center ofmasses, atomic fluctuations, buried versus accessible surface areas,hydrogen-bond frequencies) allow a clear discrimination of potent from weakMHC binders. The binding specificity of the three nonapeptides for the twoHLA alleles could be explained by the disruption of one hydrogen-bondingnetwork in the binding pocket of the HLA-B*2705 protein where the singlemutation occurs. Rearrangements of interactions in the B pocket, which bindsthe side chain of peptidic residue 2, and a weakening of interactionsinvolving the C-terminal end of the peptide also took place. In addition,extension of the peptide backbone using a β-Ala analogue did notabolish binding to any of the two HLA-B27 subtypes, but increased theselectivity for B*2703, as expected from the larger peptide binding groovein this subtype. A better understanding of the atomic details involved inpeptide selection by closely related HLA alleles is of crucial importancefor unraveling the molecular features linking particular HLA alleles toautoimmune diseases, and for the identification of antigenic peptidestriggering such pathologies.

Recovery of known T-cell epitopes by computational scanning of a viral genome

NASA Astrophysics Data System (ADS)

Logean, Antoine; Rognan, Didier

2002-04-01

A new computational method (EpiDock) is proposed for predicting peptide binding to class I MHC proteins, from the amino acid sequence of any protein of immunological interest. Starting from the primary structure of the target protein, individual three-dimensional structures of all possible MHC-peptide (8-, 9- and 10-mers) complexes are obtained by homology modelling. A free energy scoring function (Fresno) is then used to predict the absolute binding free energy of all possible peptides to the class I MHC restriction protein. Assuming that immunodominant epitopes are usually found among the top MHC binders, the method can thus be applied to predict the location of immunogenic peptides on the sequence of the protein target. When applied to the prediction of HLA-A*0201-restricted T-cell epitopes from the Hepatitis B virus, EpiDock was able to recover 92% of known high affinity binders and 80% of known epitopes within a filtered subset of all possible nonapeptides corresponding to about one tenth of the full theoretical list. The proposed method is fully automated and fast enough to scan a viral genome in less than an hour on a parallel computing architecture. As it requires very few starting experimental data, EpiDock can be used: (i) to predict potential T-cell epitopes from viral genomes (ii) to roughly predict still unknown peptide binding motifs for novel class I MHC alleles.

Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes

PubMed Central

Chuang, Jen-Zen; Vega, Carrie; Jun, Wenjin; Sung, Ching-Hwa

2004-01-01

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations. PMID:15232620

T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions

PubMed Central

Ferber, Mathias; Zoete, Vincent; Michielin, Olivier

2012-01-01

Crystallographic data about T-Cell Receptor – peptide – major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes. PMID:23251658

T-cell receptors binding orientation over peptide/MHC class I is driven by long-range interactions.

PubMed

Ferber, Mathias; Zoete, Vincent; Michielin, Olivier

2012-01-01

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

Contrasting evolutionary histories of MHC class I and class II loci in grouse—Effects of selection and gene conversion

USGS Publications Warehouse

Minias, Piotr; Bateson, Zachary W.; Whittingham, Linda A.; Johnson, Jeff A.; Oyler-McCance, Sara J.; Dunn, Peter O.

2016-01-01

Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

Contrasting evolutionary histories of MHC class I and class II loci in grouse—effects of selection and gene conversion

PubMed Central

Minias, P; Bateson, Z W; Whittingham, L A; Johnson, J A; Oyler-McCance, S; Dunn, P O

2016-01-01

Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens. PMID:26860199

Automated benchmarking of peptide-MHC class I binding predictions.

PubMed

Trolle, Thomas; Metushi, Imir G; Greenbaum, Jason A; Kim, Yohan; Sidney, John; Lund, Ole; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

2015-07-01

Numerous in silico methods predicting peptide binding to major histocompatibility complex (MHC) class I molecules have been developed over the last decades. However, the multitude of available prediction tools makes it non-trivial for the end-user to select which tool to use for a given task. To provide a solid basis on which to compare different prediction tools, we here describe a framework for the automated benchmarking of peptide-MHC class I binding prediction tools. The framework runs weekly benchmarks on data that are newly entered into the Immune Epitope Database (IEDB), giving the public access to frequent, up-to-date performance evaluations of all participating tools. To overcome potential selection bias in the data included in the IEDB, a strategy was implemented that suggests a set of peptides for which different prediction methods give divergent predictions as to their binding capability. Upon experimental binding validation, these peptides entered the benchmark study. The benchmark has run for 15 weeks and includes evaluation of 44 datasets covering 17 MHC alleles and more than 4000 peptide-MHC binding measurements. Inspection of the results allows the end-user to make educated selections between participating tools. Of the four participating servers, NetMHCpan performed the best, followed by ANN, SMM and finally ARB. Up-to-date performance evaluations of each server can be found online at http://tools.iedb.org/auto_bench/mhci/weekly. All prediction tool developers are invited to participate in the benchmark. Sign-up instructions are available at http://tools.iedb.org/auto_bench/mhci/join. mniel@cbs.dtu.dk or bpeters@liai.org Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Automated benchmarking of peptide-MHC class I binding predictions

PubMed Central

Trolle, Thomas; Metushi, Imir G.; Greenbaum, Jason A.; Kim, Yohan; Sidney, John; Lund, Ole; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

2015-01-01

Motivation: Numerous in silico methods predicting peptide binding to major histocompatibility complex (MHC) class I molecules have been developed over the last decades. However, the multitude of available prediction tools makes it non-trivial for the end-user to select which tool to use for a given task. To provide a solid basis on which to compare different prediction tools, we here describe a framework for the automated benchmarking of peptide-MHC class I binding prediction tools. The framework runs weekly benchmarks on data that are newly entered into the Immune Epitope Database (IEDB), giving the public access to frequent, up-to-date performance evaluations of all participating tools. To overcome potential selection bias in the data included in the IEDB, a strategy was implemented that suggests a set of peptides for which different prediction methods give divergent predictions as to their binding capability. Upon experimental binding validation, these peptides entered the benchmark study. Results: The benchmark has run for 15 weeks and includes evaluation of 44 datasets covering 17 MHC alleles and more than 4000 peptide-MHC binding measurements. Inspection of the results allows the end-user to make educated selections between participating tools. Of the four participating servers, NetMHCpan performed the best, followed by ANN, SMM and finally ARB. Availability and implementation: Up-to-date performance evaluations of each server can be found online at http://tools.iedb.org/auto_bench/mhci/weekly. All prediction tool developers are invited to participate in the benchmark. Sign-up instructions are available at http://tools.iedb.org/auto_bench/mhci/join. Contact: mniel@cbs.dtu.dk or bpeters@liai.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25717196

Emerging Major Histocompatibility Complex Class I-Related Functions of NLRC5.

PubMed

Chelbi, S T; Dang, A T; Guarda, G

2017-01-01

Recent evidence demonstrates a key role for the nucleotide-binding oligomerization domain-like receptor (NLR) family member NLRC5 (NLR family, CARD domain containing protein 5) in the transcriptional regulation of major histocompatibility complex (MHC) class I and related genes. Detailed information on NLRC5 target genes in various cell types and conditions is emerging. Thanks to its analogy to CIITA (class II major MHC transactivator), a NLR family member known for over 20 years to be the master regulator of MHC class II gene transcription, also the molecular mechanisms underlying NLRC5 function are being rapidly unraveled. MHC class I molecules are crucial in regulating innate and adaptive cytotoxic responses. Whereas CD8 + T cells detect antigens presented on MHC class I molecules by infected or transformed cells, natural killer (NK) lymphocytes eliminate target cells with downregulated MHC class I expression. Data uncovering the relevance of NLRC5 in homeostasis and activity of these two lymphocyte subsets have been recently reported. Given the importance of CD8 + T and NK cells in controlling infection and cancer, it is not surprising that NLRC5 is also starting to emerge as a central player in these diseases. This chapter summarizes and discusses novel insights into the molecular mechanisms underlying NLRC5 activity and its relevance to pathological conditions. A thorough understanding of both aspects is essential to evaluate the clinical significance and therapeutic potential of NLRC5. © 2017 Elsevier Inc. All rights reserved.

Structure of a Clostridium botulinum C143S thiaminase I/thiamin complex reveals active site architecture .

PubMed

Sikowitz, Megan D; Shome, Brateen; Zhang, Yang; Begley, Tadhg P; Ealick, Steven E

2013-11-05

Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II.

The most common Chinese rhesus macaque MHC class I molecule shares peptide binding repertoire with the HLA-B7 supertype

PubMed Central

Solomon, Christopher; Southwood, Scott; Hoof, Ilka; Rudersdorf, Richard; Peters, Bjoern; Sidney, John; Pinilla, Clemencia; Marcondes, Maria Cecilia Garibaldi; Ling, Binhua; Marx, Preston; Sette, Alessandro

2010-01-01

Of the two rhesus macaque subspecies used for AIDS studies, the Simian immunodeficiency virus-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection, providing both insight into pathogenesis and a system for testing novel vaccines. Despite the Chinese rhesus macaque potentially being a more relevant model for AIDS outcomes than the Indian rhesus macaque, the Chinese-origin rhesus macaques have not been well-characterized for their major histocompatibility complex (MHC) composition and function, reducing their greater utilization. In this study, we characterized a total of 50 unique Chinese rhesus macaques from several varying origins for their entire MHC class I allele composition and identified a total of 58 unique complete MHC class I sequences. Only nine of the sequences had been associated with Indian rhesus macaques, and 28/58 (48.3%) of the sequences identified were novel. From all MHC alleles detected, we prioritized Mamu-A1*02201 for functional characterization based on its higher frequency of expression. Upon the development of MHC/peptide binding assays and definition of its associated motif, we revealed that this allele shares peptide binding characteristics with the HLA-B7 supertype, the most frequent supertype in human populations. These studies provide the first functional characterization of an MHC class I molecule in the context of Chinese rhesus macaques and the first instance of HLA-B7 analogy for rhesus macaques. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0450-3) contains supplementary material, which is available to authorized users. PMID:20480161

Methods for quantifying T cell receptor binding affinities and thermodynamics

PubMed Central

Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

2013-01-01

αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

Subtle Changes in Peptide Conformation Profoundly Affect Recognition of the Non-Classical MHC Class I Molecule HLA-E by the CD94-NKG2 Natural Killer Cell Receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoare, Hilary L; Sullivan, Lucy C; Clements, Craig S

2008-03-31

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I molecule that binds peptides derived from the leader sequences of other HLA class I molecules. Natural killer cell recognition of these HLA-E molecules, via the CD94-NKG2 natural killer family, represents a central innate mechanism for monitoring major histocompatibility complex expression levels within a cell. The leader sequence-derived peptides bound to HLA-E exhibit very limited polymorphism, yet subtle differences affect the recognition of HLA-E by the CD94-NKG2 receptors. To better understand the basis for this peptide-specific recognition, we determined the structure of HLA-E in complex with two leader peptides,more » namely, HLA-Cw*07 (VMAPRALLL), which is poorly recognised by CD94-NKG2 receptors, and HLA-G*01 (VMAPRTLFL), a high-affinity ligand of CD94-NKG2 receptors. A comparison of these structures, both of which were determined to 2.5-Å resolution, revealed that allotypic variations in the bound leader sequences do not result in conformational changes in the HLA-E heavy chain, although subtle changes in the conformation of the peptide within the binding groove of HLA-E were evident. Accordingly, our data indicate that the CD94-NKG2 receptors interact with HLA-E in a manner that maximises the ability of the receptors to discriminate between subtle changes in both the sequence and conformation of peptides bound to HLA-E.« less

KIR Polymorphisms Modulate Peptide-Dependent Binding to an MHC Class I Ligand with a Bw6 Motif

PubMed Central

Colantonio, Arnaud D.; Bimber, Benjamin N.; Neidermyer, William J.; Reeves, R. Keith; Alter, Galit; Altfeld, Marcus; Johnson, R. Paul; Carrington, Mary; O'Connor, David H.; Evans, David T.

2011-01-01

Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05+ macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets. PMID:21423672

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires.

PubMed

Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F; Sette, Alessandro

2015-11-01

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires

PubMed Central

Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M.; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F.; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F.; Sette, Alessandro

2016-01-01

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif. PMID:26399241

Structural basis for NKG2A/CD94 recognition of HLA-E

PubMed Central

Kaiser, Brett K.; Pizarro, Juan Carlos; Kerns, Julie; Strong, Roland K.

2008-01-01

The NKG2x/CD94 (x = A, C, E) natural killer-cell receptors perform an important role in immunosurveillance by binding to HLA-E complexes that exclusively present peptides derived from MHC class I leader sequences, thereby monitoring MHC class I expression. We have determined the crystal structure of the NKG2A/CD94/HLA-E complex at 4.4-Å resolution, revealing two critical aspects of this interaction. First, the C-terminal region of the peptide, which displays the most variability among class I leader sequences, interacts entirely with CD94, the invariant component of these receptors. Second, residues 167–170 of NKG2A/C account for the ≈6-fold-higher affinity of the inhibitory NKG2A/CD94 receptor compared to its activating NKG2C/CD94 counterpart. These residues do not contact HLA-E or peptide directly but instead form part of the heterodimer interface with CD94. An evolutionary analysis across primates reveals that whereas CD94 is evolving under purifying selection, both NKG2A and NKG2C are evolving under positive selection. Specifically, residues at the CD94 interface have evolved under positive selection, suggesting that the evolution of these genes is driven by an interaction with pathogen-derived ligands. Consistent with this possibility, we show that NKG2C/CD94, but not NKG2A/CD94, weakly but specifically binds to the CMV MHC-homologue UL18. Thus, the evolution of the NKG2x/CD94 family of receptors has likely been shaped both by the need to bind the invariant HLA-E ligand and the need to avoid subversion by pathogen-derived decoys. PMID:18448674

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

PubMed

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

PubMed

Srikrishna, G; Panneerselvam, K; Westphal, V; Abraham, V; Varki, A; Freeze, H H

2001-04-01

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

NASA Technical Reports Server (NTRS)

Wright, A. D.; Chapes, S. K.

1999-01-01

The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis. Copyright 1999 Academic Press.

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

PubMed

Bergmann, Tobias; Lindvall, Mikaela; Moore, Erin; Moore, Eugene; Sidney, John; Miller, Donald; Tallmadge, Rebecca L; Myers, Paisley T; Malaker, Stacy A; Shabanowitz, Jeffrey; Osterrieder, Nikolaus; Peters, Bjoern; Hunt, Donald F; Antczak, Douglas F; Sette, Alessandro

2017-05-01

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

PubMed Central

Hilton, Hugo G; Parham, Peter

2013-01-01

Monoclonal antibodies with specificity for HLA class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing 3-6 HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with one of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1μg/ml) PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50μg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23, and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. PMID:23510417

A common minimal motif for the ligands of HLA-B*27 class I molecules.

PubMed

Barriga, Alejandro; Lorente, Elena; Johnstone, Carolina; Mir, Carmen; del Val, Margarita; López, Daniel

2014-01-01

CD8(+) T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.

Accurate approximation method for prediction of class I MHC affinities for peptides of length 8, 10 and 11 using prediction tools trained on 9mers.

PubMed

Lundegaard, Claus; Lund, Ole; Nielsen, Morten

2008-06-01

Several accurate prediction systems have been developed for prediction of class I major histocompatibility complex (MHC):peptide binding. Most of these are trained on binding affinity data of primarily 9mer peptides. Here, we show how prediction methods trained on 9mer data can be used for accurate binding affinity prediction of peptides of length 8, 10 and 11. The method gives the opportunity to predict peptides with a different length than nine for MHC alleles where no such peptides have been measured. As validation, the performance of this approach is compared to predictors trained on peptides of the peptide length in question. In this validation, the approximation method has an accuracy that is comparable to or better than methods trained on a peptide length identical to the predicted peptides. The algorithm has been implemented in the web-accessible servers NetMHC-3.0: http://www.cbs.dtu.dk/services/NetMHC-3.0, and NetMHCpan-1.1: http://www.cbs.dtu.dk/services/NetMHCpan-1.1

Structure of the glucagon receptor in complex with a glucagon analogue.

PubMed

Zhang, Haonan; Qiao, Anna; Yang, Linlin; Van Eps, Ned; Frederiksen, Klaus S; Yang, Dehua; Dai, Antao; Cai, Xiaoqing; Zhang, Hui; Yi, Cuiying; Cao, Can; He, Lingli; Yang, Huaiyu; Lau, Jesper; Ernst, Oliver P; Hanson, Michael A; Stevens, Raymond C; Wang, Ming-Wei; Reedtz-Runge, Steffen; Jiang, Hualiang; Zhao, Qiang; Wu, Beili

2018-01-03

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

Dynamics of major histocompatibility complex class I association with the human peptide-loading complex.

PubMed

Panter, Michaela S; Jain, Ankur; Leonhardt, Ralf M; Ha, Taekjip; Cresswell, Peter

2012-09-07

Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.

Herpes B Virus, Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

PubMed Central

Vasireddi, Mugdha

2012-01-01

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8+ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions. PMID:22973043

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

PubMed

Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas; Wiig, Jared A; Buller, Andrew R; Einsle, Oliver; Hu, Yilin; Ribbe, Markus W; Arnold, Frances H

2015-06-15

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site. © The Authors Journal Compilation © 2015 Biochemical Society.

MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris).

PubMed

Pokorny, Ina; Sharma, Reeta; Goyal, Surendra Prakash; Mishra, Sudanshu; Tiedemann, Ralph

2010-10-01

Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.

NetMHCstab – predicting stability of peptide–MHC-I complexes; impacts for cytotoxic T lymphocyte epitope discovery

PubMed Central

Jørgensen, Kasper W; Rasmussen, Michael; Buus, Søren; Nielsen, Morten

2014-01-01

Major histocompatibility complex class I (MHC-I) molecules play an essential role in the cellular immune response, presenting peptides to cytotoxic T lymphocytes (CTLs) allowing the immune system to scrutinize ongoing intracellular production of proteins. In the early 1990s, immunogenicity and stability of the peptide–MHC-I (pMHC-I) complex were shown to be correlated. At that time, measuring stability was cumbersome and time consuming and only small data sets were analysed. Here, we investigate this fairly unexplored area on a large scale compared with earlier studies. A recent small-scale study demonstrated that pMHC-I complex stability was a better correlate of CTL immunogenicity than peptide–MHC-I affinity. We here extended this study and analysed a total of 5509 distinct peptide stability measurements covering 10 different HLA class I molecules. Artificial neural networks were used to construct stability predictors capable of predicting the half-life of the pMHC-I complex. These predictors were shown to predict T-cell epitopes and MHC ligands from SYFPEITHI and IEDB to form significantly more stable MHC-I complexes compared with affinity-matched non-epitopes. Combining the stability predictions with a state-of-the-art affinity predictions NetMHCcons significantly improved the performance for identification of T-cell epitopes and ligands. For the HLA alleles included in the study, we could identify distinct sub-motifs that differentiate between stable and unstable peptide binders and demonstrate that anchor positions in the N-terminal of the binding motif (primarily P2 and P3) play a critical role for the formation of stable pMHC-I complexes. A webserver implementing the method is available at http://www.cbs.dtu.dk/services/NetMHCstab. PMID:23927693

Recognition of peptide–MHC class I complexes by activating killer immunoglobulin-like receptors

PubMed Central

Stewart, C. Andrew; Laugier-Anfossi, Fanny; Vély, Frédéric; Saulquin, Xavier; Riedmuller, Jenifer; Tisserant, Agnès; Gauthier, Laurent; Romagné, François; Ferracci, Géraldine; Arosa, Fernando A.; Moretta, Alessandro; Sun, Peter D.; Ugolini, Sophie; Vivier, Eric

2005-01-01

Inhibitory receptors for MHC class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors, including the human killer Ig-like receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein–Barr virus leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide–MHC class I complexes on Epstein–Barr virus-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer-based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self-MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK cell activation. We discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders. PMID:16141329

Recognition of peptide-MHC class I complexes by activating killer immunoglobulin-like receptors.

PubMed

Stewart, C Andrew; Laugier-Anfossi, Fanny; Vély, Frédéric; Saulquin, Xavier; Riedmuller, Jenifer; Tisserant, Agnès; Gauthier, Laurent; Romagné, François; Ferracci, Géraldine; Arosa, Fernando A; Moretta, Alessandro; Sun, Peter D; Ugolini, Sophie; Vivier, Eric

2005-09-13

Inhibitory receptors for MHC class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors, including the human killer Ig-like receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein-Barr virus leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide-MHC class I complexes on Epstein-Barr virus-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer-based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self-MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK cell activation. We discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders.

The MTA family proteins as novel histone H3 binding proteins.

PubMed

Wu, Meng; Wang, Lina; Li, Qian; Li, Jiwen; Qin, Jun; Wong, Jiemin

2013-01-03

The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

The MTA family proteins as novel histone H3 binding proteins

PubMed Central

2013-01-01

Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail. PMID:23286669

A HLA class I cis-regulatory element whose activity can be modulated by hormones.

PubMed

Sim, B C; Hui, K M

1994-12-01

To elucidate the basis of the down-regulation in major histocompatibility complex (MHC) class I gene expression and to identify possible DNA-binding regulatory elements that have the potential to interact with class I MHC genes, we have studied the transcriptional regulation of class I HLA genes in human breast carcinoma cells. A 9 base pair (bp) negative cis-regulatory element (NRE) has been identified using band-shift assays employing DNA sequences derived from the 5'-flanking region of HLA class I genes. This 9-bp element, GTCATGGCG, located within exon I of the HLA class I gene, can potently inhibit the expression of a heterologous thymidine kinase (TK) gene promoter and the HLA enhancer element. Furthermore, this regulatory element can exert its suppressive function in either the sense or anti-sense orientation. More interestingly, NRE can suppress dexamethasone-mediated gene activation in the context of the reported glucocorticoid-responsive element (GRE) in MCF-7 cells but has no influence on the estrogen-mediated transcriptional activation of MCF-7 cells in the context of the reported estrogen-responsive element (ERE). Furthermore, the presence of such a regulatory element within the HLA class I gene whose activity can be modulated by hormones correlates well with our observation that the level of HLA class I gene expression can be down-regulated by hormones in human breast carcinoma cells. Such interactions between negative regulatory elements and specific hormone trans-activators are novel and suggest a versatile form of transcriptional control.

A Signal Peptide Derived from hsp60 Binds HLA-E and Interferes with CD94/NKG2A Recognition

PubMed Central

Michaëlsson, Jakob; Teixeira de Matos, Cristina; Achour, Adnane; Lanier, Lewis L.; Kärre, Klas; Söderström, Kalle

2002-01-01

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule which presents a restricted set of nonameric peptides, derived mainly from the signal sequence of other MHC class I molecules. It interacts with CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and T cell subsets. Here we demonstrate that HLA-E also presents a peptide derived from the leader sequence of human heat shock protein 60 (hsp60). This peptide gains access to HLA-E intracellularly, resulting in up-regulated HLA-E/hsp60 signal peptide cell-surface levels on stressed cells. Notably, HLA-E molecules in complex with the hsp60 signal peptide are no longer recognized by CD94/NKG2A inhibitory receptors. Thus, during cellular stress an increased proportion of HLA-E molecules may bind the nonprotective hsp60 signal peptide, leading to a reduced capacity to inhibit a major NK cell population. Such stress induced peptide interference would gradually uncouple CD94/NKG2A inhibitory recognition and provide a mechanism for NK cells to detect stressed cells in a peptide-dependent manner. PMID:12461076

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide.

PubMed

von Ossowski, Ingemar; Oksanen, Esko; von Ossowski, Lotta; Cai, Chunlin; Sundberg, Maria; Goldman, Adrian; Keinänen, Kari

2006-11-01

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.

NetMHCcons: a consensus method for the major histocompatibility complex class I predictions.

PubMed

Karosiene, Edita; Lundegaard, Claus; Lund, Ole; Nielsen, Morten

2012-03-01

A key role in cell-mediated immunity is dedicated to the major histocompatibility complex (MHC) molecules that bind peptides for presentation on the cell surface. Several in silico methods capable of predicting peptide binding to MHC class I have been developed. The accuracy of these methods depends on the data available characterizing the binding specificity of the MHC molecules. It has, moreover, been demonstrated that consensus methods defined as combinations of two or more different methods led to improved prediction accuracy. This plethora of methods makes it very difficult for the non-expert user to choose the most suitable method for predicting binding to a given MHC molecule. In this study, we have therefore made an in-depth analysis of combinations of three state-of-the-art MHC-peptide binding prediction methods (NetMHC, NetMHCpan and PickPocket). We demonstrate that a simple combination of NetMHC and NetMHCpan gives the highest performance when the allele in question is included in the training and is characterized by at least 50 data points with at least ten binders. Otherwise, NetMHCpan is the best predictor. When an allele has not been characterized, the performance depends on the distance to the training data. NetMHCpan has the highest performance when close neighbours are present in the training set, while the combination of NetMHCpan and PickPocket outperforms either of the two methods for alleles with more remote neighbours. The final method, NetMHCcons, is publicly available at www.cbs.dtu.dk/services/NetMHCcons , and allows the user in an automatic manner to obtain the most accurate predictions for any given MHC molecule.

MHC class I–specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice

PubMed Central

Strait, Richard T.; Hicks, Wyenona; Barasa, Nathaniel; Mahler, Ashley; Khodoun, Marat; Köhl, Jörg; Stringer, Keith; Witte, David; Van Rooijen, Nico; Susskind, Brian M.

2011-01-01

Transfusion-related acute lung injury (TRALI), a form of noncardiogenic pulmonary edema that develops during or within 6 h after a blood transfusion, is the most frequent cause of transfusion-associated death in the United States. Because development of TRALI is associated with donor antibodies (Abs) reactive with recipient major histocompatibility complex (MHC), a mouse model has been studied in which TRALI-like disease is caused by injecting mice with anti–MHC class I monoclonal Ab (mAb). Previous publications with this model have concluded that disease is caused by FcR-dependent activation of neutrophils and platelets, with production of reactive oxygen species that damage pulmonary vascular endothelium. In this study, we confirm the role of reactive oxygen species in the pathogenesis of this mouse model of TRALI and show ultrastructural evidence of pulmonary vascular injury within 5 min of anti–MHC class I mAb injection. However, we demonstrate that disease induction in this model involves macrophages rather than neutrophils or platelets, activation of complement and production of C5a rather than activation of FcγRI, FcγRIII, or FcγRIV, and binding of anti–MHC class I mAb to non-BM–derived cells such as pulmonary vascular endothelium. These observations have important implications for the prevention and treatment of TRALI. PMID:22025304

ABC transporters and immunity: mechanism of self-defense.

PubMed

Hinz, Andreas; Tampé, Robert

2012-06-26

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.

Structural and Evolutionary Aspects of Antenna Chromophore Usage by Class II Photolyases*

PubMed Central

Kiontke, Stephan; Gnau, Petra; Haselsberger, Reinhard; Batschauer, Alfred; Essen, Lars-Oliver

2014-01-01

Light-harvesting and resonance energy transfer to the catalytic FAD cofactor are key roles for the antenna chromophores of light-driven DNA photolyases, which remove UV-induced DNA lesions. So far, five chemically diverse chromophores have been described for several photolyases and related cryptochromes, but no correlation between phylogeny and used antenna has been found. Despite a common protein topology, structural analysis of the distantly related class II photolyase from the archaeon Methanosarcina mazei (MmCPDII) as well as plantal orthologues indicated several differences in terms of DNA and FAD binding and electron transfer pathways. For MmCPDII we identify 8-hydroxydeazaflavin (8-HDF) as cognate antenna by in vitro and in vivo reconstitution, whereas the higher plant class II photolyase from Arabidopsis thaliana fails to bind any of the known chromophores. According to the 1.9 Å structure of the MmCPDII·8-HDF complex, its antenna binding site differs from other members of the photolyase-cryptochrome superfamily by an antenna loop that changes its conformation by 12 Å upon 8-HDF binding. Additionally, so-called N- and C-motifs contribute as conserved elements to the binding of deprotonated 8-HDF and allow predicting 8-HDF binding for most of the class II photolyases in the whole phylome. The 8-HDF antenna is used throughout the viridiplantae ranging from green microalgae to bryophyta and pteridophyta, i.e. mosses and ferns, but interestingly not in higher plants. Overall, we suggest that 8-hydroxydeazaflavin is a crucial factor for the survival of most higher eukaryotes which depend on class II photolyases to struggle with the genotoxic effects of solar UV exposure. PMID:24849603

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

PubMed Central

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J.; Kang, Seongman; Ahn, Kwangseog

2006-01-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells. PMID:16699020

A short isoform of human cytomegalovirus US3 functions as a dominant negative inhibitor of the full-length form.

PubMed

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J; Kang, Seongman; Ahn, Kwangseog

2006-06-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.

Antiviral CD8+ T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion.

PubMed

Ranasinghe, Srinika; Lamothe, Pedro A; Soghoian, Damien Z; Kazer, Samuel W; Cole, Michael B; Shalek, Alex K; Yosef, Nir; Jones, R Brad; Donaghey, Faith; Nwonu, Chioma; Jani, Priya; Clayton, Gina M; Crawford, Frances; White, Janice; Montoya, Alana; Power, Karen; Allen, Todd M; Streeck, Hendrik; Kaufmann, Daniel E; Picker, Louis J; Kappler, John W; Walker, Bruce D

2016-10-18

CD8 + T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8 + T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor β (TCRβ) analysis revealed that class II-restricted CD8 + T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8 + T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8 + T cell responses can exist in a chronic human viral infection, and may contribute to immune control. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

PubMed Central

1986-01-01

A collection of 17 monoclonal antibodies elicited against the light- harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC- II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b. PMID:3528171

Characterization of a Nonclassical Class I MHC Gene in a Reptile, the Galápagos Marine Iguana (Amblyrhynchus cristatus)

PubMed Central

Glaberman, Scott; Du Pasquier, Louis; Caccone, Adalgisa

2008-01-01

Squamates are a diverse order of vertebrates, representing more than 7,000 species. Yet, descriptions of full-length major histocompatibility complex (MHC) genes in this group are nearly absent from the literature, while the number of MHC studies continues to rise in other vertebrate taxa. The lack of basic information about MHC organization in squamates inhibits investigation into the relationship between MHC polymorphism and disease, and leaves a large taxonomic gap in our understanding of amniote MHC evolution. Here, we use both cDNA and genomic sequence data to characterize a class I MHC gene (Amcr-UA) from the Galápagos marine iguana, a member of the squamate subfamily Iguaninae. Amcr-UA appears to be functional since it is expressed in the blood and contains many of the conserved peptide-binding residues that are found in classical class I genes of other vertebrates. In addition, comparison of Amcr-UA to homologous sequences from other iguanine species shows that the antigen-binding portion of this gene is under purifying selection, rather than balancing selection, and therefore may have a conserved function. A striking feature of Amcr-UA is that both the cDNA and genomic sequences lack the transmembrane and cytoplasmic domains that are necessary to anchor the class I receptor molecule into the cell membrane, suggesting that the product of this gene is secreted and consequently not involved in classical class I antigen-presentation. The truncated and conserved character of Amcr-UA lead us to define it as a nonclassical gene that is related to the few available squamate class I sequences. However, phylogenetic analysis placed Amcr-UA in a basal position relative to other published classical MHC genes from squamates, suggesting that this gene diverged near the beginning of squamate diversification. PMID:18682845

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification.

PubMed

Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael; Stryhn, Anette; Buus, Søren; Nielsen, Morten

2015-11-01

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .

Rational design of class I MHC ligands

NASA Astrophysics Data System (ADS)

Rognan, D.; Scapozza, L.; Folkers, G.; Daser, Angelika

1995-04-01

From the knowledge of the three-dimensional structure of a class I MHC protein, several non natural peptides were designed in order to either optimize the interactions of one secondary anchor amino acid with its HLA binding pocket or to substitute the non interacting part with spacer residues. All peptides were synthesized and tested for binding to the class I MHC protein in an in vitro reconstitution assay. As predicted, the non natural peptides present an enhanced binding to the HLA-B27 molecule with respect to their natural parent peptides. This study constitutes the first step towards the rational design of non peptidic MHC ligands that should be very promising tools for the selective immunotherapy of autoimmune diseases.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

PubMed

Hilton, H G; Parham, P

2013-04-01

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels

PubMed Central

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L.

2014-01-01

SUMMARY AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine and phenylalanine which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate and serine. AvGluR1 LBD crystal structures reveal a novel scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, while in the alanine, methionine and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl− lowers affinity for these ligands, but not for glutamate, aspartate or for phenylalanine which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure based studies on iGluR-ligand interactions. PMID:23434404

The Principal Genetic Determinants for Nasopharyngeal Carcinoma in China Involve the HLA Class I Antigen Recognition Groove

PubMed Central

Tang, Minzhong; Lautenberger, James A.; Gao, Xiaojiang; Sezgin, Efe; Hendrickson, Sher L.; Troyer, Jennifer L.; David, Victor A.; Guan, Li; Mcintosh, Carl E.; Guo, Xiuchan; Zheng, Yuming; Liao, Jian; Deng, Hong; Malasky, Michael; Kessing, Bailey; Winkler, Cheryl A.; Carrington, Mary; dé The, Guy; Zeng, Yi; O'Brien, Stephen J.

2012-01-01

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA –B, and HLA -C class I genes (PHLA-A-aa-site-62 = 7.4×10−29; P HLA-B-aa-site-116 = 6.5×10−19; P HLA-C-aa-site-156 = 6.8×10−8 respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China. PMID:23209447

Distinct Conformations of Ly49 Natural Killer Cell Receptors Mediate MHC Class I Recognition in Trans and Cis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Back, J.; Malchiodi, E; Cho, S

2009-01-01

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors andmore » explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.« less

Redox modulation of plant developmental regulators from the class I TCP transcription factor family.

PubMed

Viola, Ivana L; Güttlein, Leandro N; Gonzalez, Daniel H

2013-07-01

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants.

Redox Modulation of Plant Developmental Regulators from the Class I TCP Transcription Factor Family1[W][OA

PubMed Central

Viola, Ivana L.; Güttlein, Leandro N.; Gonzalez, Daniel H.

2013-01-01

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants. PMID:23686421

Binding and activation of major histocompatibility complex class II-deficient macrophages by staphylococcal exotoxins

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Macrophages from C2D transgenic mice deficient in the expression of major histocompatibility complex (MHC) class II proteins were used to identify binding sites for superantigens distinct from the MHC class II molecule. Iodinated staphylococcal enterotoxins A and B (SEA and SEB) and exfoliative toxins A and B (ETA and ETB) bound to C2D macrophages in a concentration-dependent and competitive manner. All four toxins increased F-actin concentration within 30 s of their addition to C2D macrophages, indicating that signal transduction occurred in response to toxin in the absence of class II MHC. Furthermore, ETA, ETB, SEA, and, to a lesser extent, SEB induced C2D macrophages to produce interleukin 6. Several molecular species on C2D macrophages with molecular masses of 140, 97, 61, 52, 43, and 37 kDa bound SEA in immunoprecipitation experiments. These data indicate the presence of novel, functionally active toxin binding sites on murine macrophages distinct from MHC class II molecules.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

PubMed

Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

2011-03-01

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Histone deacetylase inhibitors with a primary amide zinc binding group display antitumor activity in xenograft model.

PubMed

Attenni, Barbara; Ontoria, Jesus M; Cruz, Jonathan C; Rowley, Michael; Schultz-Fademrecht, Carsten; Steinkühler, Christian; Jones, Philip

2009-06-01

Histone deacetylase (HDAC) inhibition causes hyperacetylation of histones leading to differentiation, growth arrest and apoptosis of malignant cells, representing a new strategy in cancer therapy. Many of the known HDAC inhibitors (HDACi) that are in clinical trials possess a hydroxamic acid, that is a strong Zn(2+) binding group, thereby inhibiting some of the class I and class II isoforms. Herein we describe the identification of a selective class I HDAC inhibitor bearing a primary carboxamide moiety as zinc binding group. This HDACi displays good antiproliferative activity against multiple cancer cell lines, and demonstrates efficacy in a xenograft model comparable to vorinostat.

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch

PubMed Central

Blees, Andreas; Reichel, Katrin; Trowitzsch, Simon; Fisette, Olivier; Bock, Christoph; Abele, Rupert; Hummer, Gerhard; Schäfer, Lars V.; Tampé, Robert

2015-01-01

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity. PMID:26611325

Natural HLA-B*2705 protein ligands with glutamine as anchor motif: implications for HLA-B27 association with spondyloarthropathy.

PubMed

Infantes, Susana; Lorente, Elena; Barnea, Eilon; Beer, Ilan; Barriga, Alejandro; Lasala, Fátima; Jiménez, Mercedes; Admon, Arie; López, Daniel

2013-04-12

The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705(+) cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides.

Natural HLA-B*2705 Protein Ligands with Glutamine as Anchor Motif

PubMed Central

Infantes, Susana; Lorente, Elena; Barnea, Eilon; Beer, Ilan; Barriga, Alejandro; Lasala, Fátima; Jiménez, Mercedes; Admon, Arie; López, Daniel

2013-01-01

The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705+ cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides. PMID:23430249

Towards peptide vaccines against Zika virus: Immunoinformatics combined with molecular dynamics simulations to predict antigenic epitopes of Zika viral proteins.

PubMed

Usman Mirza, Muhammad; Rafique, Shazia; Ali, Amjad; Munir, Mobeen; Ikram, Nazia; Manan, Abdul; Salo-Ahen, Outi M H; Idrees, Muhammad

2016-12-09

The recent outbreak of Zika virus (ZIKV) infection in Brazil has developed to a global health concern due to its likely association with birth defects (primary microcephaly) and neurological complications. Consequently, there is an urgent need to develop a vaccine to prevent or a medicine to treat the infection. In this study, immunoinformatics approach was employed to predict antigenic epitopes of Zika viral proteins to aid in development of a peptide vaccine against ZIKV. Both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted for ZIKV Envelope (E), NS3 and NS5 proteins. We further investigated the binding interactions of altogether 15 antigenic CTL epitopes with three class I major histocompatibility complex (MHC I) proteins after docking the peptides to the binding groove of the MHC I proteins. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlight the limits of rigid-body docking methods. Some of the antigenic epitopes predicted and analyzed in this work might present a preliminary set of peptides for future vaccine development against ZIKV.

A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR.

PubMed

Sgourakis, Nikolaos G; May, Nathan A; Boyd, Lisa F; Ying, Jinfa; Bax, Ad; Margulies, David H

2015-11-27

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), "mini-H2-L(d)," consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β2-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Recombinant modified vaccinia virus Ankara–simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

PubMed Central

Seth, Aruna; Ourmanov, Ilnour; Kuroda, Marcelo J.; Schmitz, Jörn E.; Carroll, Miles W.; Wyatt, Linda S.; Moss, Bernard; Forman, Meryl A.; Hirsch, Vanessa M.; Letvin, Norman L.

1998-01-01

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans. PMID:9707609

Genetically Encoded Chemical Probes In Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

PubMed Central

Coin, Irene; Katritch, Vsevolod; Sun, Tingting; Xiang, Zheng; Siu, Fai Yiu; Beyermann, Michael; Stevens, Raymond C.; Wang, Lei

2014-01-01

SUMMARY Molecular determinants regulating the activation of class B G-protein coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photo-chemical and new click-chemical probes into the receptor, 44 inter-molecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation. The experimental strategy provides unique information on full-length post-translationally modified GPCRs in the native membrane of the live cell, complementing in vitro biophysical reductionist approaches. PMID:24290358

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

CW EPR parameters reveal cytochrome P450 ligand binding modes.

PubMed

Lockart, Molly M; Rodriguez, Carlo A; Atkins, William M; Bowman, Michael K

2018-06-01

Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-H 2 O-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR. Copyright © 2018 Elsevier Inc. All rights reserved.

Downregulation of peptide transporter genes in cell lines transformed with the highly oncogenic adenovirus 12

PubMed Central

1994-01-01

The expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the high oncogenicity of this virus. In primary embryonal fibroblasts from transgenic mice that express both endogenous H-2 genes and a miniature swine class I gene (PD1), Ad12- mediated transformation results in suppression of cell surface expression of all class I antigens. Although class I mRNA levels of PD1 and H-2Db are similar to those in nonvirally transformed cells, recognition of newly synthesized class I molecules by a panel of monoclonal antibodies is impaired, presumably as a result of inefficient assembly and transport of the class I molecules. Class I expression can be partially induced by culturing cells at 26 degrees C, or by coculture of cells with class I binding peptides at 37 degrees C. Analysis of steady state mRNA levels of the TAP1 and TAP2 transporter genes for Ad12-transformed cell lines revealed that they both are significantly reduced, TAP2 by about 100-fold and TAP1 by 5-10-fold. Reconstitution of PD1 and H-2Db, but not H-2Kb, expression is achieved in an Ad12-transformed cell line by stable transfection with a TAP2, but not a TAP1, expression construct. From these data it may be concluded that suppressed expression of peptide transporter genes, especially TAP2, in Ad12-transformed cells inhibits cell surface expression of class I molecules. The failure to fully reconstitute H- 2Db and H-2Kb expression indicates that additional factors are involved in controlling class I gene expression in Ad12-transformed cells. Nevertheless, these results suggest that suppression of peptide transporter genes might be an important mechanism whereby virus- transformed cells escape immune recognition in vivo. PMID:7519239

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lach, E.; Trifilieff, A.; Landry, Y.

1991-01-01

The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin inmore » an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.« less

The Length Distribution of Class I-Restricted T Cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference.

PubMed

Trolle, Thomas; McMurtrey, Curtis P; Sidney, John; Bardet, Wilfried; Osborn, Sean C; Kaever, Thomas; Sette, Alessandro; Hildebrand, William H; Nielsen, Morten; Peters, Bjoern

2016-02-15

HLA class I-binding predictions are widely used to identify candidate peptide targets of human CD8(+) T cell responses. Many such approaches focus exclusively on a limited range of peptide lengths, typically 9 aa and sometimes 9-10 aa, despite multiple examples of dominant epitopes of other lengths. In this study, we examined whether epitope predictions can be improved by incorporating the natural length distribution of HLA class I ligands. We found that, although different HLA alleles have diverse length-binding preferences, the length profiles of ligands that are naturally presented by these alleles are much more homogeneous. We hypothesized that this is due to a defined length profile of peptides available for HLA binding in the endoplasmic reticulum. Based on this, we created a model of HLA allele-specific ligand length profiles and demonstrate how this model, in combination with HLA-binding predictions, greatly improves comprehensive identification of CD8(+) T cell epitopes. Copyright © 2016 by The American Association of Immunologists, Inc.

Characterization of MHC class I and II genes in a subantarctic seabird, the blue petrel, Halobaena caerulea (Procellariiformes).

PubMed

Strandh, Maria; Lannefors, Mimi; Bonadonna, Francesco; Westerdahl, Helena

2011-10-01

The great polymorphism observed in the major histocompatibility complex (MHC) genes is thought to be maintained by pathogen-mediated selection possibly combined with MHC-disassortative mating, guided by MHC-determined olfactory cues. Here, we partly characterize the MHC class I and II B of the blue petrel, Halobaena caerulea (Procellariiformes), a bird with significant olfactory abilities that lives under presumably low pathogen burdens in Subantarctica. Blue petrels are long-lived, monogamous birds which suggest the necessity of an accurate mate choice process. The species is ancestral to songbirds (Passeriformes; many MHC loci), although not to gamefowls (Galliformes; few MHC loci). Considering the phylogenetic relationships and the low subantarctic pathogen burden, we expected few rather than many MHC loci in the blue petrel. However, when we analysed partial MHC class I and class II B cDNA and gDNA sequences we found evidence for as many as at least eight MHC class I loci and at least two class II B loci. These class I and II B sequences showed classical MHC characteristics, e.g. high nucleotide diversity, especially in putative peptide-binding regions where signatures of positive selection was detected. Trans-species polymorphism was found between MHC class II B sequences of the blue petrel and those of thin-billed prion, Pachyptila belcheri, two species that diverged ∼25 MYA. The observed MHC allele richness in the blue petrel may well serve as a basis for mate choice, especially since olfactory discrimination of MHC types may be possible in this species.

Capture and quality control mechanisms for ATP binding

PubMed Central

Li, Li; Martinis, Susan A.

2013-01-01

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly-casting mechanism that acts up on the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates. PMID:23276298

Capture and quality control mechanisms for adenosine-5'-triphosphate binding.

PubMed

Li, Li; Martinis, Susan A; Luthey-Schulten, Zaida

2013-04-24

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly casting mechanism that acts upon the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates.

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

PubMed Central

Schafer, Jamie L.; Ries, Moritz; Guha, Natasha; Connole, Michelle; Colantonio, Arnaud D.; Wiertz, Emmanuel J.; Wilson, Nancy A.; Kaur, Amitinder; Evans, David T.

2015-01-01

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses. PMID:26333068

A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides

PubMed Central

1994-01-01

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules. PMID:8294869

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

PubMed

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

2013-03-05

AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

PubMed Central

Cho, Sunglim; Kim, Bo Young; Ahn, Kwangseog; Jun, Youngsoo

2013-01-01

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation. PMID:23951315

The C-terminal amino acid of the MHC-I heavy chain is critical for binding to Derlin-1 in human cytomegalovirus US11-induced MHC-I degradation.

PubMed

Cho, Sunglim; Kim, Bo Young; Ahn, Kwangseog; Jun, Youngsoo

2013-01-01

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.

Structure of the N-linked oligosaccharides of MHC class I molecules from cells deficient in the antigenic peptide transporter. Implications for the site of peptide association.

PubMed

Hayes, B K; Esquivel, F; Bennink, J R; Yewdell, J W; Varki, A

1995-10-15

Class I molecules are N-linked glycoproteins encoded by the MHC. They carry cytosolic protein-derived peptides to the cell surface, displaying them to enable immune surveillance of cellular processes. Peptides are delivered to class I molecules by the transporter associated with Ag processing (TAP). Peptide association is known to occur before exposure of class I molecules to the medial Golgi-processing enzyme alpha-mannosidase II, but there is limited information regarding the location or timing of peptide binding within the earlier regions of the endoplasmic reticulum (ER)-Golgi pathway. A reported association of newly synthesized class I molecules with the ER chaperonin calnexin raises the possibility of persistence of the monoglycosylated N-linked oligosaccharide (NLO) Glc1Man8GlcNAc2, known to be recognized by this lectin. To explore these matters, we determined the structure of the NLOs on the subset of newly synthesized class I molecules awaiting the loading of peptide. We pulse-labeled murine MHC H-2Db class I molecules in RMA/S cells, which lack one of the TAP subunits, causing the great majority of the molecules to be retained for prolonged periods in an early secretory compartment, awaiting peptide binding. MHC molecules pulse-labeled with [3H]glucosamine were isolated, the NLOs specifically released and structurally analyzed by a variety of techniques. Within the chosen window of biosynthetic time, most Db molecules from parental RMA cells carried mature NLOs of the biantennary complex-type, with one to two sialic acid residues. In RMA/S cells, such chains were in the minority, the majority consisting of the precursor forms Man8GlcNAc2 and Man9GlcNAc2. No glucosylated forms were detected, nor were the later processing intermediates Man5-7GlcNAc2 or GlcNAc1Man4-5GlcNAc2. Thus, most Db molecules in TAP-deficient cells are retained in an early compartment of the secretory pathway, before the point of first access to the Golgi alpha-mannosidase I, which trims alpha 1-2 linked mannose residues, but beyond the point where the alpha 1-3-linked glucose residue is finally removed by the ER glucosidase II. Thus, structural analysis of NLOs on class I molecules within a defined biosynthetic window has established a biochemical measure of the timing of peptide association.

Ligand Recognition by A-Class Eph Receptors: Crystal Structures of the EphA2 Ligand-Binding Domain and the EphA2/ephrin-A1 Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Himanen, J.; Goldgur, Y; Miao, H

2009-01-01

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B-class receptors. Here, we present the crystal structures of an A-class complex between EphA2 and ephrin-A1 and of unbound EphA2. Although these structures are similar overall to their B-class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A-class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a 'lock-and-key'-type binding mechanism, in contrast to the 'induced fit' mechanism defining the B-class molecules.more » This model is supported by structure-based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell-based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2-specific homotypic cell adhesion interactions.« less

Relationship between renal pathology and the size of circulating immune complexes in patients with systemic lupus erythematosus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wener, M.H.; Mannik, M.; Schwartz, M.M.

1987-03-01

Sera from 35 patients with biopsy-proven diffuse proliferative (WHO class IV) or membranous (WHO class V) lupus nephritis were analyzed for the presence and size of circulating immune complexes. Elevations of the C1q solid-phase assay (C1qSP) for immune complexes were found in sera from all patients with diffuse proliferative nephritis, with a mean +/- 1 SEM of 166.8 +/- 42.0 micrograms/AHG-equivalents/ml serum, and in 71.4% of the patients with membranous nephritis (83.1 +/- 26.7, p = 0.06). Using the WHO criteria for subclasses of membranous lupus nephritis, we also designated renal biopsies as nonproliferative (WHO classes Va and Vb) ormore » proliferative (WHO classes IV and Vc). Employing the latter groupings, we observed significant differences between C1qSP results of patients with nonproliferative (30.3 +/- 8.8) and proliferative (172.8 +/- 36.8, p less than 0.001) lupus nephritis. These data suggest that the presence of C1q-binding material in serum is pathophysiologically related to proliferative glomerular lesions, and that levels of C1qSP binding reflect renal lesions in SLE patients. Sucrose density gradient ultracentrifugation was performed on each serum, and gradient fractions analyzed for C1qSP-binding and total IgG, using techniques to minimize losses of immune complexes. The predominant peak of C1qSP activity sedimented with the 6.6S monomeric IgG. The 6.6S C1q-binding IgG was increased only in 1 of 10 patients with membranous lupus nephritis without proliferative changes, and was elevated in 16 of 25 patients with proliferative lesions (WHO classes IV and Vc).« less

H2-M polymorphism in mice susceptible to collagen-induced arthritis involves the peptide binding groove

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walter, W.; Loos, M.; Maeurer, M.J.

1996-12-31

The ability to develop type II collagen (CII)-induced arthritis (CIA) in mice is associated with the major histocompatibility I-A gene and with as yet poorly defined regulatory molecules of the major histocompatibility complex (MHC) class II antigen processing and presentation pathway. H2-M molecules are thought to be involved in the loading of antigenic peptides into the MHC class II binding cleft. We sequenced H2-Ma, H2-Mb1, and H2-Mb2 genes from CIA-susceptible and -resistant mouse strains and identified four different Ma and Mb2 alleles, and three different Mb1 alleles defined by polymorphic residues within the predicted peptide binding groove. Most CIA-resistant mousemore » strains share common Ma, Mb1, and Mb2 alleles. In contrast, H2-M alleles designated Ma-III, Ma-IV, Mb1-III, and Mb2-IV could be exclusively identified in the CIA-susceptible H2{sup r} and H2{sup q} haplotypes, suggesting that allelic H2-M molecules may modulate the composition of different CII peptides loaded onto MHC class II molecules, presumably presenting {open_quotes}arthritogenic{close_quotes} epitopes to T lymphocytes. 42 refs., 4 figs., 3 tabs.« less

Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6.

PubMed

Porter, Nicholas J; Wagner, Florence F; Christianson, David W

2018-05-18

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn 2+ -binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even "capless" inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1-4. Each inhibitor hydroxamate group coordinates to the catalytic Zn 2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved in the representative class I isozyme HDAC8, we employed isothermal titration calorimetry to study the thermodynamics of inhibitor binding. These measurements indicate that the entropy of inhibitor binding is generally positive for binding to HDAC6 and negative for binding to HDAC8, resulting in ≤313-fold selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl hydroxamate 2 represents a promising lead for derivatization with capping groups that may further enhance its impressive 313-fold thermodynamic selectivity for HDAC6 inhibition.

Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system

PubMed Central

2012-01-01

Background The critical role of Major Histocompatibility Complex (Mhc) genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance. Results In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major) from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities. Conclusions We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help understanding how parasite-mediated and sexual selection shape and maintain host genetic variation in nature. We believe that study systems like ours can make important contributions to the field of evolutionary biology and emphasize the necessity of integrating long-term field-based studies with detailed genetic analysis to unravel complex evolutionary processes. PMID:22587557

Analysis of Protein-RNA and Protein-Peptide Interactions in Equine Infectious Anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jae-Hyung

2007-01-01

Macromolecular interactions are essential for virtually all cellular functions including signal transduction processes, metabolic processes, regulation of gene expression and immune responses. This dissertation focuses on the characterization of two important macromolecular interactions involved in the relationship between Equine Infectious Anemia Virus (EIAV) and its host cell in horse: (1) the interaction between the EIAV Rev protein and its binding site, the Rev-responsive element (RRE) and (2) interactions between equine MHC class I molecules and epitope peptides derived from EIAV proteins. EIAV, one of the most divergent members of the lentivirus family, has a single-stranded RNA genome and carries severalmore » regulatory and structural proteins within its viral particle. Rev is an essential EIAV regulatory encoded protein that interacts with the viral RRE, a specific binding site in the viral mRNA. Using a combination of experimental and computational methods, the interactions between EIAV Rev and RRE were characterized in detail. EIAV Rev was shown to have a bipartite RNA binding domain contain two arginine rich motifs (ARMs). The RRE secondary structure was determined and specific structural motifs that act as cis-regulatory elements for EIAV Rev-RRE interaction were identified. Interestingly, a structural motif located in the high affinity Rev binding site is well conserved in several diverse lentiviral genoes, including HIV-1. Macromolecular interactions involved in the immune response of the horse to EIAV infection were investigated by analyzing complexes between MHC class I proteins and epitope peptides derived from EIAV Rev, Env and Gag proteins. Computational modeling results provided a mechanistic explanation for the experimental finding that a single amino acid change in the peptide binding domain of the quine MHC class I molecule differentially affectes the recognitino of specific epitopes by EIAV-specific CTL. Together, the findings in this dissertation provide novel insights into the strategy used by EIAV to replicate itself, and provide new details about how the host cell responds to and defends against EIAV upon the infection. Moreover, they have contributed to the understanding of the macromolecular recognition events that regulate these processes.« less

The alternative complement pathway control protein H binds to immune complexes and serves their detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.

1983-01-01

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samplesmore » from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.« less

A stable Fe{sup III}-Fe{sup IV} replacement of tyrosyl radical in a class I ribonucleotide reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voevodskaya, N.; Lendzian, F.; Graeslund, A.

2005-05-20

Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe{sup III}-Fe{sup IV} species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start themore » catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.« less

Class I HDACs control a JIP1-dependent pathway for kinesin-microtubule binding in cardiomyocytes

PubMed Central

Blakeslee, Weston W.; Lin, Ying-Hsi; Stratton, Matthew S.; Tatman, Philip D.; Hu, Tianjing; Ferguson, Bradley S.; McKinsey, Timothy A.

2018-01-01

Class I histone deacetylase (HDAC) inhibitors block hypertrophy and fibrosis of the heart by suppressing pathological signaling and gene expression programs in cardiac myocytes and fibroblasts. The impact of HDAC inhibition in unstressed cardiac cells remains poorly understood. Here, we demonstrate that treatment of cultured cardiomyocytes with small molecule HDAC inhibitors leads to dramatic induction of c-Jun amino-terminal kinase (JNK)-interacting protein-1 (JIP1) mRNA and protein expression. In contrast to prior findings, elevated levels of endogenous JIP1 in cardiomyocytes failed to significantly alter JNK signaling or cardiomyocyte hypertrophy. Instead, HDAC inhibitor-mediated induction of JIP1 was required to stimulate expression of the kinesin heavy chain family member, KIF5A. We provide evidence for an HDAC-dependent regulatory circuit that promotes formation of JIP1:KIF5A:microtubule complexes that regulate intracellular transport of cargo such as autophagosomes. These findings define a novel role for class I HDACs in the control of the JIP1/kinesin axis in cardiomyocytes, and suggest that HDAC inhibitors could be used to alter microtubule transport in the heart. PMID:28886967

Epitope predictions indicate the presence of two distinct types of epitope-antibody-reactivities determined by epitope profiling of intravenous immunoglobulins.

PubMed

Luštrek, Mitja; Lorenz, Peter; Kreutzer, Michael; Qian, Zilliang; Steinbeck, Felix; Wu, Di; Born, Nadine; Ziems, Bjoern; Hecker, Michael; Blank, Miri; Shoenfeld, Yehuda; Cao, Zhiwei; Glocker, Michael O; Li, Yixue; Fuellen, Georg; Thiesen, Hans-Jürgen

2013-01-01

Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.

DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.

PubMed

Lindow, Janet C; Dohrmann, Paul R; McHenry, Charles S

2015-07-03

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β2 processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A community resource benchmarking predictions of peptide binding to MHC-I molecules.

PubMed

Peters, Bjoern; Bui, Huynh-Hoa; Frankild, Sune; Nielson, Morten; Lundegaard, Claus; Kostem, Emrah; Basch, Derek; Lamberth, Kasper; Harndahl, Mikkel; Fleri, Ward; Wilson, Stephen S; Sidney, John; Lund, Ole; Buus, Soren; Sette, Alessandro

2006-06-09

Recognition of peptides bound to major histocompatibility complex (MHC) class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, and chimpanzee MHC class I alleles. We use this data to establish a set of benchmark predictions with one neural network method and two matrix-based prediction methods extensively utilized in our groups. In general, the neural network outperforms the matrix-based predictions mainly due to its ability to generalize even on a small amount of data. We also retrieved predictions from tools publicly available on the internet. While differences in the data used to generate these predictions hamper direct comparisons, we do conclude that tools based on combinatorial peptide libraries perform remarkably well. The transparent prediction evaluation on this dataset provides tool developers with a benchmark for comparison of newly developed prediction methods. In addition, to generate and evaluate our own prediction methods, we have established an easily extensible web-based prediction framework that allows automated side-by-side comparisons of prediction methods implemented by experts. This is an advance over the current practice of tool developers having to generate reference predictions themselves, which can lead to underestimating the performance of prediction methods they are not as familiar with as their own. The overall goal of this effort is to provide a transparent prediction evaluation allowing bioinformaticians to identify promising features of prediction methods and providing guidance to immunologists regarding the reliability of prediction tools.

The insecticide target in the PSST subunit of complex I.

PubMed

Schuler, F; Casida, J E

2001-10-01

Current insecticides have been selected by sifting and winnowing hundreds of thousands of synthetic chemicals and natural products to obtain commercial preparations of optimal effectiveness and safety. This process has often ended up with compounds of high potency as inhibitors of the electron transport chain and more specifically of complex I (NADH:ubiquinone oxidoreductase). Many classes of chemicals are involved and the enzyme is one of the most complicated known, with 43 subunits catalyzing electron transfer from NADH to ubiquinone through flavin mononucleotide and up to eight iron-sulfur clusters. We used a potent photoaffinity ligand, (trifluoromethyl)diazirinyl[3H]pyridaben, to localize the insecticide target to a single high-affinity site in the PSST subunit that couples electron transfer from iron-sulfur cluster N2 to ubiquinone. Most importantly, all of the potent complex I-inhibiting pesticides, despite their great structural diversity, compete for this same specific binding domain in PSST. Finding their common mode of action and target provides insight into shared toxicological features and potential selection for resistant pests.

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce.

PubMed

Ubhayasekera, Wimal; Rawat, Reetika; Ho, Sharon Wing Tak; Wiweger, Malgorzata; Von Arnold, Sara; Chye, Mee-Len; Mowbray, Sherry L

2009-10-01

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.

Deep convolutional neural networks for pan-specific peptide-MHC class I binding prediction.

PubMed

Han, Youngmahn; Kim, Dongsup

2017-12-28

Computational scanning of peptide candidates that bind to a specific major histocompatibility complex (MHC) can speed up the peptide-based vaccine development process and therefore various methods are being actively developed. Recently, machine-learning-based methods have generated successful results by training large amounts of experimental data. However, many machine learning-based methods are generally less sensitive in recognizing locally-clustered interactions, which can synergistically stabilize peptide binding. Deep convolutional neural network (DCNN) is a deep learning method inspired by visual recognition process of animal brain and it is known to be able to capture meaningful local patterns from 2D images. Once the peptide-MHC interactions can be encoded into image-like array(ILA) data, DCNN can be employed to build a predictive model for peptide-MHC binding prediction. In this study, we demonstrated that DCNN is able to not only reliably predict peptide-MHC binding, but also sensitively detect locally-clustered interactions. Nonapeptide-HLA-A and -B binding data were encoded into ILA data. A DCNN, as a pan-specific prediction model, was trained on the ILA data. The DCNN showed higher performance than other prediction tools for the latest benchmark datasets, which consist of 43 datasets for 15 HLA-A alleles and 25 datasets for 10 HLA-B alleles. In particular, the DCNN outperformed other tools for alleles belonging to the HLA-A3 supertype. The F1 scores of the DCNN were 0.86, 0.94, and 0.67 for HLA-A*31:01, HLA-A*03:01, and HLA-A*68:01 alleles, respectively, which were significantly higher than those of other tools. We found that the DCNN was able to recognize locally-clustered interactions that could synergistically stabilize peptide binding. We developed ConvMHC, a web server to provide user-friendly web interfaces for peptide-MHC class I binding predictions using the DCNN. ConvMHC web server can be accessible via http://jumong.kaist.ac.kr:8080/convmhc . We developed a novel method for peptide-HLA-I binding predictions using DCNN trained on ILA data that encode peptide binding data and demonstrated the reliable performance of the DCNN in nonapeptide binding predictions through the independent evaluation on the latest IEDB benchmark datasets. Our approaches can be applied to characterize locally-clustered patterns in molecular interactions, such as protein/DNA, protein/RNA, and drug/protein interactions.

Characterization of Two Classes of Small Molecule Inhibitors of Arp2/3 Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nolen, B.; Tomasevic, N; Russell, A

2009-01-01

Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2more » and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.« less

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

PubMed Central

Heyma, P; Harrison, L C

1984-01-01

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor. Images PMID:6088581

Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec

DOE Office of Scientific and Technical Information (OSTI.GOV)

Safo, Martin K., E-mail: msafo@vcu.edu; Ko, Tzu-Ping; Musayev, Faik N.

The up-and-down binding of dimeric MecI to mecA dyad DNA may account for the cooperative effect of the repressor. The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of β-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Å resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA,more » and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI–mec complex, but unlike the MecI–bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Multispectroscopic DNA-Binding studies and antimicrobial evaluation of new mixed-ligand Silver(I) complex and nanocomplex: A comparative study

NASA Astrophysics Data System (ADS)

Movahedi, Elaheh; Rezvani, Ali Reza

2018-05-01

A novel mixed-ligand Ag(I) complex, , has been synthesized and characterized by the elemental analysis, IR spectroscopy and 1HNMR. In the formula, dian and phen are N-(4,5-diazafluoren-9-ylidene)aniline and 1,10-phenanthroline, respectively. This complex also has been prepared at nano size by sonochemical technique and characterized by the FTIR and scanning electron microscopy (SEM). To evaluate the biological preferences of the Ag(I) complex and nanocomplex and verify the relationships between the structure and biological function, in vitro DNA binding and antibacterial experiments have been carried out. DNA-complex interaction has been pursued by electronic absorption titration, luminescence titration, competitive binding experiment, effect of ionic strength, thermodynamic studies, viscometric evaluation and circular dichroism spectroscopy in the physiological pH. Each compound displays significant binding trend to the CT-DNA. The mode of binding to the CT-DNA probably is a moderate intercalation mode with the partial insertion of the planar ligands between the base stacks of double-stranded DNA. The relative viscosities and circular dichroism spectra of the CT-DNA with the complex solutions, confirm the intense interactions of the Ag(I) complex and nanocomplex with DNA. An in vitro antibacterial test of the complex and nanocomplex on a series of the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and the Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) shows a remarkable antibacterial feature of the Ag(I) complex. The MIC values (minimum inhibitory concentration) of the compounds compare with silver nitrate and silver sulfadiazine. The bacterial inhibitions of the Ag(I) complex and nanocomplex are agreed to their DNA binding affinities.

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

PubMed Central

Van Eps, Ned; Thomas, Celestine J.; Hubbell, Wayne L.; Sprang, Stephen R.

2015-01-01

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF. PMID:25605908

Evolution by selection, recombination, and gene duplication in MHC class I genes of two Rhacophoridae species

PubMed Central

2013-01-01

Background Comparison of major histocompatibility complex (MHC) genes across vertebrate species can reveal molecular mechanisms underlying the evolution of adaptive immunity-related proteins. As the first terrestrial tetrapods, amphibians deserve special attention because of their exposure to probably increased spectrum of microorganisms compared with ancestral aquatic fishes. Knowledge regarding the evolutionary patterns and mechanisms associated with amphibian MHC genes remains limited. The goal of the present study was to isolate MHC class I genes from two Rhacophoridae species (Rhacophorus omeimontis and Polypedates megacephalus) and examine their evolution. Results We identified 27 MHC class I alleles spanning the region from exon 2 to 4 in 38 tree frogs. The available evidence suggests that these 27 sequences all belong to classical MHC class I (MHC Ia) genes. Although several anuran species only display one MHC class Ia locus, at least two or three loci were observed in P. megacephalus and R. omeimontis, indicating that the number of MHC class Ia loci varies among anuran species. Recombination events, which mainly involve the entire exons, played an important role in shaping the genetic diversity of the 27 MHC class Ia alleles. In addition, signals of positive selection were found in Rhacophoridae MHC class Ia genes. Amino acid sites strongly suggested by program to be under positive selection basically accorded with the putative antigen binding sites deduced from crystal structure of human HLA. Phylogenetic relationships among MHC class I alleles revealed the presence of trans-species polymorphisms. Conclusions In the two Rhacophoridae species (1) there are two or three MHC class Ia loci; (2) recombination mainly occurs between the entire exons of MHC class Ia genes; (3) balancing selection, gene duplication and recombination all contribute to the diversity of MHC class Ia genes. These findings broaden our knowledge on the evolution of amphibian MHC systems. PMID:23734729

Thermodynamics of T cell receptor – peptide/MHC interactions: progress and opportunities

PubMed Central

Armstrong, Kathryn M.; Insaidoo, Francis K.; Baker, Brian M.

2013-01-01

αβ T cell receptors (TCR) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van’t Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize commonly disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but for understanding specifics of individual interactions and the engineering of T cell receptors with desired molecular recognition properties. PMID:18496839

The Arabidopsis class I TCP transcription factor AtTCP11 is a developmental regulator with distinct DNA-binding properties due to the presence of a threonine residue at position 15 of the TCP domain.

PubMed

Viola, Ivana L; Uberti Manassero, Nora G; Ripoll, Rodrigo; Gonzalez, Daniel H

2011-04-01

The TCP domain is a DNA-binding domain present in plant transcription factors that modulate different processes. In the present study, we show that Arabidopsis class I TCP proteins are able to interact with a dyad-symmetric sequence composed of two GTGGG half-sites. TCP20 establishes symmetric interactions with the 5' half of each strand, whereas TCP11 interacts mainly with the 3' half. SELEX (systematic evolution of ligands by exponential enrichment) experiments with TCP15 and TCP20 indicated that these proteins have similar, although not identical, DNA-binding preferences and are able to interact with non-palindromic binding sites of the type GTGGGNCCNN. TCP11 shows a different DNA-binding specificity, with a preference for the sequence GTGGGCCNNN. The distinct DNA-binding properties of TCP11 are due to the presence of a threonine residue at position 15 of the TCP domain, a position that is occupied by an arginine residue in most TCP proteins. TCP11 also forms heterodimers with TCP15 that have increased DNA-binding efficiency. The expression in plants of a repressor form of TCP11 demonstrated that this protein is a developmental regulator that influences the growth of leaves, stems and petioles, and pollen development. The results suggest that changes in DNA-binding preferences may be one of the mechanisms through which class I TCP proteins achieve functional specificity.

Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors.

PubMed

Dowling, Daniel P; Gantt, Stephanie L; Gattis, Samuel G; Fierke, Carol A; Christianson, David W

2008-12-23

Metal-dependent histone deacetylases (HDACs) require Zn(2+) or Fe(2+) to regulate the acetylation of lysine residues in histones and other proteins in eukaryotic cells. Isozyme HDAC8 is perhaps the archetypical member of the class I HDAC family and serves as a paradigm for studying structure-function relationships. Here, we report the structures of HDAC8 complexes with trichostatin A and 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide (APHA) in a new crystal form. The structure of the APHA complex reveals that the hydroxamate CO group accepts a hydrogen bond from Y306 but does not coordinate to Zn(2+) with favorable geometry, perhaps due to the constraints of its extended pi system. Additionally, since APHA binds to only two of the three protein molecules in the asymmetric unit of this complex, the structure of the third monomer represents the first structure of HDAC8 in the unliganded state. Comparison of unliganded and liganded structures illustrates ligand-induced conformational changes in the L2 loop that likely accompany substrate binding and catalysis. Furthermore, these structures, along with those of the D101N, D101E, D101A, and D101L variants, support the proposal that D101 is critical for the function of the L2 loop. However, amino acid substitutions for D101 can also trigger conformational changes of Y111 and W141 that perturb the substrate binding site. Finally, the structure of H143A HDAC8 complexed with an intact acetylated tetrapeptide substrate molecule confirms the importance of D101 for substrate binding and reveals how Y306 and the active site zinc ion together bind and activate the scissile amide linkage of acetyllysine.

Oxoiron(IV) complexes as synthons for the assembly of heterobimetallic centers such as the Fe/Mn active site of Class Ic ribonucleotide reductases.

PubMed

Zhou, Ang; Crossland, Patrick M; Draksharapu, Apparao; Jasniewski, Andrew J; Kleespies, Scott T; Que, Lawrence

2018-01-01

Nonheme oxoiron(IV) complexes can serve as synthons for generating heterobimetallic oxo-bridged dimetal complexes by reaction with divalent metal complexes. The formation of Fe III -O-Cr III and Fe III -O-Mn III complexes is described herein. The latter complexes may serve as models for the Fe III -X-Mn III active sites of an emerging class of Fe/Mn enzymes represented by the Class 1c ribonucleotide reductase from Chlamydia trachomatis and the R2-like ligand-binding oxidase (R2lox) found in Mycobacterium tuberculosis. These synthetic complexes have been characterized by UV-Vis, resonance Raman, and X-ray absorption spectroscopy, as well as electrospray mass spectrometry. The Fe III -O-Cr III complexes exhibit a three-band UV-Vis pattern that differs from the simpler features associated with Fe III -O-Fe III complexes. The positions of these features are modulated by the nature of the supporting polydentate ligand on the iron center, and their bands intensify dramatically in two examples upon the binding of an axial cyanate or thiocyanate ligand trans to the oxo bridge. In contrast, the Fe III -O-Mn III complexes resemble Fe III -O-Fe III complexes more closely. Resonance Raman characterization of the Fe III -O-M III complexes reveals an 18 O-sensitive vibration in the range of 760-890 cm -1 . This feature has been assigned to the asymmetric Fe III -O-M III stretching mode and correlates reasonably with the Fe-O bond distance determined by EXAFS analysis. The likely binding of an acetate as a bridging ligand to the Fe III -O-Mn III complex 12 lays the foundation for further efforts to model the heterobimetallic active sites of Fe/Mn enzymes.

Major histocompatibility class I molecules present Urtica dioica agglutinin, a superantigen of vegetal origin, to T lymphocytes.

PubMed

Rovira, P; Buckle, M; Abastado, J P; Peumans, W J; Truffa-Bachi, P

1999-05-01

The Urtica dioica agglutinin (UDA) shares with the superantigens the property of activating T cell subsets bearing particular Vbeta segments of the TCR. However, UDA is a lectin capable of binding to many glycoproteins on cell membranes. The implication of MHC versus other glycoproteins in UDA presentation was presently studied. Using mutant mice lacking MHC class I (MHC-I), MHC class II (MHC-II) or both MHC antigens, we provided evidence that MHC-I and MHC-II molecules serve as UDA receptors. Presentation by either one of these molecules ensured similar T cell responses and co-stimulatory signals were mandatory for optimal T cell activation and proliferation both in MHC-I and MHC-II contexts. Remarkably, in the absence of MHC molecules, UDA could not be efficiently presented to T cells by other glycosylated proteins. Surface plasmon resonance studies were used to confirm the binding of UDA to MHC-I molecules using a fusion protein consisting of MHC-I domains and beta2-microglobulin. The results indicated that the interaction between UDA and MHC-I molecules implicated lectin-binding site(s) of UDA. Taken together, our data demonstrate that, in addition to MHC-II antigens, MHC-I molecules serve as an alternative ligand for UDA.

In vitro covalent binding of new brain tracer, para-125I-amphetamine, to rat liver and lung microsomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joulin, Y.; Delaforge, M.; Hoellinger, H.

1990-01-01

p-125I-amphetamine (I-Amp) is retained significantly in liver and lung during brain tomoscintigraphy. To attempt to explain this clinical observation, we have investigated the interaction of I-Amp with rat liver and lung microsomal proteins. Studies using spectral shift technique indicate that low concentration of I-Amp gives a type I complex and high concentration appears very stable type II complex with cytochrome P-450 Fe III. In the presence of NADPH, I-Amp gives rise to a 455 nm absorbing complex with similar properties to the Fe-RNO complexes. This complex formation was greatly enhanced with phenobarbital treated liver microsomes. The in vitro binding studymore » shows that I-Amp and/or its metabolites was covalently bound to macromolecules in the presence of the molecular oxygen and NADPH-generating system. Incubation in the presence of glutathione, cystein and radical scavengers decreases binding. Mixed function oxydase (MFO) inhibitors diminish the amount of covalent binding and alter the extent of metabolite formation. The total covalent binding level increased with liver microsomes from PB pretreated rats as it was observed with the 455nm complex formation. The radioactivity distribution on microsomal proteins was examinated with SDS polyacrylamide gel electrophoresis and autoradiography. This experiment proves that the radiolabelled compounds are bound on the cytochrome P-450. The radioactivity bound increased when the PB induced rat liver microsomes were used. All these results indicate that I-Amp was activated by an oxydative process dependent on the MFO system which suggests a N-oxydation of I-Amp and the formation of reactive entities which covalently bind to proteins.« less

Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nielsen, Tine Kragh; Hildmann, Christian; Riester, Daniel

2007-04-01

The crystal structure of HDAH FB188 in complex with a trifluoromethylketone at 2.2 Å resolution is reported and compared to a previously determined inhibitor complex. Histone deacetylases (HDACs) have emerged as attractive targets in anticancer drug development. To date, a number of HDAC inhibitors have been developed and most of them are hydroxamic acid derivatives, typified by suberoylanilide hydroxamic acid (SAHA). Not surprisingly, structural information that can greatly enhance the design of novel HDAC inhibitors is so far only available for hydroxamic acids in complex with HDAC or HDAC-like enzymes. Here, the first structure of an enzyme complex with amore » nonhydroxamate HDAC inhibitor is presented. The structure of the trifluoromethyl ketone inhibitor 9,9,9-trifluoro-8-oxo-N-phenylnonanamide in complex with bacterial FB188 HDAH (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes strain FB188) has been determined. HDAH reveals high sequential and functional homology to human class 2 HDACs and a high structural homology to human class 1 HDACs. Comparison with the structure of HDAH in complex with SAHA reveals that the two inhibitors superimpose well. However, significant differences in binding to the active site of HDAH were observed. In the presented structure the O atom of the trifluoromethyl ketone moiety is within binding distance of the Zn atom of the enzyme and the F atoms participate in interactions with the enzyme, thereby involving more amino acids in enzyme–inhibitor binding.« less

Structure of the Mecl Repressor from Staphylococcus aureus in Complex with the Cognate DNA Operator of mec

DOE Office of Scientific and Technical Information (OSTI.GOV)

Safo,M.; Ko, T.; Musayev, F.

The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of {beta}-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Angstroms resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtualmore » DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove

PubMed Central

Rohrer, Karin M; Haug, Markus; Schwörer, Daniela; Kalbacher, Hubert; Holzer, Ursula

2014-01-01

Heat-shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I-and II-restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA-DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA-DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA-DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild-type Hsp70 whereas HLA-DR binding remains unaffected. The removal of the C-terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA-DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA-DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA-DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA-DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70-chaperoned peptides might thereby be directly transferred into the binding groove of HLA-DR, so enabling enhanced presentation of the peptide on antigen-presenting cells and leading to an improved proliferation of responding T cells as shown previously. PMID:24428437

KIR3DL2 binds to HLA-B27 dimers and free heavy chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis

PubMed Central

Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

2013-01-01

1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC. PMID:23440420

KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis.

PubMed

Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

2013-04-01

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical β2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

Major histocompatibility complex class I evolution in songbirds: universal primers, rapid evolution and base compositional shifts in exon 3

PubMed Central

Alcaide, Miguel; Liu, Mark

2013-01-01

Genes of the Major Histocompatibility Complex (MHC) have become an important marker for the investigation of adaptive genetic variation in vertebrates because of their critical role in pathogen resistance. However, despite significant advances in the last few years the characterization of MHC variation in non-model species still remains a challenging task due to the redundancy and high variation of this gene complex. Here we report the utility of a single pair of primers for the cross-amplification of the third exon of MHC class I genes, which encodes the more polymorphic half of the peptide-binding region (PBR), in oscine passerines (songbirds; Aves: Passeriformes), a group especially challenging for MHC characterization due to the presence of large and complex MHC multigene families. In our survey, although the primers failed to amplify exon 3 from two suboscine passerine birds, they amplified exon 3 of multiple MHC class I genes in all 16 species of oscine songbirds tested, yielding a total of 120 sequences. The 16 songbird species belong to 14 different families, primarily within the Passerida, but also in the Corvida. Using a conservative approach based on the analysis of cloned amplicons (n = 16) from each species, we found between 3 and 10 MHC sequences per individual. Each allele repertoire was highly divergent, with the overall number of polymorphic sites per species ranging from 33 to 108 (out of 264 sites) and the average number of nucleotide differences between alleles ranging from 14.67 to 43.67. Our survey in songbirds allowed us to compare macroevolutionary dynamics of exon 3 between songbirds and non-passerine birds. We found compelling evidence of positive selection acting specifically upon peptide-binding codons across birds, and we estimate the strength of diversifying selection in songbirds to be about twice that in non-passerines. Analysis using comparative methods suggest weaker evidence for a higher GC content in the 3rd codon position of exon 3 in non-passerine birds, a pattern that contrasts with among-clade GC patterns found in other avian studies and may suggests different mutational mechanisms. Our primers represent a useful tool for the characterization of functional and evolutionarily relevant MHC variation across the hyperdiverse songbirds. PMID:23781408

Unfinished Business: Evolution of the MHC and the Adaptive Immune System of Jawed Vertebrates.

PubMed

Kaufman, Jim

2018-04-26

The major histocompatibility complex (MHC) is a large genetic region with many genes, including the highly polymorphic classical class I and II genes that play crucial roles in adaptive as well as innate immune responses. The organization of the MHC varies enormously among jawed vertebrates, but class I and II genes have not been found in other animals. How did the MHC arise, and are there underlying principles that can help us to understand the evolution of the MHC? This review considers what it means to be an MHC and the potential importance of genome-wide duplication, gene linkage, and gene coevolution for the emergence and evolution of an adaptive immune system. Then it considers what the original antigen-specific receptor and MHC molecule might have looked like, how peptide binding might have evolved, and finally the importance of adaptive immunity in general.

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rollins, T.E.; Siciliano, S.; Kobayashi, S.

1991-02-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42,more » 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.« less

Predicted MHC peptide binding promiscuity explains MHC class I 'hotspots' of antigen presentation defined by mass spectrometry eluted ligand data.

PubMed

Jappe, Emma Christine; Kringelum, Jens; Trolle, Thomas; Nielsen, Morten

2018-02-15

Peptides that bind to and are presented by MHC class I and class II molecules collectively make up the immunopeptidome. In the context of vaccine development, an understanding of the immunopeptidome is essential, and much effort has been dedicated to its accurate and cost-effective identification. Current state-of-the-art methods mainly comprise in silico tools for predicting MHC binding, which is strongly correlated with peptide immunogenicity. However, only a small proportion of the peptides that bind to MHC molecules are, in fact, immunogenic, and substantial work has been dedicated to uncovering additional determinants of peptide immunogenicity. In this context, and in light of recent advancements in mass spectrometry (MS), the existence of immunological hotspots has been given new life, inciting the hypothesis that hotspots are associated with MHC class I peptide immunogenicity. We here introduce a precise terminology for defining these hotspots and carry out a systematic analysis of MS and in silico predicted hotspots. We find that hotspots defined from MS data are largely captured by peptide binding predictions, enabling their replication in silico. This leads us to conclude that hotspots, to a great degree, are simply a result of promiscuous HLA binding, which disproves the hypothesis that the identification of hotspots provides novel information in the context of immunogenic peptide prediction. Furthermore, our analyses demonstrate that the signal of ligand processing, although present in the MS data, has very low predictive power to discriminate between MS and in silico defined hotspots. © 2018 John Wiley & Sons Ltd.

New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation.

PubMed

Landais, Elise; Romagnoli, Pablo A; Corper, Adam L; Shires, John; Altman, John D; Wilson, Ian A; Garcia, K Christopher; Teyton, Luc

2009-12-15

Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

PubMed Central

Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

2016-01-01

Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

Mitochondrial NADH Fluorescence is Enhanced by Complex I Binding

PubMed Central

Blinova, Ksenia; Levine, Rodney L.; Boja, Emily S.; Griffiths, Gary L.; Shi, Zhen-Dan; Ruddy, Brian; Balaban, Robert S.

2012-01-01

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state. PMID:18702505

The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex.

PubMed

Elliott, Tim; Williams, Anthony

2005-10-01

Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic beta(2) microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization.

Crystallographic and Computational Studies of a Class II MHC Complex with a Nonconforming Peptide: HLA-DRA/DRB3*0101

NASA Astrophysics Data System (ADS)

Parry, Christian S.; Gorski, Jack; Stern, Lawrence J.

2003-03-01

The stable binding of processed foreign peptide to a class II major histocompatibility (MHC) molecule and subsequent presentation to a T cell receptor is a central event in immune recognition and regulation. Polymorphic residues on the floor of the peptide binding site form pockets that anchor peptide side chains. These and other residues in the helical wall of the groove determine the specificity of each allele and define a motif. Allele specific motifs allow the prediction of epitopes from the sequence of pathogens. There are, however, known epitopes that do not satisfy these motifs: anchor motifs are not adequate for predicting epitopes as there are apparently major and minor motifs. We present crystallographic studies into the nature of the interactions that govern the binding of these so called nonconforming peptides. We would like to understand the role of the P10 pocket and find out whether the peptides that do not obey the consensus anchor motif bind in the canonical conformation observed in in prior structures of class II MHC-peptide complexes. HLA-DRB3*0101 complexed with peptide crystallized in unit cell 92.10 x 92.10 x 248.30 (90, 90, 90), P41212, and the diffraction data is reliable to 2.2ÅWe are complementing our studies with dynamical long time simulations to answer these questions, particularly the interplay of the anchor motifs in peptide binding, the range of protein and ligand conformations, and water hydration structures.

HLaffy: estimating peptide affinities for Class-1 HLA molecules by learning position-specific pair potentials.

PubMed

Mukherjee, Sumanta; Bhattacharyya, Chiranjib; Chandra, Nagasuma

2016-08-01

T-cell epitopes serve as molecular keys to initiate adaptive immune responses. Identification of T-cell epitopes is also a key step in rational vaccine design. Most available methods are driven by informatics and are critically dependent on experimentally obtained training data. Analysis of a training set from Immune Epitope Database (IEDB) for several alleles indicates that the sampling of the peptide space is extremely sparse covering a tiny fraction of the possible nonamer space, and also heavily skewed, thus restricting the range of epitope prediction. We present a new epitope prediction method that has four distinct computational modules: (i) structural modelling, estimating statistical pair-potentials and constraint derivation, (ii) implicit modelling and interaction profiling, (iii) feature representation and binding affinity prediction and (iv) use of graphical models to extract peptide sequence signatures to predict epitopes for HLA class I alleles. HLaffy is a novel and efficient epitope prediction method that predicts epitopes for any Class-1 HLA allele, by estimating the binding strengths of peptide-HLA complexes which is achieved through learning pair-potentials important for peptide binding. It relies on the strength of the mechanistic understanding of peptide-HLA recognition and provides an estimate of the total ligand space for each allele. The performance of HLaffy is seen to be superior to the currently available methods. The method is made accessible through a webserver http://proline.biochem.iisc.ernet.in/HLaffy : nchandra@biochem.iisc.ernet.in Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genetic dissection of the consensus sequence for the class 2 and class 3 flagellar promoters

PubMed Central

Wozniak, Christopher E.; Hughes, Kelly T.

2008-01-01

Summary Computational searches for DNA binding sites often utilize consensus sequences. These search models make assumptions that the frequency of a base pair in an alignment relates to the base pair’s importance in binding and presume that base pairs contribute independently to the overall interaction with the DNA binding protein. These two assumptions have generally been found to be accurate for DNA binding sites. However, these assumptions are often not satisfied for promoters, which are involved in additional steps in transcription initiation after RNA polymerase has bound to the DNA. To test these assumptions for the flagellar regulatory hierarchy, class 2 and class 3 flagellar promoters were randomly mutagenized in Salmonella. Important positions were then saturated for mutagenesis and compared to scores calculated from the consensus sequence. Double mutants were constructed to determine how mutations combined for each promoter type. Mutations in the binding site for FlhD4C2, the activator of class 2 promoters, better satisfied the assumptions for the binding model than did mutations in the class 3 promoter, which is recognized by the σ28 transcription factor. These in vivo results indicate that the activator sites within flagellar promoters can be modeled using simple assumptions but that the DNA sequences recognized by the flagellar sigma factor require more complex models. PMID:18486950

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection

PubMed Central

Rozanov, Dmitri V.; Rozanov, Nikita D.; Chiotti, Kami; Reddy, Ashok; Wilmarth, Phillip A.; David, Larry L.; Cha, Seung W.; Woo, Sunghee; Pevzner, Pavel; Bafna, Vineet; Burrows, Gregory G.; Rantala, Juha K.; Levin, Trevor; Anur, Pavana; Johnson-Camacho, Katie; Tabatabaei, Shaadi; Munson, Daniel J.; Bruno, Tullia C.; Slansky, Jill E.; Kappler, John W.; Hirano, Naoto; Boegel, Sebastian; Fox, Bernard A.; Egelston, Colt; Simons, Diana L.; Jimenez, Grecia; Lee, Peter P.; Gray, Joe W.; Spellman, Paul T.

2018-01-01

Breast cancer therapy based on amplifying a patient’s antitumor immune response depends on the availability of appropriate MHC class I-restricted, breast cancer-specific epitopes. To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cell lines. We determined the sequence of 3,196 MHC class I-bound peptides representing 1,921 proteins from a panel of 20 breast cancer cell lines including basal, luminal, and claudin-low subtypes. The data has been deposited to the ProteomeXchange with identifier PXD006406. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2,740. Of the unique peptides eluted, more than 1,750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, only 3 of these immunogenic peptides have been identified in breast cancer cells in earlier studies. MHC class I binding probability of eluted peptides was used to plot the distribution of MHC class I allele-specific peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. PMID:29331515

Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai

2012-02-21

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology ofmore » the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.« less

Dopamine transporter SPECT in patients with mitochondrial disorders

PubMed Central

Minnerop, M; Kornblum, C; Joe, A; Tatsch, K; Kunz, W; Klockgether, T; Wullner, U; Reinhardt, M

2005-01-01

Objective : To investigate the dopaminergic system in patients with known mitochondrial disorders and complex I deficiency. Methods: Dopamine transporter density was studied in 10 female patients with mitochondrial complex I deficiency by 123I-FP-CIT (N-ß-fluoropropyl-2ß-carbomethyl-3ß-(4-iodophenyl)-nortropane) SPECT. Results: No differences in 123I-FP-CIT striatal binding ratios were observed and no correlation of the degree of complex I deficiency and striatal binding ratios could be detected. Conclusions: These data argue against the possibility that mitochondrial complex I deficiency by itself is sufficient to elicit dopaminergic cell loss. PMID:15608010

Crystal Structure of HIV-1 Primary Receptor CD4 i Complex with a Potent Antiviral Antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freeman, M.M.; Hong, X.; Seaman, M.S.

2010-06-18

Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 {angstrom} resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalentmore » forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.« less

Calculation of site affinity constants and cooperativity coefficients for binding of ligands and/or protons to macromolecules. II. Relationships between chemical model and partition function algorithm.

PubMed

Fisicaro, E; Braibanti, A; Lamb, J D; Oscarson, J L

1990-05-01

The relationships between the chemical properties of a system and the partition function algorithm as applied to the description of multiple equilibria in solution are explained. The partition functions ZM, ZA, and ZH are obtained from powers of the binary generating functions Jj = (1 + kappa j gamma j,i[Y])i tau j, where i tau j = p tau j, q tau j, or r tau j represent the maximum number of sites in sites in class j, for Y = M, A, or H, respectively. Each term of the generating function can be considered an element (ij) of a vector Jj and each power of the cooperativity factor gamma ij,i can be considered an element of a diagonal cooperativity matrix gamma j. The vectors Jj are combined in tensor product matrices L tau = (J1) [J2]...[Jj]..., thus representing different receptor-ligand combinations. The partition functions are obtained by summing elements of the tensor matrices. The relationship of the partition functions with the total chemical amounts TM, TA, and TH has been found. The aim is to describe the total chemical amounts TM, TA, and TH as functions of the site affinity constants kappa j and cooperativity coefficients bj. The total amounts are calculated from the sum of elements of tensor matrices Ll. Each set of indices (pj..., qj..., rj...) represents one element of a tensor matrix L tau and defines each term of the summation. Each term corresponds to the concentration of a chemical microspecies. The distinction between microspecies MpjAqjHrj with ligands bound on specific sites and macrospecies MpAqHR corresponding to a chemical stoichiometric composition is shown. The translation of the properties of chemical model schemes into the algorithms for the generation of partition functions is illustrated with reference to a series of examples of gradually increasing complexity. The equilibria examined concern: (1) a unique class of sites; (2) the protonation of a base with two classes of sites; (3) the simultaneous binding of ligand A and proton H to a macromolecule or receptor M with four classes of sites; and (4) the binding to a macromolecule M of ligand A which is in turn a receptor for proton H. With reference to a specific example, it is shown how a computer program for least-squares refinement of variables kappa j and bj can be organized. The chemical model from the free components M, A, and H to the saturated macrospecies MpAQHR, with possible complex macrospecies MpAq and AHR, is defined first.(ABSTRACT TRUNCATED AT 250 WORDS)

NetCTLpan: pan-specific MHC class I pathway epitope predictions

PubMed Central

Larsen, Mette Voldby; Lundegaard, Claus; Nielsen, Morten

2010-01-01

Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I ligands and cytotoxic T lymphocyte (CTL) epitopes. It has been reported that MHC molecules are differentially dependent on TAP transport and proteasomal cleavage. Here, we did not find any consistent signs of such MHC dependencies, and the NetCTLpan method is implemented with fixed weights for proteasomal cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying MHC class I epitopes. Using the NetCTLpan method, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively, when compared to the NetMHCpan and NetCTL methods. The method and benchmark datasets are available at http://www.cbs.dtu.dk/services/NetCTLpan/. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0441-4) contains supplementary material, which is available to authorized users. PMID:20379710

Marsupials and monotremes possess a novel family of MHC class I genes that is lost from the eutherian lineage.

PubMed

Papenfuss, Anthony T; Feng, Zhi-Ping; Krasnec, Katina; Deakin, Janine E; Baker, Michelle L; Miller, Robert D

2015-07-22

Major histocompatibility complex (MHC) class I genes are found in the genomes of all jawed vertebrates. The evolution of this gene family is closely tied to the evolution of the vertebrate genome. Family members are frequently found in four paralogous regions, which were formed in two rounds of genome duplication in the early vertebrates, but in some species class Is have been subject to additional duplication or translocation, creating additional clusters. The gene family is traditionally grouped into two subtypes: classical MHC class I genes that are usually MHC-linked, highly polymorphic, expressed in a broad range of tissues and present endogenously-derived peptides to cytotoxic T-cells; and non-classical MHC class I genes generally have lower polymorphism, may have tissue-specific expression and have evolved to perform immune-related or non-immune functions. As immune genes can evolve rapidly and are subject to different selection pressure, we hypothesised that there may be divergent, as yet unannotated or uncharacterised class I genes. Application of a novel method of sensitive genome searching of available vertebrate genome sequences revealed a new, extensive sub-family of divergent MHC class I genes, denoted as UT, which has not previously been characterized. These class I genes are found in both American and Australian marsupials, and in monotremes, at an evolutionary chromosomal breakpoint, but are not present in non-mammalian genomes and have been lost from the eutherian lineage. We show that UT family members are expressed in the thymus of the gray short-tailed opossum and in other immune tissues of several Australian marsupials. Structural homology modelling shows that the proteins encoded by this family are predicted to have an open, though short, antigen-binding groove. We have identified a novel sub-family of putatively non-classical MHC class I genes that are specific to marsupials and monotremes. This family was present in the ancestral mammal and is found in extant marsupials and monotremes, but has been lost from the eutherian lineage. The function of this family is as yet unknown, however, their predicted structure may be consistent with presentation of antigens to T-cells.

Molecular dynamics simulations revealed structural differences among WRKY domain-DNA interaction in barley (Hordeum vulgare).

PubMed

Pandey, Bharati; Grover, Abhinav; Sharma, Pradeep

2018-02-12

The WRKY transcription factors are a class of DNA-binding proteins involved in diverse plant processes play critical roles in response to abiotic and biotic stresses. Genome-wide divergence analysis of WRKY gene family in Hordeum vulgare provided a framework for molecular evolution and functional roles. So far, the crystal structure of WRKY from barley has not been resolved; moreover, knowledge of the three-dimensional structure of WRKY domain is pre-requisites for exploring the protein-DNA recognition mechanisms. Homology modelling based approach was used to generate structures for WRKY DNA binding domain (DBD) and its variants using AtWRKY1 as a template. Finally, the stability and conformational changes of the generated model in unbound and bound form was examined through atomistic molecular dynamics (MD) simulations for 100 ns time period. In this study, we investigated the comparative binding pattern of WRKY domain and its variants with W-box cis-regulatory element using molecular docking and dynamics (MD) simulations assays. The atomic insight into WRKY domain exhibited significant variation in the intermolecular hydrogen bonding pattern, leading to the structural anomalies in the variant type and differences in the DNA-binding specificities. Based on the MD analysis, residual contribution and interaction contour, wild-type WRKY (HvWRKY46) were found to interact with DNA through highly conserved heptapeptide in the pre- and post-MD simulated complexes, whereas heptapeptide interaction with DNA was missing in variants (I and II) in post-MD complexes. Consequently, through principal component analysis, wild-type WRKY was also found to be more stable by obscuring a reduced conformational space than the variant I (HvWRKY34). Lastly, high binding free energy for wild-type and variant II allowed us to conclude that wild-type WRKY-DNA complex was more stable relative to variants I. The results of our study revealed complete dynamic and structural information about WRKY domain-DNA interactions. However, no structure base information reported to date for WRKY variants and their mechanism of interaction with DNA. Our findings highlighted the importance of selecting a sequence to generate newer transgenic plants that would be increasingly tolerance to stress conditions.

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

PubMed

Bartl, S; Weissman, I L

1994-01-04

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Identification, cloning and characterization of the tomato TCP transcription factor family.

PubMed

Parapunova, Violeta; Busscher, Marco; Busscher-Lange, Jacqueline; Lammers, Michiel; Karlova, Rumyana; Bovy, Arnaud G; Angenent, Gerco C; de Maagd, Ruud A

2014-06-06

TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.

Identification, cloning and characterization of the tomato TCP transcription factor family

PubMed Central

2014-01-01

Background TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. Results We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. Conclusions The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development. PMID:24903607

Reactivity Study of Unsymmetrical β-Diketiminato Copper(I) Complexes: Effect of the Chelating Ring.

PubMed

Chuang, Wan-Jung; Hsu, Sung-Po; Chand, Kuldeep; Yu, Fu-Lun; Tsai, Cheng-Long; Tseng, Yu-Hsuan; Lu, Yuh-Hsiu; Kuo, Jen-Yu; Carey, James R; Chen, Hsuan-Ying; Chen, Hsing-Yin; Chiang, Michael Y; Hsu, Sodio C N

2017-03-06

β-Diketiminato copper(I) complexes play important roles in bioinspired catalytic chemistry and in applications to the materials industry. However, it has been observed that these complexes are very susceptible to disproportionation. Coordinating solvents or Lewis bases are typically used to prevent disproportionation and to block the coordination sites of the copper(I) center from further decomposition. Here, we incorporate this coordination protection directly into the molecule in order to increase the stability and reactivity of these complexes and to discover new copper(I) binding motifs. Here we describe the synthesis, structural characterization, and reactivity of a series of unsymmetrical N-aryl-N'-alkylpyridyl β-diketiminato copper(I) complexes and discuss the structures and reactivity of these complexes with respect to the length of the pyridyl arm. All of the aforementioned unsymmetrical ß-diketiminato copper(I) complexes bind CO reversibly and are stable to disproportionation. The binding ability of CO and the rate of pyridyl ligand decoordination of these copper(I) complexes are directly related to the competition between the degree of puckering of the chelate system and the steric demands of the N-aryl substituent.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain*S⃞

PubMed Central

Bottomley, Matthew J.; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-01-01

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions. PMID:18614528

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

PubMed

Bottomley, Matthew J; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-09-26

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

(+)-3-( sup 123 I)Iodo-MK-801: Synthesis and characterization of binding to the N-methyl-D-aspartate receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ransom, R.W.; Wai-si Eng; Burns, H.D.

1990-01-01

Synthetic methods have been established for preparing high specific activity (+)-3-({sup 123}I)Iodo-MK-801 in high radiochemical yield. The binding of the radiotracer to rat cortical membranes has been examine to assess its potential use as an in vivo imaging agent for the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. Under the conditions of the assay, specific (+)-3-({sup 123}I)Iodo-MK-801 binding to membrane homogenates represented greater than 95% of the total binding. Several structurally distinct, noncompetitive NMDA receptor antagonists inhibited binding with potencies in accordance with their reported inhibitory activity at the receptor complex. The concentration of ({plus minus})-3-Iodo-MK-801 required to inhibit 50% of (+)-3-({supmore » 123}I)Iodo-MK-801 binding (IC{sub 50}) was 3.4 nM when using a low ionic strength assay buffer and 5.5 nM in a physiological buffer. In a thoroughly washed membrane preparation, (+)-3-({sup 123}I)Iodo-MK-801 binding was enhanced by L-glutamate and glycine at concentrations known to activate the NMDA receptor. The results indicate that (+)-3-({sup 123}I)Iodo-MK-801 specifically labels the NMDA receptor complex in rat brain membranes and the retention of high affinity under near physiological assay conditions suggests that it may be useful as a SPECT imaging agent for the receptor in vivo.« less

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group.

PubMed

Lobera, Mercedes; Madauss, Kevin P; Pohlhaus, Denise T; Wright, Quentin G; Trocha, Mark; Schmidt, Darby R; Baloglu, Erkan; Trump, Ryan P; Head, Martha S; Hofmann, Glenn A; Murray-Thompson, Monique; Schwartz, Benjamin; Chakravorty, Subhas; Wu, Zining; Mander, Palwinder K; Kruidenier, Laurens; Reid, Robert A; Burkhart, William; Turunen, Brandon J; Rong, James X; Wagner, Craig; Moyer, Mary B; Wells, Carrow; Hong, Xuan; Moore, John T; Williams, Jon D; Soler, Dulce; Ghosh, Shomir; Nolan, Michael A

2013-05-01

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jie; Lancaster, Laura; Trakhanov, Sergei

2012-03-26

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 {angstrom} crystal structure of the RF3 {center_dot} GDPNP {center_dot} ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7{sup o} rotation of the body and 14{sup o} rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. Wemore » suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.« less

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

PubMed Central

Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

2016-01-01

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

Prediction of the binding affinities of peptides to class II MHC using a regularized thermodynamic model

PubMed Central

2010-01-01

Background The binding of peptide fragments of extracellular peptides to class II MHC is a crucial event in the adaptive immune response. Each MHC allotype generally binds a distinct subset of peptides and the enormous number of possible peptide epitopes prevents their complete experimental characterization. Computational methods can utilize the limited experimental data to predict the binding affinities of peptides to class II MHC. Results We have developed the Regularized Thermodynamic Average, or RTA, method for predicting the affinities of peptides binding to class II MHC. RTA accounts for all possible peptide binding conformations using a thermodynamic average and includes a parameter constraint for regularization to improve accuracy on novel data. RTA was shown to achieve higher accuracy, as measured by AUC, than SMM-align on the same data for all 17 MHC allotypes examined. RTA also gave the highest accuracy on all but three allotypes when compared with results from 9 different prediction methods applied to the same data. In addition, the method correctly predicted the peptide binding register of 17 out of 18 peptide-MHC complexes. Finally, we found that suboptimal peptide binding registers, which are often ignored in other prediction methods, made significant contributions of at least 50% of the total binding energy for approximately 20% of the peptides. Conclusions The RTA method accurately predicts peptide binding affinities to class II MHC and accounts for multiple peptide binding registers while reducing overfitting through regularization. The method has potential applications in vaccine design and in understanding autoimmune disorders. A web server implementing the RTA prediction method is available at http://bordnerlab.org/RTA/. PMID:20089173

NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets.

PubMed

Nielsen, Morten; Andreatta, Massimo

2016-03-30

Binding of peptides to MHC class I molecules (MHC-I) is essential for antigen presentation to cytotoxic T-cells. Here, we demonstrate how a simple alignment step allowing insertions and deletions in a pan-specific MHC-I binding machine-learning model enables combining information across both multiple MHC molecules and peptide lengths. This pan-allele/pan-length algorithm significantly outperforms state-of-the-art methods, and captures differences in the length profile of binders to different MHC molecules leading to increased accuracy for ligand identification. Using this model, we demonstrate that percentile ranks in contrast to affinity-based thresholds are optimal for ligand identification due to uniform sampling of the MHC space. We have developed a neural network-based machine-learning algorithm leveraging information across multiple receptor specificities and ligand length scales, and demonstrated how this approach significantly improves the accuracy for prediction of peptide binding and identification of MHC ligands. The method is available at www.cbs.dtu.dk/services/NetMHCpan-3.0 .

Ultrasensitivity and sharp threshold theorems for multisite systems

NASA Astrophysics Data System (ADS)

Dougoud, M.; Mazza, C.; Vinckenbosch, L.

2017-02-01

This work studies the ultrasensitivity of multisite binding processes where ligand molecules can bind to several binding sites. It considers more particularly recent models involving complex chemical reactions in allosteric phosphorylation processes and for transcription factors and nucleosomes competing for binding on DNA. New statistics-based formulas for the Hill coefficient and the effective Hill coefficient are provided and necessary conditions for a system to be ultrasensitive are exhibited. It is first shown that the ultrasensitivity of binding processes can be approached using sharp-threshold theorems which have been developed in applied probability theory and statistical mechanics for studying sharp threshold phenomena in reliability theory, random graph theory and percolation theory. Special classes of binding process are then introduced and are described as density dependent birth and death process. New precise large deviation results for the steady state distribution of the process are obtained, which permits to show that switch-like ultrasensitive responses are strongly related to the multi-modality of the steady state distribution. Ultrasensitivity occurs if and only if the entropy of the dynamical system has more than one global minimum for some critical ligand concentration. In this case, the Hill coefficient is proportional to the number of binding sites, and the system is highly ultrasensitive. The classical effective Hill coefficient I is extended to a new cooperativity index I q , for which we recommend the computation of a broad range of values of q instead of just the standard one I  =  I 0.9 corresponding to the 10%-90% variation in the dose-response. It is shown that this single choice can sometimes mislead the conclusion by not detecting ultrasensitivity. This new approach allows a better understanding of multisite ultrasensitive systems and provides new tools for the design of such systems.

Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

PubMed Central

Mandic, Robert; Fackler, Oliver T.; Geyer, Matthias; Linnemann, Thomas; Zheng, Yong-Hui; Peterlin, B. Matija

2001-01-01

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity. PMID:11179428

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis*

PubMed Central

Yao, Jiangwei; Abdelrahman, Yasser M.; Robertson, Rosanna M.; Cox, John V.; Belland, Robert J.; White, Stephen W.; Rock, Charles O.

2014-01-01

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis. PMID:24958721

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

PubMed

Fusco, William G; Choudhary, Neelima R; Stewart, Shelley M; Alam, S Munir; Sempowski, Gregory D; Elkins, Christopher; Leduc, Isabelle

2015-04-01

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

PubMed Central

Haupt, V. Joachim; Schroeder, Michael; Labudde, Dirk

2018-01-01

The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes. PMID:29659563

MHC class I loci of the Bar-Headed goose (Anser indicus)

PubMed Central

2010-01-01

MHC class I proteins mediate functions in anti-pathogen defense. MHC diversity has already been investigated by many studies in model avian species, but here we chose the bar-headed goose, a worldwide migrant bird, as a non-model avian species. Sequences from exons encoding the peptide-binding region (PBR) of MHC class I molecules were isolated from liver genomic DNA, to investigate variation in these genes. These are the first MHC class I partial sequences of the bar-headed goose to be reported. A preliminary analysis suggests the presence of at least four MHC class I genes, which share great similarity with those of the goose and duck. A phylogenetic analysis of bar-headed goose, goose and duck MHC class I sequences using the NJ method supports the idea that they all cluster within the anseriforms clade. PMID:21637434

Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: a Joint ESI MS and XRD Investigation.

PubMed

Bazzicalupi, Carla; Ferraroni, Marta; Papi, Francesco; Massai, Lara; Bertrand, Benoît; Messori, Luigi; Gratteri, Paola; Casini, Angela

2016-03-18

The dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G-quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X-ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9-methylcaffein-8-ylidene)2 ](+) and Tel 23 DNA G-quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G-quadruplex recognition and its selectivity are described. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of bovine non-classical major histocompatibility complex class 1 proteins in mouse P815 and human K562 cells

USDA-ARS?s Scientific Manuscript database

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-class...

F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins.

PubMed

Abualrous, Esam T; Fritzsche, Susanne; Hein, Zeynep; Al-Balushi, Mohammed S; Reinink, Peter; Boyle, Louise H; Wellbrock, Ursula; Antoniou, Antony N; Springer, Sebastian

2015-04-01

The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genes in one megabase of the HLA class I region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, H.; Fan, Wu-Fang; Xu, Hongxia

1993-11-15

To define the gene content of the HLA class I region, cDNA selection was applied to three overlapping yeast artificial chromosomes (YACs) that spanned 1 megabase (Mb) of this region of the human major histocompatibility complex. These YACs extended from the region centromeric to HLA-E to the region telomeric to HLA-F. In additions to the recognized class I genes and pseudogenes and the anonymous non-class-I genes described recently by the authors and others, 20 additional anonymous cDNA clones were identified from this 1-Mb region. They also identified a long repetitive DNA element in the region between HLA-B and HLA-E. Homologuesmore » of this outside of the HLA complex. The portion of the HLA class I region represented by these YACs shows an average gene density as high as the class II and class III regions. Thus, the high gene density portion of the HLA complex is extended to more than 3 Mb.« less

Structural studies of bovine, equine, and leporine serum albumin complexes with naproxen.

PubMed

Bujacz, Anna; Zielinski, Kamil; Sekula, Bartosz

2014-09-01

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein-ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA-NPS), equine (ESA-NPS), and leporine (LSA-NPS) determined to 2.58 Å (C2), 2.42 Å (P61), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA-NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA-NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. © 2014 Wiley Periodicals, Inc.

Association studies to transporting proteins of fac-ReI(CO)3(pterin)(H2O) complex.

PubMed

Ragone, Fabricio; Saavedra, Héctor H Martínez; García, Pablo F; Wolcan, Ezequiel; Argüello, Gerardo A; Ruiz, Gustavo T

2017-01-01

A new synthetic route to acquire the water soluble complex fac-Re I (CO) 3 (pterin)(H 2 O) was carried out in aqueous solution. The complex has been obtained with success via the fac-[Re I (CO) 3 (H 2 O) 3 ]Cl precursor complex. Re I (CO) 3 (pterin)(H 2 O) has been found to bind strongly with bovine and human serum albumins (BSA and HSA) with intrinsic-binding constants, K b , of 6.5 × 10 5 M -1 and 5.6 × 10 5 M -1 at 310 K, respectively. The interactions of serum albumins with Re I (CO) 3 (pterin)(H 2 O) were evaluated employing UV-vis fluorescence and absorption spectroscopy and circular dichroism. The results suggest that the serum albumins-Re I (CO) 3 (pterin)(H 2 O) interactions occurred in the domain IIA-binding pocket without loss of helical stability of the proteins. The comparison of the fluorescence quenching of BSA and HSA due to the binding to the Re(I) complex suggested that local interaction around the Trp 214 residue had taken place. The analysis of the thermodynamic parameters ΔG 0 , ΔH 0 , and ΔS 0 indicated that the hydrophobic interactions played a major role in both HSA-Re(I) and BSA-Re(I) association processes. All these experimental results suggest that these proteins can be considered as good carriers for transportation of Re I (CO) 3 (pterin)(H 2 O) complex. This is of significant importance in relation to the use of this Re(I) complex in several biomedical fields, such as photodynamic therapy and radiopharmacy.

Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site

PubMed Central

West, Anthony P; Schamber, Michael; Gazumyan, Anna; Golijanin, Jovana; Seaman, Michael S; Fätkenheuer, Gerd; Klein, Florian; Nussenzweig, Michel C; Bjorkman, Pamela J

2016-01-01

HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46–derived CD4bs bNAbs, which have normal-length CDRL3s. The existence of bNAbs that combine features of VRC01-class and 8ANC131-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs. PMID:27617431

Comparative structural modeling of six old yellow enzymes (OYEs) from the necrotrophic fungus Ascochyta rabiei: insight into novel OYE classes with differences in cofactor binding, organization of active site residues and stereopreferences.

PubMed

Nizam, Shadab; Gazara, Rajesh Kumar; Verma, Sandhya; Singh, Kunal; Verma, Praveen Kumar

2014-01-01

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.

Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.

PubMed

Bago, Ruzica; Malik, Nazma; Munson, Michael J; Prescott, Alan R; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G; Alessi, Dario R

2014-11-01

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity, in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K.

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.

PubMed

Rao, Mangala; Peachman, Kristina K; Alving, Carl R; Rothwell, Stephen W

2003-12-01

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.

Molecular Determinants of Peptide Binding to Two Common Rhesus Macaque Major Histocompatibility Complex Class II Molecules

PubMed Central

Dzuris, John L.; Sidney, John; Horton, Helen; Correa, Rose; Carter, Donald; Chesnut, Robert W.; Watkins, David I.; Sette, Alessandro

2001-01-01

Major histocompatibility complex class II molecules encoded by two common rhesus macaque alleles Mamu-DRB1*0406 and Mamu-DRB*w201 have been purified, and quantitative binding assays have been established. The structural requirements for peptide binding to each molecule were characterized by testing panels of single-substitution analogs of the two previously defined epitopes HIV Env242 (Mamu-DRB1*0406 restricted) and HIV Env482 (Mamu-DRB*w201 restricted). Anchor positions of both macaque DR molecules were spaced following a position 1 (P1), P4, P6, P7, and P9 pattern. The specific binding motif associated with each molecule was distinct, but largely overlapping, and was based on crucial roles of aromatic and/or hydrophobic residues at P1, P6, and P9. Based on these results, a tentative Mamu class II DR supermotif was defined. This pattern is remarkably similar to a previously defined human HLA-DR supermotif. Similarities in binding motifs between human HLA and macaque Mamu-DR molecules were further illustrated by testing a panel of more than 60 different single-substitution analogs of the HLA-DR-restricted HA 307–319 epitope for binding to Mamu-DRB*w201 and HLA-DRB1*0101. The Mamu-DRB1*0406 and -DRB*w201 binding capacity of a set of 311 overlapping peptides spanning the entire simian immunodeficiency virus (SIV) genome was also evaluated. Ten peptides capable of binding both molecules were identified, together with 19 DRB1*0406 and 43 DRB*w201 selective binders. The Mamu-DR supermotif was found to be present in about 75% of the good binders and in 50% of peptides binding with intermediate affinity but only in approximately 25% of the peptides which did not bind either Mamu class II molecule. Finally, using flow cytometric detection of antigen-induced intracellular gamma interferon, we identify a new CD4+ T-lymphocyte epitope encoded within the Rev protein of SIV. PMID:11602736

Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agniswamy, Johnson; Louis, John M.; Roche, Julien

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker thanmore » PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.« less

Isolation of site-specific anharmonicities of individual water molecules in the I-·(H2O)2 complex using tag-free, isotopomer selective IR-IR double resonance

NASA Astrophysics Data System (ADS)

Yang, Nan; Duong, Chinh H.; Kelleher, Patrick J.; Johnson, Mark A.; McCoy, Anne B.

2017-12-01

We reveal the microscopic mechanics of iodide ion microhydration by recording the isotopomer-selective vibrational spectra of the I-·(H2O)·(D2O), I-·(HOD)·(D2O), and I-·(DOH)·(H2O) isotopologues using a new class of ion spectrometer that is optimized to carry out two-color, IR-IR photodissociation in a variety of pump-probe schemes. Using one of these, we record the linear absorption spectrum of a cryogenically cooled cluster without the use of a messenger ;tag;. In another protocol, we reveal the spectra of individual H2O and D2O molecules embedded in each of the two possible binding sites in the iodide dihydrate, as well as the bands due to individual OH and OD groups in each of the four local binding environments. Finally, we demonstrate how temperature dependent isotopic scrambling among the spectral features can be used to monitor the onset of large amplitude motion, heretofore inferred from changes in the envelope of the OH stretching vibrational manifold.

Pseudorabies virus-induced suppression of major histocompatibility complex class I antigen expression.

PubMed Central

Mellencamp, M W; O'Brien, P C; Stevenson, J R

1991-01-01

The ability of pseudorabies virus (PrV) to down-modulate expression of major histocompatibility complex class I antigens in murine and porcine cells was investigated. When quantified by flow cytometry, surface expression of class I Kk and Dk antigens on PrV-infected cells decreased by 60% or more. Down-modulation was associated with a decrease in total cellular class I antigens, indicating regulation at the transcriptional or posttranscriptional level. PrV did not suppress expression of transferrin receptor, suggesting a selective regulatory mechanism. Images PMID:1851884

An MHC class I immune evasion gene of Marek׳s disease virus.

PubMed

Hearn, Cari; Preeyanon, Likit; Hunt, Henry D; York, Ian A

2015-01-15

Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198-205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

PubMed Central

Slama-Schwok, A; Zakrzewska, K; Léger, G; Leroux, Y; Takahashi, M; Käs, E; Debey, P

2000-01-01

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation. PMID:10777751

7TM X-ray structures for class C GPCRs as new drug-discovery tools. 1. mGluR5.

PubMed

Topiol, Sid; Sabio, Michael

2016-01-15

We illustrate, with a focus on mGluR5, how the recently published, first X-ray structures of mGluR 7TM domains, specifically those of mGluR1 and mGluR5 complexed with negative allosteric modulators (NAMs), will begin to influence ligand- (e.g., drug- or sweetener-) discovery efforts involving class C GPCRs. With an extensive docking study allowing full ligand flexibility and full side chain flexibility of all residues in the ligand-binding cavity, we have predicted and analyzed the binding modes of a variety of structurally diverse mGluR5 NAM ligands, showing how the X-ray structures serve to effectively rationalize each ligand's binding characteristics. We demonstrated that the features that are inherent in our earlier overlay model are preserved in the protein structure-based docking models. We identified structurally diverse compounds, which potentially act as mGluR NAMs, and revealed binding-site differences by performing high-throughput docking using a database of approximately six million structures of commercially available compounds and the mGluR1 and mGluR5 X-ray structures. By comparing the 7TM domains of the mGluR5 and mGluR1 X-rays structures, we identified selectivity factors within group I of the mGluRs. Similarly, using homology models that we built for mGluR2 and mGluR4, we have identified the factors leading to the selectivity between group I and groups II and III for ligands occupying the deepest portion of the mGluR5 binding cavity. Finally, we have proposed a structure-based explanation of the pharmacological switching within a set of positive allosteric modulators (PAMs) and their corresponding, very close NAM analogs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) MHC class I heavy chain and β2-microglobulin.

PubMed

Pinto, Rute D; Randelli, Elisa; Buonocore, Francesco; Pereira, Pedro J B; dos Santos, Nuno M S

2013-03-01

In this work, the gene and cDNA of sea bass (Dicentrarchus labrax) β2-microglobulin (Dila-β2m) and several cDNAs of MHC class I heavy chain (Dila-UA) were characterized. While Dila-β2m is single-copy, numerous Dila-UA transcripts were identified per individual with variability at the peptide-binding domain (PBD), but also with unexpected diversity from the connective peptide (CP) through the 3' untranslated region (UTR). Phylogenetic analysis segregates Dila-β2m and Dila-UA into each subfamily cluster, placing them in the fish class and branching Dila-MHC-I with lineage U. The α1 domains resemble those of the recently proposed L1 trans-species lineage. Although no Dila-specific α1, α2 or α3 sub-lineages could be observed, two highly distinct sub-lineages were identified at the CP/TM/CYT regions. The three-dimensional homology model of sea bass MHC-I complex is consistent with other characterized vertebrate structures. Furthermore, basal tissue-specific expression profiles were determined for both molecules, and expression of β2m was evaluated after poly I:C stimulus. Results suggest these molecules are orthologues of other β2m and teleost classical MHC-I and their basic structure is evolutionarily conserved, providing relevant information for further studies on antigen presentation in this fish species. Copyright © 2012 Elsevier Ltd. All rights reserved.

Electrostatic occlusion and quaternary structural ion pairing are key determinants of Cu(I)-mediated allostery in the copper-sensing operon repressor (CsoR).

PubMed

Chang, Feng-Ming James; Martin, Julia E; Giedroc, David P

2015-04-21

The copper-sensing operon repressor (CsoR) is an all-α-helical disc-shaped D2-symmetric homotetramer that forms a 2:1 tetramer/DNA operator complex and represses the expression of copper-resistance genes in a number of bacteria. A previous bioinformatics analysis of CsoR-family repressors distributes Cu(I)-sensing CsoRs in four of seven distinct clades on the basis of global sequence similarity. In this work, we define energetically important determinants of DNA binding in the apo-state (ΔΔGbind), and for allosteric negative coupling of Cu(I) binding to DNA binding (ΔΔGc) in a model clade IV CsoR from Geobacillus thermodenitrificans (Gt) of known structure, by selectively targeting for mutagenesis those charged residues uniquely conserved in clade IV CsoRs. These include a folded N-terminal "tail" and a number of Cu(I)-sensor and clade-specific residues that when mapped onto a model of Cu(I)-bound Gt CsoR define a path across one face of the tetramer. We find that Cu(I)-binding prevents formation of the 2:1 "sandwich" complex rather than DNA binding altogether. Folding of the N-terminal tail (residues R18, E22, R74) upon Cu-binding to the periphery of the tetramer inhibits assembly of the 2:1 apoprotein-DNA complex. In contrast, Ala substitution of residues that surround the central "hole" (R65, K101) in the tetramer, as well R48, impact DNA binding. We also identify a quaternary structural ion-pair, E73-K101″, that crosses the tetramer interface, charge-reversal of which restores DNA binding activity, allosteric regulation by Cu(I), and transcriptional derepression by Cu(I) in cells. These findings suggest an "electrostatic occlusion" model, in which basic residues important for DNA binding and/or allostery become sequestered via ion-pairing specifically in the Cu(I)-bound state, and this aids in copper-dependent disassembly of a repression complex.

Multiheteromacrocycles that Complex Metal Ions. Ninth Progress Report (includes results of last three years), 1 May 1980 -- 30 April 1983

DOE R&D Accomplishments Database

Cram, D. J.

1982-09-15

The overall objective of this research is to design, synthesize, and evaluate cyclic and polycyclic host organic compounds for the abilities to complex and lipophilize guest metal ions, their complexes, and their clusters. Host organic compounds consist of strategically placed solvating, coordinating, and ion-pairing sites tied together by covalent bonds through hydrocarbon units around cavities shaped to be occupied by guest metal ions, or by metal ions plus their ligands. Specificity in complexation is sought by matching the following properties of host and guest: cavity and metal ion sizes; geometric arrangements of binding sites; numbers of binding sites; characters of binding sites; and valences. The hope is to synthesize new classes of compounds useful in the separation of metal ions, their complexes, and their clusters.

Structural analysis of a putative SAM-dependent methyltransferase, YtqB, from Bacillus subtilis.

PubMed

Park, Sun Cheol; Song, Wan Seok; Yoon, Sung-il

2014-04-18

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB. Copyright © 2014 Elsevier Inc. All rights reserved.

Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

DOE PAGES

Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; ...

2014-11-07

The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

Characterisation of major histocompatibility complex class I transcripts in an Australian dragon lizard.

PubMed

Hacking, Jessica; Bertozzi, Terry; Moussalli, Adnan; Bradford, Tessa; Gardner, Michael

2018-07-01

Characterisation of squamate major histocompatibility complex (MHC) genes has lagged behind other taxonomic groups. MHC genes encode cell-surface glycoproteins that present self- and pathogen-derived peptides to T cells and play a critical role in pathogen recognition. Here we characterise MHC class I transcripts for an agamid lizard (Ctenophorus decresii) and investigate the evolution of MHC class I in Iguanian lizards. An iterative assembly strategy was used to identify six full-length C. decresii MHC class I transcripts, which were validated as likely to encode classical class I MHC molecules. Evidence for exon shuffling recombination was uncovered for C. decresii transcripts and Bayesian phylogenetic analysis of Iguanian MHC class I sequences revealed a pattern expected under a birth-and-death mode of evolution. This work provides a stepping stone towards further research on the agamid MHC class I region. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular modeling reveals binding interface of γ-tubulin with GCP4 and interactions with noscapinoids.

PubMed

Suri, Charu; Joshi, Harish C; Naik, Pradeep Kumar

2015-05-01

The initiation of microtubule assembly within cells is guided by a cone shaped multi-protein complex, γ-tubulin ring complex (γTuRC) containing γ-tubulin and atleast five other γ-tubulin-complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ-tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/β-tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ-tubulin and GCP4 through computational techniques. We simulated two complexes of γ-tubulin-GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of -158.82 and -170.19 kcal/mol for dimer1 and -79.53 and -101.50 kcal/mol for dimer2 using MM-PBSA and MM-GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ-tubulin. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ-tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ-tubulin-GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ-tubulin-GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo-noscapine followed by noscapine and amino-noscapine. This offers a novel chemical scaffold for γ-tubulin binding drugs near γ-tubulin-GCP4 interface. © 2015 Wiley Periodicals, Inc.

Alkoxy bridged binuclear rhenium (I) complexes as a potential sensor for β-amyloid aggregation.

PubMed

Sathish, Veerasamy; Babu, Eththilu; Ramdass, Arumugam; Lu, Zong-Zhan; Velayudham, Murugesan; Thanasekaran, Pounraj; Lu, Kuang-Lieh; Rajagopal, Seenivasan

2014-12-01

Alkoxy bridged binuclear rhenium(I) complexes are used as a probe for the selective and sensitive detection of aggregation of β-amyloid fibrils that are consorted with Alzheimer's disease (AD). The strong binding of the complexes is affirmed by the fluorescence enhancement and calculated binding constant value in the order of 10(5)M(-1) is obtained from the Scatchard plots. The binding of β-amyloid can be attributed to π-π stacking interaction of naphthalene moiety present in rhenium(I) complexes, and it is supported by docking studies. The selectivity is quite high towards other proteins and the formation of fibrils can be observed in the range of 30-40 nm through the AFM and TEM techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

NASA Astrophysics Data System (ADS)

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

2012-05-01

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

The coactivator CBP stimulates human T-cell lymphotrophic virus type I Tax transactivation in vitro.

PubMed

Kashanchi, F; Duvall, J F; Kwok, R P; Lundblad, J R; Goodman, R H; Brady, J N

1998-12-18

Tax interacts with the cellular cyclic AMP-responsive element binding protein (CREB) and facilitates the binding of the coactivator CREB binding protein (CBP), forming a multimeric complex on the cyclic AMP-responsive element (CRE)-like sites in the human T-cell lymphotrophic virus type I (HTLV-I) promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I long terminal repeat, but there has been no direct evidence that CBP is required for Tax-mediated transactivation. We present evidence that Tax and CBP activate transcription from the HTLV-I 21 base pair repeats on naked DNA templates. Transcriptional activation of the HTLV-I sequences required both Tax and CBP and could be mediated by either the N-terminal activation domain of CBP or the full-length protein. Fluorescence polarization binding assays indicated that CBP does not markedly enhance the affinity of Tax for the trimeric complex. Transcription analyses suggest that CBP activates Tax-dependent transcription by promoting transcriptional initiation and reinitiation. The ability of CBP to activate the HTLV-I promoter does not involve the stabilization of Tax binding, but rather depends upon gene activation properties of the co-activator that function in the context of a naked DNA template.

Supramolecular Ex plorations: Ex hibiting the Ex tent of Ex tended Cationic Cyclophanes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dale, Edward J.; Vermeulen, Nicolaas A.; Juricek, Michal

Acting as hosts, cationic cyclophanes, consisting of π-electron-poor bipyridinium units, are capable of entering into strong donor–acceptor interactions to form host–guest complexes with various guests when the size and electronic constitution are appropriately matched. A synthetic protocol has been developed that utilizes catalytic quantities of tetrabutylammonium iodide to make a wide variety of cationic pyridinium-based cyclophanes in a quick and easy manner. Members of this class of cationic cyclophanes with boxlike geometries, dubbed ExnBoxm4+ for short, have been prepared by altering a number of variables: (i) n, the number of “horizontal” p-phenylene spacers between adjoining pyridinium units, to modulate themore » “length” of the cavity; (ii) m, the number of “vertical” p-phenylene spacers, to modulate the “width” of the cavity; and (iii) the aromatic linkers, namely, 1,4-di- and 1,3,5-trisubstituted units for the construction of macrocycles (ExBoxes) and macrobicycles (ExCages), respectively. This Account serves as an exploration of the properties that emerge from these structural modifications of the pyridinium-based hosts, coupled with a call for further investigation into the wealth of properties inherent in this class of compounds. By variation of only the aforementioned components, the role of these cationic receptors covers ground that spans (i) synthetic methodology, (ii) extraction and sequestration, (iii) catalysis, (iv) molecular electronics, (v) physical organic chemistry, and (vi) supramolecular chemistry. Ex1Box4+ (or simply ExBox4+) has been shown to be a multipurpose receptor capable of binding a wide range of polycyclic aromatic hydrocarbons (PAHs), while also being a suitable component in switchable mechanically interlocked molecules. Additionally, the electronic properties of some host–guest complexes allow the development of artificial photosystems. Ex2Box4+ boasts the ability to bind both π-electron-rich and -poor aromatic guests in different binding sites located within the same cavity. ExBox24+ forms complexes with C60 in which discrete arrays of aligned fullerenes result in single cocrystals, leading to improved material conductivities. When the substitution pattern of the ExnBox4+ series is changed to 1,3,5-trisubstituted benzenoid cores, the hexacationic cagelike compound, termed ExCage6+, exhibits different kinetics of complexation with guests of varying sizes—a veritable playground for physical organic chemists. The organization of functionality with respect to structure becomes valuable as the number of analogues continues to grow. With each of these minor structural modifications, a wealth of properties emerge, begging the question as to what discoveries await and what properties will be realized with the continued exploration of this area of supramolecular chemistry based on a unique class of receptor molecules.« less

Supramolecular Ex plorations: Ex hibiting the Ex tent of Ex tended Cationic Cyclophanes

DOE PAGES

Dale, Edward J.; Vermeulen, Nicolaas A.; Juricek, Michal; ...

2016-01-19

Acting as hosts, cationic cyclophanes, consisting of π-electron-poor bipyridinium units, are capable of entering into strong donor–acceptor interactions to form host–guest complexes with various guests when the size and electronic constitution are appropriately matched. A synthetic protocol has been developed that utilizes catalytic quantities of tetrabutylammonium iodide to make a wide variety of cationic pyridinium-based cyclophanes in a quick and easy manner. Members of this class of cationic cyclophanes with boxlike geometries, dubbed ExnBoxm4+ for short, have been prepared by altering a number of variables: (i) n, the number of “horizontal” p-phenylene spacers between adjoining pyridinium units, to modulate themore » “length” of the cavity; (ii) m, the number of “vertical” p-phenylene spacers, to modulate the “width” of the cavity; and (iii) the aromatic linkers, namely, 1,4-di- and 1,3,5-trisubstituted units for the construction of macrocycles (ExBoxes) and macrobicycles (ExCages), respectively. This Account serves as an exploration of the properties that emerge from these structural modifications of the pyridinium-based hosts, coupled with a call for further investigation into the wealth of properties inherent in this class of compounds. By variation of only the aforementioned components, the role of these cationic receptors covers ground that spans (i) synthetic methodology, (ii) extraction and sequestration, (iii) catalysis, (iv) molecular electronics, (v) physical organic chemistry, and (vi) supramolecular chemistry. Ex1Box4+ (or simply ExBox4+) has been shown to be a multipurpose receptor capable of binding a wide range of polycyclic aromatic hydrocarbons (PAHs), while also being a suitable component in switchable mechanically interlocked molecules. Additionally, the electronic properties of some host–guest complexes allow the development of artificial photosystems. Ex2Box4+ boasts the ability to bind both π-electron-rich and -poor aromatic guests in different binding sites located within the same cavity. ExBox24+ forms complexes with C60 in which discrete arrays of aligned fullerenes result in single cocrystals, leading to improved material conductivities. When the substitution pattern of the ExnBox4+ series is changed to 1,3,5-trisubstituted benzenoid cores, the hexacationic cagelike compound, termed ExCage6+, exhibits different kinetics of complexation with guests of varying sizes—a veritable playground for physical organic chemists. The organization of functionality with respect to structure becomes valuable as the number of analogues continues to grow. With each of these minor structural modifications, a wealth of properties emerge, begging the question as to what discoveries await and what properties will be realized with the continued exploration of this area of supramolecular chemistry based on a unique class of receptor molecules.« less

Class-first analysis in a continuum: an approach to the complexities of schools, society, and insurgent science

NASA Astrophysics Data System (ADS)

Valdiviezo, Laura Alicia

2010-06-01

This essay addresses Katherine Richardson Bruna's paper: Mexican Immigrant Transnational Social Capital and Class Transformation: Examining the Role of Peer Mediation in Insurgent Science, through five main points . First, I offer a comparison between the traditional analysis of classism in Latin America and Richardson Bruna's call for a class-first analysis in the North American social sciences where there has been a tendency to obviate the specific examination of class relations and class issues. Secondly, I discuss that a class-first analysis solely cannot suffice to depict the complex dimensions in the relations of schools and society. Thus, I suggest a continuum in the class-first analysis. Third, I argue that social constructions surrounding issues of language, ethnicity, and gender necessarily intersect with issues of class and that, in fact, those other constructions offer compatible epistemologies that aid in representing the complexity of social and institutional practices in the capitalist society. Richardson Bruna's analysis of Augusto's interactions with his teacher and peers in the science class provides a fourth point of discussion in this essay. As a final point in my response I discuss Richardson Bruna's idea of making accessible class-first analysis knowledge to educators and especially to science teachers.

Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR.

PubMed

Horváth, Gergő; Bencsura, Ákos; Simon, Ágnes; Tochtrop, Gregory P; DeKoster, Gregory T; Covey, Douglas F; Cistola, David P; Toke, Orsolya

2016-02-01

Besides aiding digestion, bile salts are important signal molecules exhibiting a regulatory role in metabolic processes. Human ileal bile acid binding protein (I-BABP) is an intracellular carrier of bile salts in the epithelial cells of the distal small intestine and has a key role in the enterohepatic circulation of bile salts. Positive binding cooperativity combined with site selectivity of glycocholate and glycochenodeoxycholate, the two most abundant bile salts in the human body, make human I-BABP a unique member of the family of intracellular lipid binding proteins. Solution NMR structure of the ternary complex of human I-BABP with glycocholate and glycochenodeoxycholate reveals an extensive network of hydrogen bonds and hydrophobic interactions stabilizing the bound bile salts. Conformational changes accompanying bile salt binding affects four major regions in the protein including the C/D, E/F and G/H loops as well as the helical segment. Most of these protein regions coincide with a previously described network of millisecond time scale fluctuations in the apo protein, a motion absent in the bound state. Comparison of the heterotypic doubly ligated complex with the unligated form provides further evidence of a conformation selection mechanism of ligand entry. Structural and dynamic aspects of human I-BABP-bile salt interaction are discussed and compared with characteristics of ligand binding in other members of the intracellular lipid binding protein family. The coordinates of the 10 lowest energy structures of the human I-BABP : GCDA : GCA complex as well as the distance restraints used to calculate the final ensemble have been deposited in the Brookhaven Protein Data Bank with accession number 2MM3. © 2015 FEBS.

Type II fatty acid synthesis is essential for the replication of Chlamydia trachomatis.

PubMed

Yao, Jiangwei; Abdelrahman, Yasser M; Robertson, Rosanna M; Cox, John V; Belland, Robert J; White, Stephen W; Rock, Charles O

2014-08-08

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Screening and structure-based modeling of T-cell epitopes of Nipah virus proteome: an immunoinformatic approach for designing peptide-based vaccine.

PubMed

Kamthania, Mohit; Sharma, D K

2015-12-01

Identification of Nipah virus (NiV) T-cell-specific antigen is urgently needed for appropriate diagnostic and vaccination. In the present study, prediction and modeling of T-cell epitopes of Nipah virus antigenic proteins nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, L protein, W protein, V protein and C protein followed by the binding simulation studies of predicted highest binding scorers with their corresponding MHC class I alleles were done. Immunoinformatic tool ProPred1 was used to predict the promiscuous MHC class I epitopes of viral antigenic proteins. The molecular modelings of the epitopes were done by PEPstr server. And alleles structure were predicted by MODELLER 9.10. Molecular dynamics (MD) simulation studies were performed through the NAMD graphical user interface embedded in visual molecular dynamics. Epitopes VPATNSPEL, NPTAVPFTL and LLFVFGPNL of Nucleocapsid, V protein and Fusion protein have considerable binding energy and score with HLA-B7, HLA-B*2705 and HLA-A2MHC class I allele, respectively. These three predicted peptides are highly potential to induce T-cell-mediated immune response and are expected to be useful in designing epitope-based vaccines against Nipah virus after further testing by wet laboratory studies.

Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor Binding Site

DTIC Science & Technology

2016-06-14

characterize FVM04 binding to EBOV GP, FVM04 Fab in complex with GP∆Muc was analyzed by negative stain electron microscopy. The binding location of...FVM04 revealed an epitope consistent with the crest region residues derived by mutagenesis studies. The class averages suggest that only one FVM04 Fab ...binds to each GP trimer (Figure 3A-B). It is likely that the binding orientation and proximity to the threefold axis precludes additional FVM04 Fabs

The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

PubMed

Ho, Ruoya; Stroupe, Christopher

2016-10-01

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA

PubMed Central

Fusco, William G.; Choudhary, Neelima R.; Stewart, Shelley M.; Alam, S. Munir; Sempowski, Gregory D.; Elkins, Christopher

2015-01-01

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrAI) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine. PMID:25897604

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

PubMed Central

Antoniou, Antony N.; Ford, Stuart; Alphey, Magnus; Osborne, Andrew; Elliott, Tim; Powis, Simon J.

2002-01-01

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation. PMID:12032078

NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11.

PubMed

Lundegaard, Claus; Lamberth, Kasper; Harndahl, Mikkel; Buus, Søren; Lund, Ole; Nielsen, Morten

2008-07-01

NetMHC-3.0 is trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles. As only the MHC class I prediction server is available, predictions are possible for peptides of length 8-11 for all 122 alleles. artificial neural network predictions are given as actual IC(50) values whereas PSSM predictions are given as a log-odds likelihood scores. The output is optionally available as download for easy post-processing. The training method underlying the server is the best available, and has been used to predict possible MHC-binding peptides in a series of pathogen viral proteomes including SARS, Influenza and HIV, resulting in an average of 75-80% confirmed MHC binders. Here, the performance is further validated and benchmarked using a large set of newly published affinity data, non-redundant to the training set. The server is free of use and available at: http://www.cbs.dtu.dk/services/NetMHC.

Characterization of mGluR5R, a novel, metabotropic glutamate receptor 5-related gene.

PubMed

Bates, Brian; Xie, Yuhong; Taylor, Noel; Johnson, Jeremy; Wu, Leeying; Kwak, Seung; Blatcher, Maria; Gulukota, Kamalakar; Paulsen, Janet E

2002-12-30

We report here the isolation of a novel gene termed mGluR5R (mGluR5-related). The N-terminus of mGluR5R is highly similar to the extracellular domain of metabotropic glutamate receptor 5 (mGluR5) whereas the C-terminus bears similarity to the testis-specific gene, RNF18. mGluR5R is expressed in the human CNS in a coordinate fashion with mGluR5. Although the sequence suggests that mGluR5R may be a secreted glutamate binding protein, we found that when expressed in HEK293 cells it was membrane associated and not secreted. Furthermore, mGluR5R was incapable of binding the metabotropic glutamate receptor class I selective agonist, quisqualate. Although mGluR5R could not form disulfide-mediated covalent homodimers, it was able to form a homomeric complex, presumably through noncovalent interactions. mGluR5R also formed noncovalent heteromeric associations with an engineered construct of the extracellular domain of mGluR5 as well as with full-length mGluR5 and mGluR1alpha. The ability of mGluR5R to associate with mGluR1alpha and mGluR5 suggests that it may be a modulator of class I metabotropic glutamate receptor function.

NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence

PubMed Central

Nielsen, Morten; Lundegaard, Claus; Blicher, Thomas; Lamberth, Kasper; Harndahl, Mikkel; Justesen, Sune; Røder, Gustav; Peters, Bjoern; Sette, Alessandro; Lund, Ole; Buus, Søren

2007-01-01

Background Binding of peptides to Major Histocompatibility Complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC class I system (HLA-I) is extremely polymorphic. The number of registered HLA-I molecules has now surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. Principal Findings Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account, and generates quantitative predictions of the affinity of any peptide-HLA-I interaction. Prospective experimental validation of peptides predicted to bind to previously untested HLA-I molecules, cross-validation, and retrospective prediction of known HIV immune epitopes and endogenous presented peptides, all successfully validate this method. We further demonstrate that the method can be applied to perform a clustering analysis of MHC specificities and suggest using this clustering to select particularly informative novel MHC molecules for future biochemical and functional analysis. Conclusions Encompassing all HLA molecules, this high-throughput computational method lends itself to epitope searches that are not only genome- and pathogen-wide, but also HLA-wide. Thus, it offers a truly global analysis of immune responses supporting rational development of vaccines and immunotherapy. It also promises to provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan. PMID:17726526

Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP.

PubMed

Xu, Shuping; Hori, Roderick T

2004-09-01

RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110. Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized. This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF. The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48. Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays. When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP. By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes [Transcription Factor IID (TFIID) and TFIIIB] were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48. The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.

The D0 immunoglobulin-like domain plays a central role for the stronger binding of KIR3DL2 to B27 free heavy chain dimers

PubMed Central

Hatano, Hiroko; Shaw, Jacqueline; Marquardt, Kaitlin; Zhang, Zhiyong; Gauthier, Laurent; Chanteux, Stephanie; Rossi, Benjamin; Li, Demin; Mitchell, Julie; Kollnberger, Simon

2015-01-01

We have proposed that the killer cell immunoglobulin-like receptor KIR3DL2 binding more strongly to HLA-B27 (B27) β2m-free heavy chain (FHC) dimers regulates lymphocyte function in arthritis and infection. We compared the function of B27 FHC dimers with other class I heavy chains and identified contact residues in KIR3DL2. B27 FHC dimers interacted functionally with KIR3DL2 on NK and reporter cells more strongly than other class I FHC. Mutagenesis identified key residues in the D0 and other immunoglobulin-like domains which were shared and distinct from KIR3DL1, for KIR3DL2 binding to B27 and other class I FHC. We modeled B27 dimer binding to KIR3DL2 and compared experimental mutagenesis data with computational “hot spot” predictions. Modelling predicts the stronger binding of B27 dimers to KIR3DL2 is mediated by non-symmetrical complementary contacts of the D0 and D1 domains with the α1, α2 and α3 domains of both B27 heavy chains. By contrast, the D2 domain primarily contacts residues in the α2 domain of one B27 heavy chain. These findings both provide novel insights about the molecular basis of KIR3DL2 binding to HLA-B27 and other ligands and suggest an important role for KIR3DL2 HLA-B27 interactions in controlling the function of NK cells in HLA-B27+ individuals. PMID:25582852

High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

PubMed

Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

2010-12-01

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.

PubMed

Essen, L O; Perisic, O; Lynch, D E; Katan, M; Williams, R L

1997-03-11

We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishino, Tomonori G.; Department of Biotechnology, University of Tokyo, Bunkyo-ku, Tokyo 113-8657; Miyazaki, Masaya

Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a mannermore » dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.« less

HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4+ T Cell Responses

PubMed Central

Wang, Mingjun; Larsen, Mette V.; Nielsen, Morten; Harndahl, Mikkel; Justesen, Sune; Dziegiel, Morten H.; Buus, Søren; Tang, Sheila T.; Lund, Ole; Claesson, Mogens H.

2010-01-01

Background Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. Methodology/Principal Findings In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNγ ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4+ or CD8+ T cells prior to the ELISPOT culture revealed that effectors are either CD4+ (the majority of reactivities) or CD8+ T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4+ T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. Conclusions/Significance HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4+ T cell responses restricted by HLA-II molecules. PMID:20479886

Docosahexaenoic acid modifies the clustering and size of lipid rafts and the lateral organization and surface expression of MHC class I of EL4 cells.

PubMed

Shaikh, Saame Raza; Rockett, Benjamin Drew; Salameh, Muhammad; Carraway, Kristen

2009-09-01

An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data.

PubMed

Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten

2017-11-01

Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I molecules. Peptide binding to MHC molecules is the single most selective step in the Ag-presentation pathway. Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing number of MHC-presented peptides identified by mass spectrometry have been reported containing information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging the information from both data types. Large-scale benchmarking of the method demonstrates an increase in predictive performance compared with state-of-the-art methods when it comes to identification of naturally processed ligands, cancer neoantigens, and T cell epitopes. Copyright © 2017 by The American Association of Immunologists, Inc.

Bioinorganic Chemistry of Parkinson's Disease: Affinity and Structural Features of Cu(I) Binding to the Full-Length β-Synuclein Protein.

PubMed

Miotto, Marco C; Pavese, Mayra D; Quintanar, Liliana; Zweckstetter, Markus; Griesinger, Christian; Fernández, Claudio O

2017-09-05

Alterations in the levels of copper in brain tissue and formation of α-synuclein (αS)-copper complexes might play a key role in the amyloid aggregation of αS and the onset of Parkinson's disease (PD). Recently, we demonstrated that formation of the high-affinity Cu(I) complex with the N-terminally acetylated form of the protein αS substantially increases and stabilizes local conformations with α-helical secondary structure and restricted motility. In this work, we performed a detailed NMR-based structural characterization of the Cu(I) complexes with the full-length acetylated form of its homologue β-synuclein (βS), which is colocalized with αS in vivo and can bind copper ions. Our results show that, similarly to αS, the N-terminal region of βS constitutes the preferential binding interface for Cu(I) ions, encompassing two independent and noninteractive Cu(I) binding sites. According to these results, βS binds the metal ion with higher affinity than αS, in a coordination environment that involves the participation of Met-1, Met-5, and Met-10 residues (site 1). Compared to αS, the shift of His from position 50 to 65 in the N-terminal region of βS does not change the Cu(I) affinity features at that site (site 2). Interestingly, the formation of the high-affinity βS-Cu(I) complex at site 1 in the N-terminus promotes a short α-helix conformation that is restricted to the 1-5 segment of the AcβS sequence, which differs with the substantial increase in α-helix conformations seen for N-terminally acetylated αS upon Cu(I) complexation. Our NMR data demonstrate conclusively that the differences observed in the conformational transitions triggered by Cu(I) binding to AcαS and AcβS find a correlation at the level of their backbone dynamic properties; added to the potential biological implications of these findings, this fact opens new avenues of investigations into the bioinorganic chemistry of PD.

The measles virus N(TAIL)-XD complex: an illustrative example of fuzziness.

PubMed

Longhi, Sonia

2012-01-01

In this chapter, I focus on the biochemical and structural characterization of the complex between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) and the C-terminal X domain (XD) of the viral phosphoprotein (P). I summarize the main experimental data available so far pointing out the prevalently disordered nature of N(TAIL) even after complex formation and the role of the flexible C-terminal appendage in the binding reaction. I finally discuss the possible functional role of these residual disordered regions within the complex in terms of their ability to capture other regulatory, binding partners.

Improvement of Peptide-Based Tumor Immunotherapy Using pH-Sensitive Fusogenic Polymer-Modified Liposomes.

PubMed

Yoshizaki, Yuta; Yuba, Eiji; Komatsu, Toshihiro; Udaka, Keiko; Harada, Atsushi; Kono, Kenji

2016-09-26

To establish peptide vaccine-based cancer immunotherapy, we investigated the improvement of antigenic peptides by encapsulation with pH-sensitive fusogenic polymer-modified liposomes for induction of antigen-specific immunity. The liposomes were prepared by modification of egg yolk phosphatidylcholine and l-dioleoyl phosphatidylethanolamine with 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG) and were loaded with antigenic peptides derived from ovalbumin (OVA) OVA-I (SIINFEKL), and OVA-II (PSISQAVHAAHAEINEAP β A), which bind, respectively, to major histocompatibility complex (MHC) class I and class II molecules on dendritic cell (DCs). The peptide-loaded liposomes were taken up efficiently by DCs. The peptides were delivered into their cytosol. Administration of OVA-I-loaded MGlu-HPG-modified liposomes to mice bearing OVA-expressing E.G7-OVA tumors induced the activation of OVA-specific CTLs much more efficiently than the administration of free OVA-I peptide did. Mice strongly rejected E.G7-OVA cells after immunization with OVA-I peptide-loaded MGlu-HPG liposomes, although mice treated with free OVA-I peptide only slightly rejected the cells. Furthermore, efficient suppression of tumor volume was observed when tumor-bearing mice were immunized with OVA-I-peptide-loaded liposomes. Immunization with OVA-II-loaded MGlu-HPG-modified liposomes exhibited much lower tumor-suppressive effects. Results indicate that MGlu-HPG liposomes might be useful for improvement of CTL-inducing peptides for efficient cancer immunotherapy.

Rational Design Synthesis and Evaluation of First Generation Inhibitors of the Giardia Lamblia Fructose-1 6-biphosphate Aldolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Z Li; Z Liu; D Cho

2011-12-31

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn{sup 2+} binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K{sub i} = 14 {micro}M) that is comparable tomore » that of FBP (K{sub m} = 2 {micro}M) or its inert analog TBP (K{sub i} = 1 {micro}M). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3 {angstrom}) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn{sup 2+}. The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn{sup 2+} are not consistent with a strong interaction. To determine if Zn{sup 2+} coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn{sup 2+} coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn{sup 2+} coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn{sup 2+} coordination to binding of 8 to GlFBPA.« less

Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

PubMed

Li, Zhimin; Liu, Zhengang; Cho, Dae Won; Zou, Jiwen; Gong, Maozhen; Breece, Robert M; Galkin, Andrey; Li, Ling; Zhao, Hong; Maestas, Gabriel D; Tierney, David L; Herzberg, Osnat; Dunaway-Mariano, Debra; Mariano, Patrick S

2011-04-01

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn(2+) binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K(i)=14μM) that is comparable to that of FBP (K(m)=2μM) or its inert analog TBP (K(i)=1μM). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn(2+). The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn(2+) are not consistent with a strong interaction. To determine if Zn(2+)coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn(2+) coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn(2+) coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn(2+) coordination to binding of 8 to GlFBPA. Copyright © 2010 Elsevier Inc. All rights reserved.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

PubMed

Chandrasekaran, E V; Xue, Jun; Xia, Jie; Khaja, Siraj D; Piskorz, Conrad F; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

2016-10-01

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Compactly supported Wannier functions and algebraic K -theory

NASA Astrophysics Data System (ADS)

Read, N.

2017-03-01

In a tight-binding lattice model with n orbitals (single-particle states) per site, Wannier functions are n -component vector functions of position that fall off rapidly away from some location, and such that a set of them in some sense span all states in a given energy band or set of bands; compactly supported Wannier functions are such functions that vanish outside a bounded region. They arise not only in band theory, but also in connection with tensor-network states for noninteracting fermion systems, and for flat-band Hamiltonians with strictly short-range hopping matrix elements. In earlier work, it was proved that for general complex band structures (vector bundles) or general complex Hamiltonians—that is, class A in the tenfold classification of Hamiltonians and band structures—a set of compactly supported Wannier functions can span the vector bundle only if the bundle is topologically trivial, in any dimension d of space, even when use of an overcomplete set of such functions is permitted. This implied that, for a free-fermion tensor network state with a nontrivial bundle in class A, any strictly short-range parent Hamiltonian must be gapless. Here, this result is extended to all ten symmetry classes of band structures without additional crystallographic symmetries, with the result that in general the nontrivial bundles that can arise from compactly supported Wannier-type functions are those that may possess, in each of d directions, the nontrivial winding that can occur in the same symmetry class in one dimension, but nothing else. The results are obtained from a very natural usage of algebraic K -theory, based on a ring of polynomials in e±i kx,e±i ky,..., which occur as entries in the Fourier-transformed Wannier functions.

Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, D.; Kern, R; Puthenveedu, M

2009-01-01

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formedmore » by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.« less

Postprocessing of docked protein-ligand complexes using implicit solvation models.

PubMed

Lindström, Anton; Edvinsson, Lotta; Johansson, Andreas; Andersson, C David; Andersson, Ida E; Raubacher, Florian; Linusson, Anna

2011-02-28

Molecular docking plays an important role in drug discovery as a tool for the structure-based design of small organic ligands for macromolecules. Possible applications of docking are identification of the bioactive conformation of a protein-ligand complex and the ranking of different ligands with respect to their strength of binding to a particular target. We have investigated the effect of implicit water on the postprocessing of binding poses generated by molecular docking using MM-PB/GB-SA (molecular mechanics Poisson-Boltzmann and generalized Born surface area) methodology. The investigation was divided into three parts: geometry optimization, pose selection, and estimation of the relative binding energies of docked protein-ligand complexes. Appropriate geometry optimization afforded more accurate binding poses for 20% of the complexes investigated. The time required for this step was greatly reduced by minimizing the energy of the binding site using GB solvation models rather than minimizing the entire complex using the PB model. By optimizing the geometries of docking poses using the GB(HCT+SA) model then calculating their free energies of binding using the PB implicit solvent model, binding poses similar to those observed in crystal structures were obtained. Rescoring of these poses according to their calculated binding energies resulted in improved correlations with experimental binding data. These correlations could be further improved by applying the postprocessing to several of the most highly ranked poses rather than focusing exclusively on the top-scored pose. The postprocessing protocol was successfully applied to the analysis of a set of Factor Xa inhibitors and a set of glycopeptide ligands for the class II major histocompatibility complex (MHC) A(q) protein. These results indicate that the protocol for the postprocessing of docked protein-ligand complexes developed in this paper may be generally useful for structure-based design in drug discovery.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

PubMed

Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

2016-03-28

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Probing binding hot spots at protein-RNA recognition sites.

PubMed

Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

2016-01-29

We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

PubMed

Hilton, Hugo G; Moesta, Achim K; Guethlein, Lisbeth A; Blokhuis, Jeroen; Parham, Peter; Norman, Paul J

2015-10-01

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I. Copyright © 2015 Elsevier B.V. All rights reserved.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

PubMed Central

Urata, Shuzo; Yokosawa, Hideyoshi; Yasuda, Jiro

2007-01-01

Background HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins. Results Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding. Conclusion These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus. PMID:17601348

Mechanism for the Inhibition of the Carboxyl-transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

L Yu; Y Kim; L Tong

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and have been targeted for drug development against obesity, diabetes, and other diseases. The carboxyltransferase (CT) domain of this enzyme is the site of action for three different classes of herbicides, as represented by haloxyfop, tepraloxydim, and pinoxaden. Our earlier studies have demonstrated that haloxyfop and tepraloxydim bind in the CT active site at the interface of its dimer. However, the two compounds probe distinct regions of the dimer interface, sharing primarily only two common anchoring points of interaction with the enzyme. We report here the crystal structure of the CT domain ofmore » yeast ACC in complex with pinoxaden at 2.8-{angstrom} resolution. Despite their chemical diversity, pinoxaden has a similar binding mode as tepraloxydim and requires a small conformational change in the dimer interface for binding. Crystal structures of the CT domain in complex with all three classes of herbicides confirm the importance of the two anchoring points for herbicide binding. The structures also provide a foundation for understanding the molecular basis of the herbicide resistance mutations and cross resistance among the herbicides, as well as for the design and development of new inhibitors against plant and human ACCs.« less

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.

2012-05-15

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized bymore » interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.« less

Structural and functional organization of the ESCRT-I trafficking complex

PubMed Central

Kostelansky, Michael S.; Sun, Ji; Lee, Sangho; Kim, Jaewon; Ghirlando, Rodolfo; Hierro, Aitor; Emr, Scott D.; Hurley, James H.

2006-01-01

Summary The Endosomal Sorting Complex Required for Transport (ESCRT) complexes are central to receptor downregulation, lysosome biogenesis, and budding of HIV. The yeast ESCRT-I complex contains the Vps23, Vps28, and Vps37 proteins and its assembly is directed by the C-terminal steadiness box of Vps23, the N-terminal half of Vps28, and the C-terminal half of Vps37. The crystal structures of a Vps23:Vps28 core subcomplex and the Vps23:Vps28:Vps37 core were solved at 2.1 and 2.8 Å resolution. Each subunit contains a structurally similar pair of helices that form the core. The N-terminal domain of Vps28 has a hydrophobic binding site on its surface that is conformationally dynamic. The C-terminal domain of Vps28 binds the ESCRT-II complex. The structure shows how ESCRT-I is assembled by a compact core from which the Vps23 UEVdomain, the Vps28 C-domain, and other domains project to bind their partners. PMID:16615894

BiodMHC: an online server for the prediction of MHC class II-peptide binding affinity.

PubMed

Wang, Lian; Pan, Danling; Hu, Xihao; Xiao, Jinyu; Gao, Yangyang; Zhang, Huifang; Zhang, Yan; Liu, Juan; Zhu, Shanfeng

2009-05-01

Effective identification of major histocompatibility complex (MHC) molecules restricted peptides is a critical step in discovering immune epitopes. Although many online servers have been built to predict class II MHC-peptide binding affinity, they have been trained on different datasets, and thus fail in providing a unified comparison of various methods. In this paper, we present our implementation of seven popular predictive methods, namely SMM-align, ARB, SVR-pairwise, Gibbs sampler, ProPred, LP-top2, and MHCPred, on a single web server named BiodMHC (http://biod.whu.edu.cn/BiodMHC/index.html, the software is available upon request). Using a standard measure of AUC (Area Under the receiver operating characteristic Curves), we compare these methods by means of not only cross validation but also prediction on independent test datasets. We find that SMM-align, ProPred, SVR-pairwise, ARB, and Gibbs sampler are the five best-performing methods. For the binding affinity prediction of class II MHC-peptide, BiodMHC provides a convenient online platform for researchers to obtain binding information simultaneously using various methods.

Antibody-mediated cofactor-driven reactions

DOEpatents

Schultz, Peter G.

1993-01-01

Chemical reactions capable of being rate-enhanced by auxiliary species which interact with the reactants but do not become chemically bound to them in the formation of the final product are performed in the presence of antibodies which promote the reactions. The antibodies contain regions within their antigen binding sites which recognize the auxiliary species in a conformation which promotes the reaction. The antigen binding site frequently recognizes a particular transition state complex or other high energy complex along the reaction coordinate, thereby promoting the progress of the reaction along the desired route as opposed to other less favorable routes. Various classes of reaction together with appropriate antigen binding site specificities tailored for each are disclosed.

Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of beta-tubulins.

PubMed

Tommasi, Stefania; Mangia, Anita; Lacalamita, Rosanna; Bellizzi, Antonia; Fedele, Vita; Chiriatti, Annalisa; Thomssen, Christopher; Kendzierski, Nancy; Latorre, Agnese; Lorusso, Vito; Schittulli, Francesco; Zito, Francesco; Kavallaris, Maria; Paradiso, Angelo

2007-05-15

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the beta subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I beta-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of beta-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of beta-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC(50). The Class I beta-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III beta-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III beta-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between beta-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III beta-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III beta-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients. (c) 2007 Wiley-Liss, Inc.

Antagonists of the miRNA-Argonaute 2 Protein Complex: Anti-miR-AGOs.

PubMed

Schmidt, Marco F; Korb, Oliver; Abell, Chris

2017-01-01

microRNAs (miRNAs) have been identified as high-value drug targets. A widely applied strategy in miRNA inhibition is the use of antisense agents. However, it has been shown that oligonucleotides are poorly cell permeable because of their complex chemical structure and due to their negatively charged backbone. Consequently, the general application of oligonucleotides in therapy is limited. Since miRNAs' functions are executed exclusively by the Argonaute 2 protein, we therefore describe a protocol for the design of a novel miRNA inhibitor class: antagonists of the miRNA-Argonaute 2 protein complex, so-called anti-miR-AGOs, that not only block the crucial binding site of the target miRNA but also bind to the protein's active site. Due to their lower molecular weight and, thus, more drug-like chemical structure, the novel inhibitor class may show better pharmacokinetic properties than reported oligonucleotide inhibitors, enabling them for potential therapeutic use.

Rapid assessment of the antigenic integrity of tetrameric HLA complexes by human monoclonal HLA antibodies.

PubMed

Eijsink, Chantal; Kester, Michel G D; Franke, Marry E I; Franken, Kees L M C; Heemskerk, Mirjam H M; Claas, Frans H J; Mulder, Arend

2006-08-31

The ability of tetrameric major histocompatibility complex (MHC) class I-peptide complexes (tetramers) to detect antigen-specific T lymphocyte responses has yielded significant information about the generation of in vivo immunity in numerous antigenic systems. Here we present a novel method for rapid validation of tetrameric HLA molecules based on the presence of allodeterminants. Human monoclonal antibodies (mAbs) recognizing polymorphic determinants on HLA class I were immobilized on polystyrene microparticles and used to probe the structural integrity of tetrameric HLA class I molecules by flow cytometry. A total of 22 tetramers, based on HLA-A1, A2, A3, A24, B7 and B8 were reactive with their counterpart mAbs, thus confirming their antigenic integrity. A positive outcome of this mAb test ensures that tetrameric HLA class I can be used with greater confidence in subsequent functional assays.

Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

PubMed

Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

2016-05-01

Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Structural Aspects for Evolution of [beta]-Lactamases from Penicillin-Binding Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meroueh, Samy O.; Minasov, George; Lee, Wenlin

Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and {beta}-lactamases, resistance enzymes to {beta}-lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for {beta}-lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7{beta}-[N-Acetyl-L-alanyl-{gamma}-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporinmore » bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC {beta}-lactamase from Escherichia coli was solved at 1.71 {angstrom} resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the {beta}-lactamase active site. Furthermore, insertion of a peptide in the {beta}-lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the {beta}-lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.« less

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex

PubMed Central

Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

2011-01-01

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics. PMID:21969598

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

PubMed Central

Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

2014-01-01

ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an HIV protein partially determines which epitopes are dominant, most likely by controlling the breakdown of HIV into peptides. Moreover, some types of signals from CD4+ T cells are affected by the HIV protein 3D structure; and thus the protectiveness of a particular peptide vaccine could be related to its location in the 3D structure. PMID:24920818

The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cash, Jennifer N.; Rejon, Carlis A.; McPherron, Alexandra C.

2009-09-29

Myostatin is a member of the transforming growth factor-{beta} (TGF-{beta}) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-{beta} class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuousmore » electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.« less

Human Cytomegalovirus UL18 Utilizes US6 for Evading the NK and T-Cell Responses

PubMed Central

Kim, Youngkyun; Park, Boyoun; Cho, Sunglim; Shin, Jinwook; Cho, Kwangmin; Jun, Youngsoo; Ahn, Kwangseog

2008-01-01

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses. PMID:18688275

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins.

PubMed

Piscitelli, Alessandra; Cicatiello, Paola; Gravagnuolo, Alfredo Maria; Sorrentino, Ilaria; Pezzella, Cinzia; Giardina, Paola

2017-06-26

Class I hydrophobins produced from fungi are amongst the first proteins recognized as functional amyloids. They are amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins. Herein, the modification of diverse types of surfaces with Class I hydrophobins is reported, highlighting the applications of the coated surfaces. Indeed, these coatings can be exploited in several fields, spanning from biomedical to industrial applications, which include biosensing and textile manufacturing.

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase).

PubMed

Rice, Amy J; Lei, Hao; Santarsiero, Bernard D; Lee, Hyun; Johnson, Michael E

2016-10-01

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sathiyamoorthy, Karthik; Jiang, Jiansen; Mohl, Britta S.

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studiedmore » the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. Furthermore, these data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.« less

Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sathiyamoorthy, Karthik; Jiang, Jiansen; Möhl, Britta S.

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studiedmore » the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.« less

Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

DOE PAGES

Sathiyamoorthy, Karthik; Jiang, Jiansen; Mohl, Britta S.; ...

2017-09-22

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studiedmore » the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. Furthermore, these data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.« less

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

PubMed

Harris, Kimberly A; Zhou, Zhiyuan; Peters, Michelle L; Wilkins, Sarah G; Breaker, Ronald R

2018-06-18

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans , OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA ( ∆ole ) or OapA ( ∆oapA ) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans .

Identification and quantification of human kidney atrial natriuretic peptide receptors.

PubMed

Kahana, L; Yechiely, H; Mecz, Y; Lurie, A

1995-04-01

The present study determined 125I-label atrial natriuretic peptide (ANP) binding sites in human kidney glomerular and papillary membranes. The membranes were prepared from non-malignant renal tissue obtained at nephrectomy of patients with renal carcinoma. To evaluate the proportion of ANP receptor classes ANP-R1 (ANPR-A, -B) versus ANP-R2 (ANPR-C), competitive binding studies were performed using [125I]-ANP in the presence of increasing concentrations of ANP or an internally ring-deleted analog, des(Gln116, Ser117, Gly118, Leu119, Gly120)ANP(102-121), called C-ANP, which binds selectively to ANPR-C receptors. Analysis of the competitive binding curve with ANP in glomerular membranes suggested the presence of one group of high-affinity receptors with dissociation constant Kd = 26 +/- 12 pmol/l and density Bmax = 101 +/- 47 nmol/kg protein. A decrease of 10-30% in Bmax with no change in Kd was obtained in the presence of excess (10(-6) mol/l) C-ANP, suggesting the existence of a small amount of a second class of receptors, the ANPR-C class. The densities of ANPR-A, -B versus ANPR-C receptors in human glomeruli, calculated from competitive inhibition experiments, were 75 +/- 42 and 22 +/- 16 nmol/kg protein (N = 8). Autoradiography of the sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions showed two bands: a highly labeled 130kD band and a weakly labeled 66 kD band, both displaced by ANP. Only the 66-kD band was displaced by the C-ANP analog. Human papilla membrane, as shown by competition binding studies and SDS gel electrophoresis, presented only one class of receptors with Kd = 40 +/- 23 pmol/l (mean +/- SD, N = 3) and Bmax = 17 +/- 6.3 nmol/kg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Evolution of the nuclear receptor gene superfamily.

PubMed Central

Laudet, V; Hänni, C; Coll, J; Catzeflis, F; Stéhelin, D

1992-01-01

Nuclear receptor genes represent a large family of genes encoding receptors for various hydrophobic ligands such as steroids, vitamin D, retinoic acid and thyroid hormones. This family also contains genes encoding putative receptors for unknown ligands. Nuclear receptor gene products are composed of several domains important for transcriptional activation, DNA binding (C domain), hormone binding and dimerization (E domain). It is not known whether these genes have evolved through gene duplication from a common ancestor or if their different domains came from different independent sources. To test these possibilities we have constructed and compared the phylogenetic trees derived from two different domains of 30 nuclear receptor genes. The tree built from the DNA binding C domain clearly shows a common progeny of all nuclear receptors, which can be grouped into three subfamilies: (i) thyroid hormone and retinoic acid receptors, (ii) orphan receptors and (iii) steroid hormone receptors. The tree constructed from the central part of the E domain which is implicated in transcriptional regulation and dimerization shows the same distribution in three subfamilies but two groups of receptors are in a different position from that in the C domain tree: (i) the Drosophila knirps family genes have acquired very different E domains during evolution, and (ii) the vitamin D and ecdysone receptors, as well as the FTZ-F1 and the NGF1B genes, seem to have DNA binding and hormone binding domains belonging to different classes. These data suggest a complex evolutionary history for nuclear receptor genes in which gene duplication events and swapping between domains of different origins took place. PMID:1312460

Proton NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bertini, I.; Luchinat, C.; Messori, L.

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N/sub 3//sup -/, NCO/sup -/, and NCS/sup -/) has been studied by /sup 1/H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E/times/I, E/times/I/sub 2/, and E/times/I/times/X, each with characteristic NMR features, have been identified. The NMR data suggest that when the carboxylate group of a substrate of inhibitor binds atmore » the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The /sup 1/H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes. These signals are relatively constant for all E/times/I and E/times/I/times/X complexes, indicating that this ligand is in a relatively fixed or buried conformation. However in the 2:1 carboxylate inhibitor (E/times/I/sub 2/) complexes, both signals are shifted upfield, suggesting a disturbance in the interaction of this histidine with the metal.« less

Pancreatic acini possess endothelin receptors whose internalization is regulated by PLC-activating agents.

PubMed

Hildebrand, P; Mrozinski, J E; Mantey, S A; Patto, R J; Jensen, R T

1993-05-01

Endothelin-1 (ET-1) and ET-3 mRNA have been found in the pancreas. We investigated the ability of ET-1, ET-2, and ET-3 to interact with and alter dispersed rat pancreatic acinar cell function. Radiolabeled ETs bound in a time- and temperature-dependent fashion, which was specific and saturable. Analysis demonstrated two classes of receptors, one class (ETA receptor) had a high affinity for ET-1 but a low affinity for ET-3, and the other class (ETB receptor) had equally high affinities for ET-1 and ET-3. No specific receptor for ET-2 was identified. Pancreatic secretagogues that activate phospholipase C (PLC) inhibited binding of 125I-labeled ET-1 (125I-ET-1) or 125I-ET-3, whereas agents that act through adenosine 3',5'-cyclic monophosphate (cAMP) did not. A23187 had no effect on 125I-ET-1 or 125I-ET-3 binding, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate reduced binding. The effect of cholecystokinin octapeptide (CCK-8) was mediated through its own receptor. Stripping of surface bound ligand studies demonstrated that both 125I-labeled ET-1 and 125I-labeled ET-3 were rapidly internalized. CCK-8 decreased the internalization but did not change the amount of surface bound ligand. Endothelins neither stimulate nor alter changes in enzyme secretion, intracellular calcium, cAMP, or [3H]inositol trisphosphate (IP3). This study demonstrates the presence of ETA and ETB receptors on rat pancreatic acini; occupation of both receptors resulted in rapid internalization, which is regulated by PLC-activating secretagogues. Occupation of either ET receptor did not alter intracellular calcium, cAMP, IP3, or stimulate amylase release.

Characterization of the binding of 8-anilinonaphthalene sulphonate to rat class Mu GST M1-1

PubMed Central

Kinsley, Nichole; Sayed, Yasien; Armstrong, Richard N.; Dirr, Heini W.

2008-01-01

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulphonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5–30 °C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a ΔCp of −80 ± 4 cal K−1 mol−1 indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins. PMID:18703268

Structure of a class I tagatose-1,6-bisphosphate aldolase: investigation into an apparent loss of stereospecificity.

PubMed

LowKam, Clotilde; Liotard, Brigitte; Sygusch, Jurgen

2010-07-02

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (alpha/beta)(8) fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys(205), different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase

PubMed Central

LowKam, Clotilde; Liotard, Brigitte; Sygusch, Jurgen

2010-01-01

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)8 fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys205, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2–C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase. PMID:20427286

The binding of platinum hexahalides (Cl, Br and I) to hen egg-white lysozyme and the chemical transformation of the PtI{sub 6} octahedral complex to a PtI{sub 3} moiety bound to His15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanley, Simon W. M.; Starkey, Laurina-Victoria; Lamplough, Lucinda

The platinum hexahalides have an octahedral arrangement of six halogen atoms bound to a Pt centre, thus having an octahedral shape that could prove to be useful in interpreting poor electron-density maps. In a detailed characterization, PtI{sub 6} chemically transformed to a square-planar PtI{sub 3} complex bound to the N{sup δ} atom of His15 of HEWL was also observed, which was not observed for PtBr{sub 6} or PtCl{sub 6}. This study examines the binding and chemical stability of the platinum hexahalides K{sub 2}PtCl{sub 6}, K{sub 2}PtBr{sub 6} and K{sub 2}PtI{sub 6} when soaked into pre-grown hen egg-white lysozyme (HEWL) crystalsmore » as the protein host. Direct comparison of the iodo complex with the chloro and bromo complexes shows that the iodo complex is partly chemically transformed to a square-planar PtI{sub 3} complex bound to the N{sup δ} atom of His15, a chemical behaviour that is not exhibited by the chloro or bromo complexes. Each complex does, however, bind to HEWL in its octahedral form either at one site (PtI{sub 6}) or at two sites (PtBr{sub 6} and PtCl{sub 6}). As heavy-atom derivatives of a protein, the octahedral shape of the hexahalides could be helpful in cases of difficult-to-interpret electron-density maps as they would be recognisable ‘objects’.« less

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

PubMed Central

Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

2014-01-01

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

Metal complexes as DNA intercalators.

PubMed

Liu, Hong-Ke; Sadler, Peter J

2011-05-17

DNA has a strong affinity for many heterocyclic aromatic dyes, such as acridine and its derivatives. Lerman in 1961 first proposed intercalation as the source of this affinity, and this mode of DNA binding has since attracted considerable research scrutiny. Organic intercalators can inhibit nucleic acid synthesis in vivo, and they are now common anticancer drugs in clinical therapy. The covalent attachment of organic intercalators to transition metal coordination complexes, yielding metallointercalators, can lead to novel DNA interactions that influence biological activity. Metal complexes with σ-bonded aromatic side arms can act as dual-function complexes: they bind to DNA both by metal coordination and through intercalation of the attached aromatic ligand. These aromatic side arms introduce new modes of DNA binding, involving mutual interactions of functional groups held in close proximity. The biological activity of both cis- and trans-diamine Pt(II) complexes is dramatically enhanced by the addition of σ-bonded intercalators. We have explored a new class of organometallic "piano-stool" Ru(II) and Os(II) arene anticancer complexes of the type [(η(6)-arene)Ru/Os(XY)Cl](+). Here XY is, for example, ethylenediamine (en), and the arene ligand can take many forms, including tetrahydroanthracene, biphenyl, or p-cymene. Arene-nucleobase stacking interactions can have a significant influence on both the kinetics and thermodynamics of DNA binding. In particular, the cytotoxic activity, conformational distortions, recognition by DNA-binding proteins, and repair mechanisms are dependent on the arene. A major difficulty in developing anticancer drugs is cross-resistance, a phenomenon whereby a cell that is resistant to one drug is also resistant to another drug in the same class. These new complexes are non-cross-resistant with cisplatin towards cancer cells: they constitute a new class of anticancer agents, with a mechanism of action that differs from the anticancer drug cisplatin and its analogs. The Ru-arene complexes with dual functions are more potent towards cancer cells than their nonintercalating analogs. In this Account, we focus on recent studies of dual-function organometallic Ru(II)- and Os(II)-arene complexes and the methods used to detect arene-DNA intercalation. We relate these interactions to the mechanism of anticancer activity and to structure-activity relationships. The interactions between these complexes and DNA show close similarities to those of covalent polycyclic aromatic carcinogens, especially to N7-alkylating intercalation compounds. However, Ru-arene complexes exhibit some new features. Classical intercalation and base extrusion next to the metallated base is observed for {(η(6)-biphenyl)Ru(ethylenediamine)}(2+) adducts of a 14-mer duplex, while penetrating arene intercalation occurs for adducts of the nonaromatic bulky intercalator {(η(6)-tetrahydroanthracene)Ru(ethylenediamine)}(2+) with a 6-mer duplex. The introduction of dual-function Ru-arene complexes introduces new mechanisms of antitumor activity, novel mechanisms for attack on DNA, and new concepts for developing structure- activity relationships. We hope this discussion will stimulate thoughtful and focused research on the design of anticancer chemotherapeutic agents using these unique approaches.

Synthesis, structures, nuclease activity, cytotoxicity, DFT and molecular docking studies of two nitrato bridged homodinuclear (Cu-Cu, Zn-Zn) complexes containing 2,2'-bipyridine and a chalcone derivative.

PubMed

Gaur, Ruchi; Choubey, Diksha Kumari; Usman, Mohammad; Ward, Benzamin D; Roy, Jagat Kumar; Mishra, Lallan

2017-08-01

Nitrato briged dinuclear complexes of type [Cu 2 (L) 2 (bpy) 2 (NO 3 )](NO 3 )·4H 2 O, 1 and [Zn 2 (L) 2 (bpy) 2 (NO 3 )](NO 3 )·4H 2 O, 2 (L=deprotonated form of free ligand LH, [1-(2-hydroxyphenyl)-3-(9-anthracenyl) propenone; bpy=2,2'bipyridine] are synthesized and characterized using a battery of physicochemical techniques and X-ray crystallography. A distorted square pyramidal geometry is assigned to them with N 2 O 3 coordination core around the metal ion. The co-ligand L binds the metal ions through its O,O' atoms in anti-syn mode. The metal centers in complexes 1 and 2 are separated via bridging nitrato group at a distance of 6.073Å and 5.635Å respectively. Their structures and absorption spectra are supported by the computational studies using density functional theory (DFT) and TD-DFT. Both complexes exhibit nuclease activity and cleave supercoiled (form I) DNA. The complex 1 preferentially binds major groove of DNA and follows an oxidative pathway whereas complex 2 binds with minor groove of DNA via hydrolytic pathway. Both complexes inhibit topoisomerase I relaxation activity with IC 50 values of 7 and 35μM. Molecular docking studies support the groove binding and topoisomerase I binding of the complexes. The complex 1 showed a significant cytotoxicity against HeLa cell lines (a cervical cancer cell lines) in vitro with IC 50 value calculated as 2.9±0.021μM as compared to 28.2±0. 044μΜ for complex 2. Complex 2 induces the cell apoptosis at a later-stage as compared to complex 1. The cell apoptosis and topoisomerase inhibition by complexes enable them to be potential candidates as future anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crofton, K.M.; Reiter, L.W.; Mailman, R.B.

1987-01-01

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on /sup 3/H-flunitrazepam (FLU), /sup 3/H-muscimol (MUS), and (/sup 35/S-t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with /sup 3/H-FLU and /sup 3/H-MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of (/sup 35/S-TBPS at concentrations as high as 50 pM. Type II pyrethroids inhibited (/sup 35/S-TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 pM. The data presented suggest that the interaction of Type II pyrethroids with the GABA receptor-ionophore complex ismore » restricted to a site near the TBPS/picrotoxinin binding site.« less

Presence of strong association of the major histocompatibility complex (MHC) class I allele HLA-A*26:01 with idiopathic hypoparathyroidism.

PubMed

Goswami, Ravinder; Singh, Archana; Gupta, Nandita; Rani, Rajni

2012-09-01

The pathogenesis of isolated hypoparathyroidism, also referred to as idiopathic hypoparathyroidism (IH), is not clear. There is a paucity of information related to the immunogenetic basis of the disease due to its rarity. A recurrent theme of several autoimmune disorders is aberrant antigen presentation. We investigated for the association of alleles of the human leukocyte antigen (HLA) class I and II loci with IH. A total of 134 patients with IH and 902 healthy controls from the same ethnic background participated in the study. There was a significant increase of HLA class I alleles HLA-A*26:01 [P < 1.71 × 10(-34); odds ratio (OR) = 9.29; 95% confidence interval (CI) = 6.08-14.16] and HLA-B*08:01 (P < 8.19 × 10(-6); OR = 2.59; 95% CI = 1.63-4.04) in patients with IH compared to healthy controls. However, the association of A*26:01 was primary because B*08:01 was in linkage disequilibrium with A*26:01. Although the major histocompatibility complex (MHC) is very polymorphic, several alleles of HLA loci share key residues at anchor positions in the peptide binding pockets such that similar peptides may be presented by different MHC molecules encoded by the same locus. These allelic forms with similar anchoring amino acids have been clustered in supertypes. An analysis of HLA-A locus supertypes A01, A02, A03, and A04 revealed that supertype A01 was significantly increased (P < 9.18 × 10(-9); OR = 2.95) in IH compared to controls. However, this increase in the supertype A01 was contributed by A*26:01 because 68.7% of the A01 samples had A*26:01. Other alleles of the supertype did not show any significant differences. The strong association of HLA-A*26:01 suggests an important role of MHC class I-mediated presentation of autoantigenic peptides to CD8(+) cytotoxic T cells in the pathogenesis of IH. These data provide evidence for the autoimmune etiology of IH akin to other autoimmune disorders like type 1 diabetes and rheumatoid arthritis.

Crystal structures of botulinum neurotoxin DC in complex with its protein receptors synaptotagmin I and II.

PubMed

Berntsson, Ronnie Per-Arne; Peng, Lisheng; Svensson, Linda Marie; Dong, Min; Stenmark, Pål

2013-09-03

Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human Cytomegalovirus Protein US2 Interferes with the Expression of Human HFE, a Nonclassical Class I Major Histocompatibility Complex Molecule That Regulates Iron Homeostasis

PubMed Central

Ben-Arieh, Sayeh Vahdati; Zimerman, Baruch; Smorodinsky, Nechama I.; Yaacubovicz, Margalit; Schechter, Chana; Bacik, Igor; Gibbs, Jim; Bennink, Jack R.; Yewdell, Jon W.; Coligan, John E.; Firat, Hüseyin; Lemonnier, François; Ehrlich, Rachel

2001-01-01

HFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/β2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses. PMID:11581431

Genetic Polymorphism in Extracellular Regulators of Wnt Signaling Pathway

PubMed Central

Sharma, Ashish Ranjan; Seo, Eun-Min; Nam, Ju-Suk

2015-01-01

The Wnt signaling pathway is mediated by a family of secreted glycoproteins through canonical and noncanonical mechanism. The signaling pathways are regulated by various modulators, which are classified into two classes on the basis of their interaction with either Wnt or its receptors. Secreted frizzled-related proteins (sFRPs) are the member of class that binds to Wnt protein and antagonizes Wnt signaling pathway. The other class consists of Dickkopf (DKK) proteins family that binds to Wnt receptor complex. The present review discusses the disease related association of various polymorphisms in Wnt signaling modulators. Furthermore, this review also highlights that some of the sFRPs and DKKs are unable to act as an antagonist for Wnt signaling pathway and thus their function needs to be explored more extensively. PMID:25945348

Molecular docking studies of curcumin natural derivatives with DNA topoisomerase I and II-DNA complexes.

PubMed

Kumar, Anil; Bora, Utpal

2014-12-01

DNA topoisomerase I (topo I) and II (topo II) are essential enzymes that solve the topological problems of DNA by allowing DNA strands or double helices to pass through each other during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Their critical roles make topoisomerases an attractive drug target against cancer. The present molecular docking study provides insights into the inhibition of topo I and II by curcumin natural derivatives. The binding modes suggested that curcumin natural derivatives docked at the site of DNA cleavage parallel to the axis of DNA base pairing. Cyclocurcumin and curcumin sulphate were predicted to be the most potent inhibitors amongst all the curcumin natural derivatives docked. The binding modes of cyclocurcumin and curcumin sulphate were similar to known inhibitors of topo I and II. Residues like Arg364, Asn722 and base A113 (when docked to topo I-DNA complex) and residues Asp479, Gln778 and base T9 (when docked to topo II-DNA complex) seem to play important role in the binding of curcumin natural derivatives at the site of DNA cleavage.

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes

PubMed Central

Yu, Xinchao; Breitman, Marianna; Goldberg, Jonathan

2012-01-01

Summary Budding of COPI-coated vesicles from Golgi membranes requires an Arf-family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P2-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules. PMID:22304919

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

Characterizing the Specificity and Co-operation of Aminopeptidases in the Cytosol and ER During MHC Class I antigen Presentation1

PubMed Central

Hearn, Arron; York, Ian A.; Bishop, Courtney; Rock, Kenneth L.

2010-01-01

Many MHC class I binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can be subsequently trimmed by aminopeptidases in the cytosol and/or the ER to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for antigen presentation is not well defined. Here we investigate this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of antigen presentation. In many cases MHC class I binding peptides are produced through sequential trimming in both the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Since N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented. PMID:20351195

Identification of a better Homo sapiens Class II HDAC inhibitor through binding energy calculations and descriptor analysis

PubMed Central

2010-01-01

Human papillomaviruses (HPVs) are the most common on sexually transmitted viruses in the world. HPVs are responsible for a large spectrum of deseases, both benign and malignant. The certain types of HPV are involved in the development of cervical cancer. In attemps to find additional drugs in the treatment of cervical cancer, inhibitors of the histone deacetylases (HDAC) have received much attention due to their low cytotoxic profiles and the E6/E7 oncogene function of human papilomavirus can be completely by passed by HDAC inhibition. The histone deacetylase inhibitors can induce growth arrest, differentiation and apoptosis of cancer cells. HDAC class I and class II are considered the main targets for cancer. Therefore, the six HDACs class II was modeled and about two inhibitors (SAHA and TSA) were docked using AutoDock4.2, to each of the inhibitor in order to identify the pharmacological properties. Based on the results of docking, SAHA and TSA were able to bind with zinc ion in HDACs models as a drug target. SAHA was satisfied almost all the properties i.e., binding affinity, the Drug-Likeness value and Drug Score with 70% oral bioavailability and the carbonyl group of these compound fits well into the active site of the target where the zinc is present. Hence, SAHA could be developed as potential inhibitors of class II HDACs and valuable cervical cancer drug candidate. PMID:21106123

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related.

PubMed

Ekhlasi-Hundrieser, Mahnaz; Schäfer, Bettina; Philipp, Ute; Kuiper, Heidi; Leeb, Tosso; Mehta, Meenal; Kirchhoff, Christiane; Töpfer-Petersen, Edda

2007-05-01

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11-q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

Identification of a better Homo sapiens Class II HDAC inhibitor through binding energy calculations and descriptor analysis.

PubMed

Tambunan, Usman Sumo Friend; Wulandari, Evi Kristin

2010-10-15

Human papillomaviruses (HPVs) are the most common on sexually transmitted viruses in the world. HPVs are responsible for a large spectrum of deseases, both benign and malignant. The certain types of HPV are involved in the development of cervical cancer. In attemps to find additional drugs in the treatment of cervical cancer, inhibitors of the histone deacetylases (HDAC) have received much attention due to their low cytotoxic profiles and the E6/E7 oncogene function of human papilomavirus can be completely by passed by HDAC inhibition. The histone deacetylase inhibitors can induce growth arrest, differentiation and apoptosis of cancer cells. HDAC class I and class II are considered the main targets for cancer. Therefore, the six HDACs class II was modeled and about two inhibitors (SAHA and TSA) were docked using AutoDock4.2, to each of the inhibitor in order to identify the pharmacological properties. Based on the results of docking, SAHA and TSA were able to bind with zinc ion in HDACs models as a drug target. SAHA was satisfied almost all the properties i.e., binding affinity, the Drug-Likeness value and Drug Score with 70% oral bioavailability and the carbonyl group of these compound fits well into the active site of the target where the zinc is present. Hence, SAHA could be developed as potential inhibitors of class II HDACs and valuable cervical cancer drug candidate.

Structural basis of cargo recognitions for class V myosins

PubMed Central

Wei, Zhiyi; Liu, Xiaotian; Yu, Cong; Zhang, Mingjie

2013-01-01

Class V myosins (MyoV), the most studied unconventional myosins, recognize numerous cargos mainly via the motor’s globular tail domain (GTD). Little is known regarding how MyoV-GTD recognizes such a diverse array of cargos specifically. Here, we solved the crystal structures of MyoVa-GTD in its apo-form and in complex with two distinct cargos, melanophilin and Rab interacting lysosomal protein-like 2. The apo-MyoVa-GTD structure indicates that most mutations found in patients with Griscelli syndrome, microvillus inclusion disease, or cancers or in “dilute” rodents likely impair the folding of GTD. The MyoVa-GTD/cargo complex structure reveals two distinct cargo-binding surfaces, one primarily via charge–charge interaction and the other mainly via hydrophobic interactions. Structural and biochemical analysis reveal the specific cargo-binding specificities of various isoforms of mammalian MyoV as well as very different cargo recognition mechanisms of MyoV between yeast and higher eukaryotes. The MyoVa-GTD structures resolved here provide a framework for future functional studies of vertebrate class V myosins. PMID:23798443

Comparative genomic analysis of the MHC: the evolution of class I duplication blocks, diversity and complexity from shark to man.

PubMed

Kulski, Jerzy K; Shiina, Takashi; Anzai, Tatsuya; Kohara, Sakae; Inoko, Hidetoshi

2002-12-01

The major histocompatibility complex (MHC) genomic region is composed of a group of linked genes involved functionally with the adaptive and innate immune systems. The class I and class II genes are intrinsic features of the MHC and have been found in all the jawed vertebrates studied so far. The MHC genomic regions of the human and the chicken (B locus) have been fully sequenced and mapped, and the mouse MHC sequence is almost finished. Information on the MHC genomic structures (size, complexity, genic and intergenic composition and organization, gene order and number) of other vertebrates is largely limited or nonexistent. Therefore, we are mapping, sequencing and analyzing the MHC genomic regions of different human haplotypes and at least eight nonhuman species. Here, we review our progress with these sequences and compare the human MHC structure with that of the nonhuman primates (chimpanzee and rhesus macaque), other mammals (pigs, mice and rats) and nonmammalian vertebrates such as birds (chicken and quail), bony fish (medaka, pufferfish and zebrafish) and cartilaginous fish (nurse shark). This comparison reveals a complex MHC structure for mammals and a relatively simpler design for nonmammalian animals with a hypothetical prototypic structure for the shark. In the mammalian MHC, there are two to five different class I duplication blocks embedded within a framework of conserved nonclass I and/or nonclass II genes. With a few exceptions, the class I framework genes are absent from the MHC of birds, bony fish and sharks. Comparative genomics of the MHC reveal a highly plastic region with major structural differences between the mammalian and nonmammalian vertebrates. Additional genomic data are needed on animals of the reptilia, crocodilia and marsupial classes to find the origins of the class I framework genes and examples of structures that may be intermediate between the simple and complex MHC organizations of birds and mammals, respectively.

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

PubMed

Tussey, L G; Frelinger, J A

1991-11-01

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.

RNA polymerase I-Rrn3 complex at 4.8 Å resolution

NASA Astrophysics Data System (ADS)

Engel, Christoph; Plitzko, Jürgen; Cramer, Patrick

2016-07-01

Transcription of ribosomal DNA by RNA polymerase I (Pol I) requires the initiation factor Rrn3. Here we report the cryo-EM structure of the Pol I-Rrn3 complex at 4.8 Å resolution. The structure reveals how Rrn3 binding converts an inactive Pol I dimer into an initiation-competent monomeric complex and provides insights into the mechanisms of Pol I-specific initiation and regulation.

Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.

PubMed

Pomel, Vincent; Klicic, Jasna; Covini, David; Church, Dennis D; Shaw, Jeffrey P; Roulin, Karen; Burgat-Charvillon, Fabienne; Valognes, Delphine; Camps, Montserrat; Chabert, Christian; Gillieron, Corinne; Françon, Bernard; Perrin, Dominique; Leroy, Didier; Gretener, Denise; Nichols, Anthony; Vitte, Pierre Alain; Carboni, Susanna; Rommel, Christian; Schwarz, Matthias K; Rückle, Thomas

2006-06-29

Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using ({sup 125}I)melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37{degree}C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of ({sup 125}I)melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8more » fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of ({sup 125}I)melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the ({sup 125}I)melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.« less

Directional control of WAVE2 membrane targeting by EB1 and phosphatidylinositol 3,4,5-triphosphate.

PubMed

Takahashi, Kazuhide; Tanaka, Tacu; Suzuki, Katsuo

2010-03-01

Membrane targeting of WAVE2 along microtubules is mediated by a motor protein kinesin and requires Pak1, a downstream effector of Rac1. However, the mechanism by which WAVE2 targeting to the leading edge is directionally controlled remains largely unknown. Here we demonstrate that EB1, a microtubule plus-end-binding protein, constitutively associates with stathmin, a microtubule-destabilizing protein, in human breast cancer cells. Stimulation of the cells with insulin-like growth factor I (IGF-I) induced Pak1-dependent binding of the EB1-stathmin complex to microtubules that bear WAVE2 and colocalization of the complex with WAVE2 at the leading edge. Depletion of EB1 by small interfering RNA (siRNA) abrogated the IGF-I-induced WAVE2 targeting and stathmin binding to microtubules. On the other hand, chemotaxis chamber assays indicated that the IGF-I receptor (IGF-IR) was locally activated in the region facing toward IGF-I. In addition, IGF-I caused phosphatidylinositol 3-kinase (PI 3-kinase)-dependent production of phosphatidylinositol 3,4,5-triphosphate (PIP3) near activated IGF-IR and WAVE2 colocalization with it. Collectively, WAVE2-membrane targeting is directionally controlled by binding of the EB1-stathmin complex to WAVE2-bearing microtubules and by the interaction between WAVE2 and PIP3 produced near IGF-IR that is locally activated by IGF-I.

Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

PubMed

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-04-10

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K(i) = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K(m) = 0.1 mm). The crystal structure at 1.7-A resolution revealed that the dual mutation causes a shift of residue Gly(96) toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile(97) points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr(97) and Pro(101) induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.

Predicted structure of MIF/CD74 and RTL1000/CD74 complexes.

PubMed

Meza-Romero, Roberto; Benedek, Gil; Leng, Lin; Bucala, Richard; Vandenbark, Arthur A

2016-04-01

Macrophage migration inhibitory factor (MIF) is a key cytokine in autoimmune and inflammatory diseases that attracts and then retains activated immune cells from the periphery to the tissues. MIF exists as a homotrimer and its effects are mediated through its primary receptor, CD74 (the class II invariant chain that exhibits a highly structured trimerization domain), present on class II expressing cells. Although a number of binding residues have been identified between MIF and CD74 trimers, their spatial orientation has not been established. Using a docking program in silico, we have modeled binding interactions between CD74 and MIF as well as CD74 and a competitive MIF inhibitor, RTL1000, a partial MHC class II construct that is currently in clinical trials for multiple sclerosis. These analyses revealed 3 binding sites on the MIF trimer that each were predicted to bind one CD74 trimer through interactions with two distinct 5 amino acid determinants. Surprisingly, predicted binding of one CD74 trimer to a single RTL1000 antagonist utilized the same two 5 residue determinants, providing strong suggestive evidence in support of the MIF binding regions on CD74. Taken together, our structural modeling predicts a new MIF(CD74)3 dodecamer that may provide the basis for increased MIF potency and the requirement for ~3-fold excess RTL1000 to achieve full antagonism.

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation.

PubMed

Akiyama, Y; Zicht, R; Ferrone, S; Bonnard, G D; Herberman, R B

1985-04-01

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.

A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

PubMed

Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

2014-12-01

We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

Computational dissection of allosteric inhibition of the SH2 domain of Bcr-Abl kinase by the monobody inhibitor AS25.

PubMed

Ji, Mingfei; Zheng, Guodong; Li, Xiaolong; Zhang, Zhongqin; Jv, Guanqun; Wang, Xiaowei; Wang, Jialin

2017-06-01

The deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein represents an attractive pharmacological target for the treatment of chronic myeloid leukemia (CML). The high affinity of monobody AS25 was designed to target the Src homology 2 (SH2) domain of Bcr-Abl, leading to allosteric inhibition of Bcr-Abl through formation of protein-protein interactions. An I164E mutation in the SH2 domain disrupts AS25 binding to the SH2 domain of Bcr-Abl. The detailed mechanisms, however, remain to be unresolved. Here, molecular dynamics (MD) simulations and binding free energy calculations were performed to explore the conformational and energetic differences between the wild-type (WT) complexes of Bcr-Abl SH2 domain and AS25 (SH2 WT -AS25) as well as the mutated complexes (SH2 I164E -AS25). The results revealed that I164E mutation not only caused an increase in the conformational flexibility of SH2-AS25 complexes, but also weakened the binding affinity of AS25 to SH2. The comparative binding modes of SH2-AS25 complexes between WT and the I164E mutant were comprehensively analyzed to unravel the disruption of hydrophobic and hydrogen bonding interactions in the interface of the SH2-AS25 complex triggered by the I164E mutation. The results obtained may help to design the next generation of higher affinity Bcr-Abl SH2-specific peptide inhibitors.

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increasedmore » the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.« less

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

PubMed Central

Peter, Daniel; Weber, Ramona; Köne, Carolin; Chung, Min-Yi; Ebertsch, Linda; Truffault, Vincent; Weichenrieder, Oliver; Igreja, Cátia; Izaurralde, Elisa

2015-01-01

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation. PMID:26294658

A novel role of HLA class I in the pathology of medulloblastoma.

PubMed

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-07-12

MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.

A novel role of HLA class I in the pathology of medulloblastoma

PubMed Central

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-01-01

Background MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. Methods We investigated expression of four essential components of MHC class I (heavy chain, β2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. Results The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of β2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. Conclusion MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or β2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility. PMID:19594892

Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs.

PubMed

Hickey, Scott F; Hammond, Ming C

2014-03-20

Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Populus ARBORKNOX1 homeodomain transcription factor regulates woody growth through binding to evolutionarily conserved target genes of diverse function

Treesearch

Lijun Liu; Matthew S. Zinkgraf; H. Earl Petzold; Eric P. Beers; Vladimir Filkov; Andrew Groover

2014-01-01

The class I KNOX homeodomain transcription factor ARBORKNOX1 (ARK1) is a key regulator of vascular cambium maintenance and cell differentiation in Populus. Currently, basic information is lacking concerning the distribution, functional characteristics, and evolution of ARK1 binding in the Populus genome.

Identifying Marine Copper-Binding Ligands in Seawater

NASA Astrophysics Data System (ADS)

Whitby, H.; Hollibaugh, J. T.; Maldonado, M. T.; Ouchi, S.; van den Berg, S. M.

2016-02-01

Complexation reactions are important because they affect the bioavailability of trace metals such as copper and iron. For example, organic complexation can determine whether copper is a limiting or a toxic micronutrient at natural levels. Copper competes with iron for complexing ligands, and when iron is limiting, copper can also substitute for iron in some metabolic pathways. The speciation of copper can be measured using complexing capacity titrations, which provide the concentration of individual ligand classes (L1, L2 etc.) and the complex stabilities (log K). Using methods recently developed in our laboratory, we show that the ligands within these classes can be measured independently of titrations, thus confirming the titration method and simultaneously identifying the ligands within each class. Thiols were identified as the L1 ligand class and humic compounds as the weaker L2 class in samples from coastal Georgia, USA, collected monthly from April to December. Log K values of the ligand complexes were consistent with values expected for thiols and humic substances. Recent results from culture studies and from samples collected along Line P, a coastal - oceanic transect in the HNLC region of the NE subarctic Pacific, will be presented in comparison to the estuarine results. This comparison will help to broaden our perspective on copper complexation and the ligands responsible, furthering our understanding of ligand sources and life cycles.

TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis

PubMed Central

Shim, Jae Sung; Song, Yong Hun; Laboy Cintrón, Dianne; Koyama, Tomotsugu; Ohme-Takagi, Masaru; Pruneda-Paz, Jose L.; Kay, Steve A.; MacCoss, Michael J.

2017-01-01

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation. PMID:28628608

TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis.

PubMed

Kubota, Akane; Ito, Shogo; Shim, Jae Sung; Johnson, Richard S; Song, Yong Hun; Breton, Ghislain; Goralogia, Greg S; Kwon, Michael S; Laboy Cintrón, Dianne; Koyama, Tomotsugu; Ohme-Takagi, Masaru; Pruneda-Paz, Jose L; Kay, Steve A; MacCoss, Michael J; Imaizumi, Takato

2017-06-01

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding.

PubMed

Casino, Patricia; Niks, Dimitri; Ngo, Huu; Pan, Peng; Brzovic, Peter; Blumenstein, Lars; Barends, Thomas Reinier; Schlichting, Ilme; Dunn, Michael F

2007-07-03

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.

DNA binding, anti-inflammatory and analgesic evaluation of metal complexes of N/S/O donor ligands; Synthesis, spectral characterization

NASA Astrophysics Data System (ADS)

Kumar Naik, K. H.; Ashok, B.; Naik, Nagaraja; Mulla, Jameel Ahmed S.; Prakasha, Avinash

2015-04-01

Transition metal complexes containing tri-dentate NSN donor ligands i.e., 5-((1(aminomethyl)cyclohexyl)methyl)-1,3,4-thiadiazol-2-amine (AMTA) (2) and 5-(2-aminophenyl)-1,3,4-thiadiazol-2-amine (ATA) (4i-ii) have been synthesized. The newly synthesized ligands and their respective complexes were characterized by elemental analysis, molar conductance measurement and various spectral studies [infrared (IR), electronic, and NMR (for ligands only)]. Metal complexes are like [M(AMTA)2], [M(ATA)2] type, where M = Mn(II), Co(II) and Cu(II). The proposed geometries of the complexes are octahedral in nature. The synthesized ligands and their complexes were exhibits effective anti-inflammatory, analgesic and DNA binding activities. All the tested compounds exhibited significant analgesic activity, whereas the compound 4i, 4(ia) and 4(iib) is equipotent with Diclofenac sodium.

Role of halogen and hydrogen bonds for stabilization of antithyroid drugs with hypohalous acids (HOX, X = I, Br, and Cl) adducts

NASA Astrophysics Data System (ADS)

El-Sheshtawy, Hamdy S.; El-Mehasseb, Ibrahim

2017-11-01

The mechanism for the inhibition of thyroid hormones by the thioamide-like antithyroid drug is a key process in the thyroid gland function. Therefore, in this study theoretical investigation of the molecular interaction between two antithyroid drugs, namely methimazol (MMI) and thiazoline-2-thione (T2T), with the hypohalous acids (HOX, X = I, Br, and Cl), which act as heme-linked halogenated species to tyrosine residue was discussed. The calculations were performed by M06-2X and MP2 using aug-cc-pVDZ level of theory. In addition, wB97xd/6-31G* level of theory was used in order to account for the dispersion forces. The results show the possible formation of three adducts, which is stabilized by halogen bond (I), both halogen and hydrogen bonds (II), two hydrogen bonds (III). The binding energies of the complexes reveals stabilization in the order III > II > I. The binding energies of the complexes was increased with increasing the electron affinity and polarizability of halogen atom, the dipole moment of the complexes (I and II), the electrostatic potential on halogen atom (Vmax:i.e σ-hole), and the charge-transfer process through the halogen bond in I. On the other hand, the binding energies of the complexes decreased with increasing the halogen atom electronegativity and the dipole moment of complex III. Natural bond orbital (NBO) analysis was used to investigate the molecular orbital interactions and the charge transfer process upon complexation.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

PubMed

Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

A Gene Encoding a Hevein-Like Protein from Elderberry Fruits Is Homologous to PR-4 and Class V Chitinase Genes1

PubMed Central

Van Damme, Els J.M.; Charels, Diana; Roy, Soma; Tierens, Koenraad; Barre, Annick; Martins, José C.; Rougé, Pierre; Van Leuven, Fred; Does, Mirjam; Peumans, Willy J.

1999-01-01

We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene. PMID:10198114

gamma. -Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dam, T.V.; Takeda, Y.; Krause, J.E.

1990-01-01

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide (gamma-PPT-(72-92)-NH2), a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeledmore » Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.« less

Imbalance in chemical space: How to facilitate the identification of protein-protein interaction inhibitors.

PubMed

Kuenemann, Mélaine A; Labbé, Céline M; Cerdan, Adrien H; Sperandio, Olivier

2016-04-01

Protein-protein interactions (PPIs) play vital roles in life and provide new opportunities for therapeutic interventions. In this large data analysis, 3,300 inhibitors of PPIs (iPPIs) were compared to 17 reference datasets of collectively ~566,000 compounds (including natural compounds, existing drugs, active compounds on conventional targets, etc.) using a chemoinformatics approach. Using this procedure, we showed that comparable classes of PPI targets can be formed using either the similarity of their ligands or the shared properties of their binding cavities, constituting a proof-of-concept that not only can binding pockets be used to group PPI targets, but that these pockets certainly condition the properties of their corresponding ligands. These results demonstrate that matching regions in both chemical space and target space can be found. Such identified classes of targets could lead to the design of PPI-class-specific chemical libraries and therefore facilitate the development of iPPIs to the stage of drug candidates.

Crystallization and preliminary X-ray diffraction analysis of the two distinct types of zebrafish β2-microglobulin

PubMed Central

Chen, Zhaosan; Zhang, Nianzhi; Lu, Shuangshuang; Tariq, Mansoor; Wang, Junya; Xia, Chun

2015-01-01

β2-Microglobulin (β2m) noncovalently associates with the heavy chain of major histocompatibility complex class I (MHC I) molecules, which bind foreign antigen peptides to control the cytotoxic T lymphocyte (CTL) immune response. In contrast to mammals, there are distinct types of β2ms derived from two loci in a number of teleost species. In order to clarify the structures of the β2ms, the zebrafish (Danio rerio) β2ms Dare-β2m-I and Dare-β2m-II were expressed in Escherichia coli, purified and crystallized, and diffraction data were collected to 1.6 and 1.9 Å resolution, respectively. Both crystals belonged to space group P212121. The unit-cell parameters were determined to be a = 38.2, b = 50.4, c = 50.9 Å for Dare-β2m-I and a = 38.9, b = 52.7, c = 65.8 Å for Dare-β2m-II. Each asymmetric unit was constituted of one molecule, with Matthews coefficients of 2.22 and 3.01 Å3 Da−1 and solvent contents of 45 and 59% for Dare-β2m-I and Dare-β2m-II, respectively. These two β2m structures will provide relevant information for further studies of the structures of the MHC I complex. PMID:26057815

The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins

PubMed Central

Eaton, James R. O.; Alenazi, Yara; Singh, Kamayani; Davies, Graham; Geis-Asteggiante, Lucia; Kessler, Benedikt; Robinson, Carol V.; Kawamura, Akane; Bhattacharya, Shoumo

2018-01-01

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus. We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm. The CC chemokine–binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties. PMID:29487134

Semi-empirical quantum evaluation of peptide - MHC class II binding

NASA Astrophysics Data System (ADS)

González, Ronald; Suárez, Carlos F.; Bohórquez, Hugo J.; Patarroyo, Manuel A.; Patarroyo, Manuel E.

2017-01-01

Peptide presentation by the major histocompatibility complex (MHC) is a key process for triggering a specific immune response. Studying peptide-MHC (pMHC) binding from a structural-based approach has potential for reducing the costs of investigation into vaccine development. This study involved using two semi-empirical quantum chemistry methods (PM7 and FMO-DFTB) for computing the binding energies of peptides bonded to HLA-DR1 and HLA-DR2. We found that key stabilising water molecules involved in the peptide binding mechanism were required for finding high correlation with IC50 experimental values. Our proposal is computationally non-intensive, and is a reliable alternative for studying pMHC binding interactions.

Immunological Functions of the Membrane Proximal Region of MHC Class II Molecules

PubMed Central

Harton, Jonathan; Jin, Lei; Hahn, Amy; Drake, Jim

2016-01-01

Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Engineering allostery.

PubMed

Raman, Srivatsan; Taylor, Noah; Genuth, Naomi; Fields, Stanley; Church, George M

2014-12-01

Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. This approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Engineering allostery

DOE PAGES

Raman, Srivatsan; Taylor, Noah; Genuth, Naomi; ...

2014-10-08

Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, in this papermore » we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. Finally, this approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases.« less

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

PubMed Central

Al-Khouri, Anna Maria; Paule, Marvin R.

2002-01-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

PubMed

Al-Khouri, Anna Maria; Paule, Marvin R

2002-02-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

B and T Cell Epitope-Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets.

PubMed

Yasmin, T; Nabi, A H M Nurun

2016-05-01

Ebola virus (EBV) has become a serious threat to public health. Different approaches were applied to predict continuous and discontinuous B cell epitopes as well as T cell epitopes from the sequence-based and available three-dimensional structural analyses of each protein of EBV. Peptides '(79) VPSATKRWGFRSGVPP(94) ' from GP1 and '(515) LHYWTTQDEGAAIGLA(530) ' from GP2 of Ebola were found to be the consensus peptidic sequences predicted as linear B cell epitope of which the latter contains a region (519) TTQDEG(524) that fulfilled all the criteria of accessibility, hydrophilicity, flexibility and beta turn region for becoming an ideal B cell epitope. Different nonamers as T cell epitopes were obtained that interacted with different numbers of MHC class I and class II alleles with a binding affinity of <100 nm. Interestingly, these alleles also bound to the MHC class I alleles mostly prevalent in African and South Asian regions. Of these, 'LANETTQAL' and 'FLYDRLAST' nonamers were predicted to be the most potent T cell epitopes and they, respectively, interacted with eight and twelve class I alleles that covered 63.79% and 54.16% of world population, respectively. These nonamers were found to be the core sequences of 15mer peptides that interacted with the most common class II allele, HLA-DRB1*01:01. They were further validated for their binding to specific class I alleles using docking technique. Thus, these predicted epitopes may be used as vaccine targets against EBV and can be validated in model hosts to verify their efficacy as vaccine. © 2016 The Foundation for the Scandinavian Journal of Immunology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneousmore » leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.« less

Role of Su(Hw) zinc finger 10 and interaction with CP190 and Mod(mdg4) proteins in recruiting the Su(Hw) complex to chromatin sites in Drosophila.

PubMed

Melnikova, Larisa; Kostyuchenko, Margarita; Parshikov, Alexander; Georgiev, Pavel; Golovnin, Anton

2018-01-01

Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, we have examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Δ10 and CP190. Having studied insulator complex formation in different mutant backgrounds, we conclude that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.

Improved methods for predicting peptide binding affinity to MHC class II molecules.

PubMed

Jensen, Kamilla Kjaergaard; Andreatta, Massimo; Marcatili, Paolo; Buus, Søren; Greenbaum, Jason A; Yan, Zhen; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

2018-07-01

Major histocompatibility complex class II (MHC-II) molecules are expressed on the surface of professional antigen-presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC-II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T-cell epitopes. We here present updated versions of two MHC-II-peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC-peptide binding affinity data obtained from the Immune Epitope Database covering HLA-DR, HLA-DQ, HLA-DP and H-2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2. © 2018 John Wiley & Sons Ltd.

Noncanonical expression of a murine cytomegalovirus early protein CD8 T-cell epitope as an immediate early epitope based on transcription from an upstream gene.

PubMed

Fink, Annette; Büttner, Julia K; Thomas, Doris; Holtappels, Rafaela; Reddehase, Matthias J; Lemmermann, Niels A W

2014-02-14

Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I) glycoproteins, are often identified by "reverse immunology", a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs) based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype) mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E) phase protein, the m164 epitope is presented already during the Immediate Early (IE) phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

Characterization of MHC-I in the blue tit (Cyanistes caeruleus) reveals low levels of genetic diversity and trans-population evolution across European populations.

PubMed

Schut, Elske; Aguilar, Juan Rivero-de; Merino, Santiago; Magrath, Michael J L; Komdeur, Jan; Westerdahl, Helena

2011-08-01

The major histcompatibility complex (MHC) is a vital component of the adaptive immune system in all vertebrates. This study is the first to characterize MHC class I (MHC-I) in blue tits (Cyanistes caeruleus), and we use MHC-I exon 3 sequence data from individuals originating from three locations across Europe: Spain, the Netherlands to Sweden. Our phylogeny of the 17 blue tit MHC-I alleles contains one allele cluster with low nucleotide diversity compared to the remaining more diverse alleles. We found a significant evidence for balancing selection in the peptide-binding region in the diverse allele group only. No separation according to geographic location was found in the phylogeny of alleles. Although the number of MHC-I loci of the blue tit is comparable to that of other passerine species, the nucleotide diversity of MHC-I appears to be much lower than that of other passerine species, including the closely related great tit (Parus major) and the severely inbred Seychelles warbler (Acrocephalus sechellensis). We believe that this initial MHC-I characterization in blue tits provides an important step towards understanding the mechanisms shaping MHC-I diversity in natural populations.

Exploiting three kinds of interface propensities to identify protein binding sites.

PubMed

Liu, Bin; Wang, Xiaolong; Lin, Lei; Dong, Qiwen; Wang, Xuan

2009-08-01

Predicting the binding sites between two interacting proteins provides important clues to the function of a protein. In this study, we present a building block of proteins called order profiles to use the evolutionary information of the protein sequence frequency profiles and apply this building block to produce a class of propensities called order profile interface propensities. For comparisons, we revisit the usage of residue interface propensities and binary profile interface propensities for protein binding site prediction. Each kind of propensities combined with sequence profiles and accessible surface areas are inputted into SVM. When tested on four types of complexes (hetero-permanent complexes, hetero-transient complexes, homo-permanent complexes and homo-transient complexes), experimental results show that the order profile interface propensities are better than residue interface propensities and binary profile interface propensities. Therefore, order profile is a suitable profile-level building block of the protein sequences and can be widely used in many tasks of computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the protein remote homology detection.

Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli*S⃞

PubMed Central

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-01-01

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (Ki = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (Km = 0.1 mm). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr97 and Pro101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS. PMID:19211556

New modulated design and synthesis of quercetin-Cu(II)/Zn(II)-Sn2(IV) scaffold as anticancer agents: in vitro DNA binding profile, DNA cleavage pathway and Topo-I activity.

PubMed

Tabassum, Sartaj; Zaki, Mehvash; Afzal, Mohd; Arjmand, Farukh

2013-07-21

New molecular topologies quercetin-Cu(II)-Sn2(IV) and Zn(II)-Sn2(IV)1 and 2 were designed and synthesized to act as potential cancer chemotherapeutic agents. Their interaction with CT DNA by UV-vis and fluorescence spectroscopy was evaluated revealing an electrostatic mode of binding. Quercetin complexes are capable of promoting DNA cleavage involving both single and double strand breaks. Complex 1 cleaved pBR322 DNA via an oxidative mechanism while 2 followed a hydrolytic pathway, accessible to the minor groove of the DNA double helix in accordance with molecular docking studies with the DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer demonstrating that the complex was stabilized by additional electrostatic and hydrogen bonding interactions with the DNA. ROS such as OH˙, H2O2 and O2˙(-) are the major metabolites responsible for chronic diseases such as cancer, respiratory disorders, HIV, and diabetes etc., therefore eliminating ROS by molecular scaffolds involving SOD enzymatic activity has emerged as a potential way to develop a novel class of drugs. Therefore, in vitro superoxide dismutase activity of redox active complex 1 was evaluated by using a xanthine/xanthine oxidase-NBT assay which showed an IC50 value of 2.26 μM. Moreover, the cytotoxicity of both the complexes were screened on a panel of human carcinoma cell lines (GI50 values <8.7 μM) which revealed that 1 has a better prospect of acting as a cancer chemotherapeutic agent, and to elucidate the mechanism of tumor inhibition, Topo-I enzymatic activity was carried out. Furthermore, molecular modeling studies were carried out to understand molecular features important for drug-enzyme interactions which offer new insights into the experimental model observations.

Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17β-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps

NASA Astrophysics Data System (ADS)

Negri, Matthias; Recanatini, Maurizio; Hartmann, Rolf W.

2011-09-01

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17β-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the βFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17β-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17β-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.

Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules.

PubMed

Chapman, Daniel C; Stocki, Pawel; Williams, David B

2015-01-01

Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin.

Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules

PubMed Central

Chapman, Daniel C.; Stocki, Pawel; Williams, David B.

2015-01-01

Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin. PMID:26691022

The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

PubMed Central

Shen, Xiao Z.; Billet, Sandrine; Lin, Chentao; Okwan-Duodu, Derick; Chen, Xu; Lukacher, Aron E.; Bernstein, Kenneth E.

2011-01-01

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell mediated adaptive immune responses. Aminopeptidases are implicated in the editing of peptides for MHC class I loading, but C-terminal editing is thought due to proteasome cleavage. By comparing genetically deficient, wild-type and over-expressing mice, we now identify the dipeptidase angiotensin-converting enzyme (ACE) as playing a physiologic role in peptide processing for MHC class I. ACE edits the C-termini of proteasome-produced class I peptides. The lack of ACE exposes novel antigens but also abrogates some self-antigens. ACE has major effects on surface MHC class I expression in a haplotype-dependent manner. We propose a revised model of MHC class I peptide processing by introducing carboxypeptidase activity. PMID:21964607

Mechanisms of virus immune evasion lead to development from chronic inflammation to cancer formation associated with human papillomavirus infection.

PubMed

Senba, Masachika; Mori, Naoki

2012-10-02

Human papillomavirus (HPV) has developed strategies to escape eradication by innate and adaptive immunity. Immune response evasion has been considered an important aspect of HPV persistence, which is the main contributing factor leading to HPV-related cancers. HPV-induced cancers expressing viral oncogenes E6 and E7 are potentially recognized by the immune system. The major histocompatibility complex (MHC) class I molecules are patrolled by natural killer cells and CD8+ cytotoxic T lymphocytes, respectively. This system of recognition is a main target for the strategies of immune evasion deployed by viruses. The viral immune evasion proteins constitute useful tools to block defined stages of the MHC class I presentation pathway, and in this way HPV avoids the host immune response. The long latency period from initial infection to persistence signifies that HPV evolves mechanisms to escape the immune response. It has now been established that there are oncogenic mechanisms by which E7 binds to and degrades tumor suppressor Rb, while E6 binds to and inactivates tumor suppressor p53. Therefore, interaction of p53 and pRb proteins can give rise to an increased immortalization and genomic instability. Overexpression of NF-κB in cervical and penile cancers suggests that NF-κB activation is a key modulator in driving chronic inflammation to cancer. HPV oncogene-mediated suppression of NF-κB activity contributes to HPV escape from the immune system. This review focuses on the diverse mechanisms of the virus immune evasion with HPV that leads to chronic inflammation and cancer.

Preferential reduction of binding of sup 125 I-iodopindolol to beta-1 adrenoceptors in the amygdala of rat after antidepressant treatments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ordway, G.A.; Gambarana, C.; Tejani-Butt, S.M.

1991-05-01

This study utilized quantitative receptor autoradiography to examine the effects of repeated administration of antidepressants to rats on the binding of the beta adrenoceptor antagonist, {sup 125}I-iodopindolol ({sup 125}I-IPIN) to either beta-1 or beta-2 adrenoceptors in various regions of brain. Antidepressants were selected to represent various chemical and pharmacological classes including tricyclic compounds (desipramine and protriptyline), monoamine oxidase inhibitors (clorgyline, phenelzine and tranylcypromine), atypical antidepressants (mianserin and trazodone) and selective inhibitors of the uptake of serotonin (citalopram and sertraline). Additionally, rats were treated with various psychotropic drugs that lack antidepressant efficacy (cocaine, deprenyl, diazepam and haloperidol). Repeated treatment of ratsmore » with desipramine, protriptyline, clorgyline, phenelzine, tranylcypromine or mianserin reduced the binding of {sup 125}I-IPIN to beta-1 adrenoceptors in many brain areas. Only in the basolateral and lateral nuclei of the amygdala did all six of these antidepressants significantly reduce {sup 125}I-IPIN binding to beta-1 adrenoceptors. In these amygdaloid nuclei, the magnitude of the reduction in the binding of {sup 125}I-IPIN caused by each of these drugs was comparable to or greater than the reduction in binding produced in any other region of brain. Reductions of binding of {sup 125}I-IPIN after antidepressant treatments were not consistently observed in the cortex, the area of brain examined most often in homogenate binding studies. Only the monoamine oxidase inhibitors caused reductions in the binding of {sup 125}I-IPIN to beta-2 adrenoceptors, and this effect was generally localized to the amygdala and hypothalamus.« less

Functional characterization of JMJD2A, a histone deacetylase- and retinoblastoma-binding protein.

PubMed

Gray, Steven G; Iglesias, Antonio H; Lizcano, Fernando; Villanueva, Raul; Camelo, Sandra; Jingu, Hisaka; Teh, Bin T; Koibuchi, Noriyuki; Chin, William W; Kokkotou, Efi; Dangond, Fernando

2005-08-05

To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G(2)/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis.

Complexes of plexin-A4 and plexin-D1 convey semaphorin-3C signals to induce cytoskeletal collapse in the absence of neuropilins.

PubMed

Smolkin, Tatyana; Nir-Zvi, Inbal; Duvshani, Nerri; Mumblat, Yelena; Kessler, Ofra; Neufeld, Gera

2018-05-04

Class-3 semaphorin guidance factors bind to receptor complexes containing neuropilin and plexin receptors. A semaphorin may bind to several receptor complexes containing somewhat different constituents, resulting in diverse effects on cell migration. U87MG glioblastoma cells express both neuropilins and the four class-A plexins. Here, we show that these cells respond to Sema3A or Sema3B by cytoskeletal collapse and cell contraction but fail to contract in response to Sema3C, Sema3D, Sema3G or Sema3E, even when class-A plexins are overexpressed in the cells. In contrast, expression of recombinant plexin-D1 enabled contraction in response to these semaphorins. Surprisingly, unlike Sema3D and Sema3G, Sema3C also induced the contraction and repulsion of plexin-D1-expressing U87MG cells in which both neuropilins were knocked out using CRISPR/Cas9. In the absence of neuropilins, the EC50 of Sema3C was 5.5 times higher, indicating that the neuropilins function as enhancers of plexin-D1-mediated Sema3C signaling but are not absolutely required for Sema3C signal transduction. Interestingly, in the absence of neuropilins, plexin-A4 formed complexes with plexin-D1, and was required in addition to plexin-D1 to enable Sema3C-induced signal transduction. © 2018. Published by The Company of Biologists Ltd.

Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution.

PubMed

Ahmad, Malik Shoaib; Rasheed, Saima; Falke, Sven; Khaliq, Binish; Perbandt, Markus; Choudhary, M Iqbal; Markiewicz, Wojciech T; Barciszewski, Jan; Betzel, Christian

2018-05-23

Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified via in-silico docking. The high-resolution structure was refined subsequently to analyze particularly the active site conformation and binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. In comparison to other reported ML-I complexes we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition and cytotoxicity of the ML-I A-chain and related RIPs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quantifying Na(I)-insulin and K(I)-insulin non-covalent complexes by ESI-MS method and calculation of their equilibrium constants.

PubMed

Gülfen, Mustafa; Özdemir, Abdil; Lin, Jung-Lee; Chen, Chung-Hsuan

2017-10-01

In this study, the dissociation and formation equilibrium constants of Na(I)-insulin and K(I)-insulin complexes have been calculated after the quantifying them on ESI mass spectrometer. The ESI-MS spectra of the complexes were measured by using the solvents as 50% MeOH in water and 100% water. The effect of pH on the Na(I)-insulin and K(I)-insulin complex formation were examined. Serial binding of Na(I) and K(I) ions to the insulin molecule were observed in the ESI-MS measurements. The first formation equilibrium constants were calculated as K f1 : 5.48×10 3 1/M for Na(I)-insulin complex and K f1 : 4.87×10 3 1/M for K(I)-insulin in water. The binding capability of Na(I) ions to insulin molecule is higher than the capability of K(I) ions. In case of a comparison together with Ca(II)-insulin and Mg(II)-insulin, the formation equilibrium constants (K f1 ) are in order of Ca(II)-insulin>Mg(II)-insulin>Na(I)-insulin>K(I)-insulin in water. The results showed that Na(I) and K(I) ions are involved in the formation of the non-covalent complexes with insulin molecule, since high extracellular and intracellular concentrations of them in the body. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of the Bacillus subtilis replication termination protein in complex with the 37-base-pair TerI-binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vivian, J. P.; Porter, C.; Wilce, J. A.

2006-11-01

A preparation of replication terminator protein (RTP) of B. subtilis and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. The replication terminator protein (RTP) of Bacillus subtilis binds to specific DNA sequences that halt the progression of the replisome in a polar manner. These terminator complexes flank a defined region of the chromosome into which they allow replication forks to enter but not exit. Forcing the fusion of replication forks in a specific zone is thought to allow the coordination of post-replicative processes. The functional terminator complex comprises two homodimers each of 29more » kDa bound to overlapping binding sites. A preparation of RTP and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. A data set to 3.9 Å resolution with 97.0% completeness and an R{sub sym} of 12% was collected from a single flash-cooled crystal using synchrotron radiation. The diffraction data are consistent with space group P622, with unit-cell parameters a = b = 118.8, c = 142.6 Å.« less

The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaakola, Veli-Pekka; Griffith, Mark T.; Hanson, Michael A.

2009-01-15

The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A{sub 2A} adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extendedmore » conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.« less

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

PubMed Central

Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O’Hanlon; Gordon, Sandra; Mortensen, Anne Louise; Clausen, Johannes D.; Pallin, Thomas David; Hansen, John Bondo; Fuglsang, Anja Thoe; Dalby-Brown, William

2018-01-01

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents. PMID:29293507

Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

PubMed Central

Nielsen, Morten; Lundegaard, Claus; Lund, Ole

2007-01-01

Background Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion The SMM-align method was shown to outperform other state of the art MHC class II prediction methods. The method predicts quantitative peptide:MHC binding affinity values, making it ideally suited for rational epitope discovery. The method has been trained and evaluated on the, to our knowledge, largest benchmark data set publicly available and covers the nine HLA-DR supertypes suggested as well as three mouse H2-IA allele. Both the peptide benchmark data set, and SMM-align prediction method (NetMHCII) are made publicly available. PMID:17608956

Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method.

PubMed

Nielsen, Morten; Lundegaard, Claus; Lund, Ole

2007-07-04

Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. The SMM-align method was shown to outperform other state of the art MHC class II prediction methods. The method predicts quantitative peptide:MHC binding affinity values, making it ideally suited for rational epitope discovery. The method has been trained and evaluated on the, to our knowledge, largest benchmark data set publicly available and covers the nine HLA-DR supertypes suggested as well as three mouse H2-IA allele. Both the peptide benchmark data set, and SMM-align prediction method (NetMHCII) are made publicly available.

Theoretical study of X⁻ · 1 · YF (1 = triazine, X = Cl, Br and I, Y = H, Cl, Br, I, PH₂ and AsH₂): noncovalently electron-withdrawing effects on anion-arene interactions.

PubMed

Chen, Yishan; Yao, Lifeng

2014-01-01

The ternary complexes X(-) · 1 · YF (1 = triazine, X = Cl, Br and I, Y = H, Cl, Br, I, PH2 and AsH2) have been investigated by MP2 calculations to understand the noncovalently electron-withdrawing effects on anion-arene interactions. The results indicate that in binary complexes (1 · X(-)), both weak σ-type and anion-π complexes can be formed for Cl(-) and Br(-), but only anion-π complex can be formed for I(-). Moreover, the hydrogen-bonding complex is the global minimum for all three halides in binary complexes. However, in ternary complexes, anion-π complex become unstable and only σ complex can retain in many cases for Cl(-) and Br(-). Anion-π complex keeps stable only when YF = HF. In contrast with binary complexes, σ complex become the global minimum for Cl(-) and Br(-) in ternary complexes. These changes in binding mode and strength are consistent with the results of covalently electron-withdrawing effects. However, in contrast with the covalently electron-withdrawing substituents, Cl(-) and Br(-) can attack the aromatic carbon atom to form a strong σ complex when the noncovalently electron-withdrawing effect is induced by halogen bonding. The binding behavior for I(-) is different from that for Cl(-) and Br(-) in two aspects. First, the anion-π complex for I(-) can also keep stable when the noncovalent interaction is halogen bonding. Second, the anion-π complex for I(-) is the global minimum when it can retain as a stable structure.

Part I. Cobalt thiolate complexes modeling the active site of cobalt nitrile hydratase. Part II. Formation of inorganic nanoparticles on protein scaffolding in Escherichia coli glutamine synthetase

NASA Astrophysics Data System (ADS)

Kung, Irene Yuk Man

Part I. A series of novel cobalt dithiolate complexes with mixed imine/amine ligand systems is presented here as electronic and structural models for the active site in the bacterial enzyme class, nitrile hydratase (NHase). Pentadentate cobalt(II) complexes with S2N 3 ligand environments are first studied as precursors to the more relevant cobalt(III) complexes. Adjustment of the backbone length by removal of a methylene group increases the reactivity of the system; whereas reduction of the two backbone imine bonds to allow free rotation about those bonds may decrease reactivity. Reactivity change due to the replacement of the backbone amine proton with a more sterically challenging methyl group is not yet clear. Upon oxidation, the monocationic pentadentate cobalt(III) complex, 1b, shows promising reactivity similar to that of NHase. The metal's open coordination site allows reversible binding of the endogenous, monoanionic ligands, N 3- and NCS-. Oxygenation of the thiolate sulfur atoms by exposure to O2 and H2O 2 produces sulfenate and sulfinate ligands in complex 8, which resembles the crystal structure of "deactivated" Fe NHase. However, its lack of reactivity argues against the oxygenated enzyme structure as the active form. Six-coordinate cobalt(III) complexes with S2N4 amine/amine ligand systems are also presented as analogues of previously reported iron(III) compounds, which mimic the spectroscopic properties of Fe NHase. The cobalt complexes do not seem to similarly model Co NHase. However, the S = 0 cobalt(III) center can be spectroscopically silent and difficult to detect, making comparison with synthetic models using common techniques hard. Part II. Dodecameric Escherichia coli glutamine synthetase mutant, E165C, stacks along its six-fold axis to produce tubular nanostructures in the presence of some divalent metal ions, as does the wild type enzyme. The centrally located, engineered Cys-165 residues appear to bind to various species and may serve as scaffolding for inorganic mineralization. US nanoclusters of discreet size seem to grow in the presence of E165C in aqueous solution spontaneously. Commercially available mono(maleimido)undecagold seem to bind only to E165C through the reactive cysteine side chains. Reduction of Au3+ to elemental gold in solution with E165C, generates long, linear structures of approximately 100-nm diameter.

Yeast hexokinase. A fluorescence temperature-jump study of the kinetics of the binding of glucose to the monomer forms of hexokinases P-I and P-II.

PubMed

Hoggett, J G; Kellett, G L

1976-09-15

The binding of glucose to the monomeric forms of hexokinases P-I and P-II in Tris and phosphate buffers at pH 8.0 in the presence of 1 mol l-1 KCl has been studied using the fluorescence temperature-jump technique. For both isozymes only one relaxation time was observed; values of tau-1 increased linearly with increasing concentration of free reacting partners. The apparent second-order rate constant for association was about 2 X 10(6) 1 mol-1 s-1 for both isozymes; the differences in the stabilities of the complexes with P-I and P-II are entirely attributable to the fact that glucose dissociates more slowly from its complex with P-I than P-II (approximately 300 s-1 and 1100 s-1 respectively). Although the kinetic data are compatible with a single-step mechanism for glucose binding the association rate constant was much lower than that expected for a diffusion-limited rate of encounter. Other mechanisms for describing an induced-fit are discussed. It is shown that the data are incompatible with a slow 'prior-isomerization' pathway of substrate binding, but are consistent with a 'substrate-guided' pathway involving isomerization of the enzyme-substrate complex.

sc-PDB: a database for identifying variations and multiplicity of 'druggable' binding sites in proteins.

PubMed

Meslamani, Jamel; Rognan, Didier; Kellenberger, Esther

2011-05-01

The sc-PDB database is an annotated archive of druggable binding sites extracted from the Protein Data Bank. It contains all-atoms coordinates for 8166 protein-ligand complexes, chosen for their geometrical and physico-chemical properties. The sc-PDB provides a functional annotation for proteins, a chemical description for ligands and the detailed intermolecular interactions for complexes. The sc-PDB now includes a hierarchical classification of all the binding sites within a functional class. The sc-PDB entries were first clustered according to the protein name indifferent of the species. For each cluster, we identified dissimilar sites (e.g. catalytic and allosteric sites of an enzyme). SCOPE AND APPLICATIONS: The classification of sc-PDB targets by binding site diversity was intended to facilitate chemogenomics approaches to drug design. In ligand-based approaches, it avoids comparing ligands that do not share the same binding site. In structure-based approaches, it permits to quantitatively evaluate the diversity of the binding site definition (variations in size, sequence and/or structure). The sc-PDB database is freely available at: http://bioinfo-pharma.u-strasbg.fr/scPDB.

Structural and biochemical studies of RIG-I antiviral signaling.

PubMed

Feng, Miao; Ding, Zhanyu; Xu, Liang; Kong, Liangliang; Wang, Wenjia; Jiao, Shi; Shi, Zhubing; Greene, Mark I; Cong, Yao; Zhou, Zhaocai

2013-02-01

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.

AgI -Induced Switching of DNA Binding Modes via Formation of a Supramolecular Metallacycle.

PubMed

Basak, Shibaji; Léon, J Christian; Ferranco, Annaleizle; Sharma, Renu; Hebenbrock, Marian; Lough, Alan; Müller, Jens; Kraatz, Heinz-Bernhard

2018-03-12

The histidine derivative L1 of the DNA intercalator naphthalenediimide (NDI) forms a triangular Ag I complex (C2). The interactions of L1 and of C2 with DNA were studied by circular dichroism (CD) and UV/Vis spectroscopy and by viscosity studies. Different binding modes were observed for L1 and for C2, as the Ag I complex C2 is too large in size to act as an intercalator. If Ag I is added to the NDI molecule that is already intercalated into a duplex, higher order complexes are formed within the DNA duplex and cause disruptions in the helical duplex structure, which leads to a significant decrease in the characteristic CD features of B-DNA. Thus, via addition of a metal we show how a classic and well-known organic intercalator unit can be turned into a partial metallo insertor. We also show how electrochemical impedance spectroscopy (EIS) can be used to probe DNA binding modes on DNA films that are immobilized on gold surfaces. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distinguishing functional polymorphism from random variation in the sequences of >10,000 HLA-A, -B and -C alleles.

PubMed

Robinson, James; Guethlein, Lisbeth A; Cereb, Nezih; Yang, Soo Young; Norman, Paul J; Marsh, Steven G E; Parham, Peter

2017-06-01

HLA class I glycoproteins contain the functional sites that bind peptide antigens and engage lymphocyte receptors. Recently, clinical application of sequence-based HLA typing has uncovered an unprecedented number of novel HLA class I alleles. Here we define the nature and extent of the variation in 3,489 HLA-A, 4,356 HLA-B and 3,111 HLA-C alleles. This analysis required development of suites of methods, having general applicability, for comparing and analyzing large numbers of homologous sequences. At least three amino-acid substitutions are present at every position in the polymorphic α1 and α2 domains of HLA-A, -B and -C. A minority of positions have an incidence >1% for the 'second' most frequent nucleotide, comprising 70 positions in HLA-A, 85 in HLA-B and 54 in HLA-C. The majority of these positions have three or four alternative nucleotides. These positions were subject to positive selection and correspond to binding sites for peptides and receptors. Most alleles of HLA class I (>80%) are very rare, often identified in one person or family, and they differ by point mutation from older, more common alleles. These alleles with single nucleotide polymorphisms reflect the germ-line mutation rate. Their frequency predicts the human population harbors 8-9 million HLA class I variants. The common alleles of human populations comprise 42 core alleles, which represent all selected polymorphism, and recombinants that have assorted this polymorphism.

Distinguishing functional polymorphism from random variation in the sequences of >10,000 HLA-A, -B and -C alleles

PubMed Central

Cereb, Nezih; Yang, Soo Young; Marsh, Steven G. E.; Parham, Peter

2017-01-01

HLA class I glycoproteins contain the functional sites that bind peptide antigens and engage lymphocyte receptors. Recently, clinical application of sequence-based HLA typing has uncovered an unprecedented number of novel HLA class I alleles. Here we define the nature and extent of the variation in 3,489 HLA-A, 4,356 HLA-B and 3,111 HLA-C alleles. This analysis required development of suites of methods, having general applicability, for comparing and analyzing large numbers of homologous sequences. At least three amino-acid substitutions are present at every position in the polymorphic α1 and α2 domains of HLA-A, -B and -C. A minority of positions have an incidence >1% for the ‘second’ most frequent nucleotide, comprising 70 positions in HLA-A, 85 in HLA-B and 54 in HLA-C. The majority of these positions have three or four alternative nucleotides. These positions were subject to positive selection and correspond to binding sites for peptides and receptors. Most alleles of HLA class I (>80%) are very rare, often identified in one person or family, and they differ by point mutation from older, more common alleles. These alleles with single nucleotide polymorphisms reflect the germ-line mutation rate. Their frequency predicts the human population harbors 8–9 million HLA class I variants. The common alleles of human populations comprise 42 core alleles, which represent all selected polymorphism, and recombinants that have assorted this polymorphism. PMID:28650991

Ran-dependent nuclear export mediators: a structural perspective

PubMed Central

Güttler, Thomas; Görlich, Dirk

2011-01-01

Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP–exportin–cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions. PMID:21878989

The Crystal Structure Analysis of Group B Streptococcus Sortase C1: A Model for the ;Lid; Movement upon Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu

2012-02-07

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible 'lid' anchored in the active site. However, the mechanistic details of the 'lid' displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its 'lid' mutant and proposed a role of the 'lid' as a protectormore » of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.« less

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S-adenosyl-l-methionine-dependent methyltransferase.

PubMed

Im, Ha Na; Kim, Hyoun Sook; An, Doo Ri; Jang, Jun Young; Kim, Jieun; Yoon, Hye-Jin; Yang, Jin Kuk; Suh, Se Won

2016-03-01

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site. Copyright © 2016 Elsevier Inc. All rights reserved.

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

PubMed

Ogawara, Hiroshi

2016-09-01

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro.

PubMed

Phumyen, Achara; Jantasorn, Siriporn; Jumnainsong, Amonrat; Leelayuwat, Chanvit

2014-01-01

Cancer therapy by systemic administration of anticancer drugs, besides the effectiveness shown on cancer cells, demonstrated the side effects and cytotoxicity on normal cells. The targeted drug-carrying nanoparticles may decrease the required drug concentration at the site and the distribution of drugs to normal tissues. Overexpression of major histocompatibility complex class I chain-related A (MICA) in cancer is useful as a targeted molecule for the delivery of doxorubicin to MICA-expressing cell lines. The application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) chemistry was employed to conjugate the major coat protein of bacteriophages carrying anti-MICA and doxorubicin in a mildly acid condition. Doxorubicin (Dox) on phages was determined by double fluorescence of phage particles stained by M13-fluorescein isothiocyanate (FITC) and drug autofluorescence by flow cytometry. The ability of anti-MICA on phages to bind MICA after doxorubicin conjugation was evaluated by indirect enzyme-linked immunosorbent assay. One cervical cancer and four cholangiocarcinoma cell lines expressing MICA were used as models to evaluate targeting activity by cell cytotoxicity test. Flow cytometry and indirect enzyme-linked immunosorbent assay demonstrated that most of the phages (82%) could be conjugated with doxorubicin, and the Dox-carrying phage-displaying anti-MICA (Dox-phage) remained the binding activity against MICA. Dox-phage was more efficient than free drugs in killing all the cell lines tested. The half maximal inhibitory concentration (IC50) values of Dox-phage were lower than those of free drugs at approximately 1.6-6 times depending on MICA expressions and the cell lines tested. Evidently, the application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide chemistry is effective to conjugate doxorubicin and major coat protein of bacteriophages without destroying binding activity of MICA antibodies. Dox-carrying bacteriophages targeting MICA have been successfully developed and may enable a broad range of applications in cancer-targeting chemotherapy.

A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

PubMed Central

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-01-01

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

Biochemical and Structural Analysis of Inhibitors Targeting the ADC-7 Cephalosporinase of Acinetobacter baumannii

DOE PAGES

Powers, Rachel A.; Swanson, Hollister C.; Taracila, Magdalena A.; ...

2014-11-07

β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (K i =more » 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C 3/C 4 carboxylate found in β-lactams. The C 3/C 4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. In conclusion, the ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.« less

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

PubMed Central

Hoggard, Timothy; Shor, Erika; Müller, Carolin A.; Nieduszynski, Conrad A.; Fox, Catherine A.

2013-01-01

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time. PMID:24068963

[125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

PubMed Central

Dumont, Yvan; Quirion, Rémi

2000-01-01

GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]-GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]-GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [125I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]-GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]-GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. PMID:10694200

Quantitation of IgE antibody specific for ragweed and grass allergens: binding of radiolabeled allergens by solid-phase bond IgE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiss, C.R.; Levitz, D.; Suszko, I.M.

1978-08-01

IgE antibody specific for multiple allergens extracted from grass and ragweed pollens was measured by radioimmunoassay. The assay depends on the interaction between IgE antibody bound to a polystyrene solid phase, /sup 125/I-labeled grass allergens (GA), and ragweed allergens (RW). The binding of /sup 125/I RW by serum IgE antibody from 37 allergic patients ranged from 0.2 ng to 75 ng RW protein (P) bound per ml. This binding of /sup 125/I RW by patient's IgE was paralleled by their IgE binding of /sup 125/I antigen E (AgE), a purified allergen from ragweed pollen (r = 0.90, p less thanmore » 0.001). Inhibition of patient's IgE binding of /sup 125/I RW by highly purified AgE ranged from 25 to 85% indicated individual differences in patient's IgE response to inhaled ragweed pollen. The binding of /sup 125/I GA by serum IgE antibody from 7 grass-sensitive patients ranged from 0.6 ng GA P bound per ml to 15 ng. This assay should be useful in the study of IgE responses to environmental agents containing multiple allergens and has the advantage that other antibody classes cannot interfere with the interaction between IgE antibody and labeled allergens.« less

A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

PubMed Central

Koehler, Jeffrey W.; Smith, Jeffrey M.; Ripoll, Daniel R.; Spik, Kristin W.; Taylor, Shannon L.; Badger, Catherine V.; Grant, Rebecca J.; Ogg, Monica M.; Wallqvist, Anders; Guttieri, Mary C.; Garry, Robert F.; Schmaljohn, Connie S.

2013-01-01

For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors. PMID:24069485

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi *

PubMed Central

Jia, Min; Li, Jianchao; Zhu, Jinwei; Wen, Wenyu; Zhang, Mingjie; Wang, Wenning

2012-01-01

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gαi/o during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gαi·GDP. The crystal structures of Gαi·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gαi interaction features when compared with the only high resolution structure known with GL/Gαi interaction between RGS14 and Gαi1. Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gαi·GDP. A highly conserved “double Arg finger” sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gαi. Together with the sequence alignment, we suggest that the LGN GL/Gαi interaction represents a general binding mode between GL motifs and Gαi. We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors. PMID:22952234

PSSMHCpan: a novel PSSM-based software for predicting class I peptide-HLA binding affinity

PubMed Central

Liu, Geng; Li, Dongli; Li, Zhang; Qiu, Si; Li, Wenhui; Chao, Cheng-chi; Yang, Naibo; Li, Handong; Cheng, Zhen; Song, Xin; Cheng, Le; Zhang, Xiuqing; Wang, Jian; Yang, Huanming

2017-01-01

Abstract Predicting peptide binding affinity with human leukocyte antigen (HLA) is a crucial step in developing powerful antitumor vaccine for cancer immunotherapy. Currently available methods work quite well in predicting peptide binding affinity with HLA alleles such as HLA-A*0201, HLA-A*0101, and HLA-B*0702 in terms of sensitivity and specificity. However, quite a few types of HLA alleles that are present in the majority of human populations including HLA-A*0202, HLA-A*0203, HLA-A*6802, HLA-B*5101, HLA-B*5301, HLA-B*5401, and HLA-B*5701 still cannot be predicted with satisfactory accuracy using currently available methods. Furthermore, currently the most popularly used methods for predicting peptide binding affinity are inefficient in identifying neoantigens from a large quantity of whole genome and transcriptome sequencing data. Here we present a Position Specific Scoring Matrix (PSSM)-based software called PSSMHCpan to accurately and efficiently predict peptide binding affinity with a broad coverage of HLA class I alleles. We evaluated the performance of PSSMHCpan by analyzing 10-fold cross-validation on a training database containing 87 HLA alleles and obtained an average area under receiver operating characteristic curve (AUC) of 0.94 and accuracy (ACC) of 0.85. In an independent dataset (Peptide Database of Cancer Immunity) evaluation, PSSMHCpan is substantially better than the popularly used NetMHC-4.0, NetMHCpan-3.0, PickPocket, Nebula, and SMM with a sensitivity of 0.90, as compared to 0.74, 0.81, 0.77, 0.24, and 0.79. In addition, PSSMHCpan is more than 197 times faster than NetMHC-4.0, NetMHCpan-3.0, PickPocket, sNebula, and SMM when predicting neoantigens from 661 263 peptides from a breast tumor sample. Finally, we built a neoantigen prediction pipeline and identified 117 017 neoantigens from 467 cancer samples of various cancers from TCGA. PSSMHCpan is superior to the currently available methods in predicting peptide binding affinity with a broad coverage of HLA class I alleles. PMID:28327987

Discovering amino acid patterns on binding sites in protein complexes

PubMed Central

Kuo, Huang-Cheng; Ong, Ping-Lin; Lin, Jung-Chang; Huang, Jen-Peng

2011-01-01

Discovering amino acid (AA) patterns on protein binding sites has recently become popular. We propose a method to discover the association relationship among AAs on binding sites. Such knowledge of binding sites is very helpful in predicting protein-protein interactions. In this paper, we focus on protein complexes which have protein-protein recognition. The association rule mining technique is used to discover geographically adjacent amino acids on a binding site of a protein complex. When mining, instead of treating all AAs of binding sites as a transaction, we geographically partition AAs of binding sites in a protein complex. AAs in a partition are treated as a transaction. For the partition process, AAs on a binding site are projected from three-dimensional to two-dimensional. And then, assisted with a circular grid, AAs on the binding site are placed into grid cells. A circular grid has ten rings: a central ring, the second ring with 6 sectors, the third ring with 12 sectors, and later rings are added to four sectors in order. As for the radius of each ring, we examined the complexes and found that 10Å is a suitable range, which can be set by the user. After placing these recognition complexes on the circular grid, we obtain mining records (i.e. transactions) from each sector. A sector is regarded as a record. Finally, we use the association rule to mine these records for frequent AA patterns. If the support of an AA pattern is larger than the predetermined minimum support (i.e. threshold), it is called a frequent pattern. With these discovered patterns, we offer the biologists a novel point of view, which will improve the prediction accuracy of protein-protein recognition. In our experiments, we produced the AA patterns by data mining. As a result, we found that arginine (arg) most frequently appears on the binding sites of two proteins in the recognition protein complexes, while cysteine (cys) appears the fewest. In addition, if we discriminate the shape of binding sites between concave and convex further, we discover that patterns {arg, glu, asp} and {arg, ser, asp} on the concave shape of binding sites in a protein more frequently (i.e. higher probability) make contact with {lys} or {arg} on the convex shape of binding sites in another protein. Thus, we can confidently achieve a rate of at least 78%. On the other hand {val, gly, lys} on the convex surface of binding sites in proteins is more frequently in contact with {asp} on the concave site of another protein, and the confidence achieved is over 81%. Applying data mining in biology can reveal more facts that may otherwise be ignored or not easily discovered by the naked eye. Furthermore, we can discover more relationships among AAs on binding sites by appropriately rotating these residues on binding sites from a three-dimension to two-dimension perspective. We designed a circular grid to deposit the data, which total to 463 records consisting of AAs. Then we used the association rules to mine these records for discovering relationships. The proposed method in this paper provides an insight into the characteristics of binding sites for recognition complexes. PMID:21464838

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pröpper, Kevin; Instituto de Biologia Molecular de Barcelona; Meindl, Kathrin

2014-06-01

The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite themore » fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.« less

Radiosynthesis and evaluation of novel acetylcholine receptor radioligands

NASA Astrophysics Data System (ADS)

Pimlott, Sally L.

Neuroreceptor single photon emission computed tomography (SPECT) imaging provides a powerful tool for the evaluation of the function of a neurotransmitter system in normal and or disease states in the living human brain. The cholinergic system is involved in the control of a variety of complex functions including learning, memory and modulation of behaviour. Deficits in the cholinergic system have been found in a number of neurological diseases, such as Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease and Epilepsy. Acetylcholine receptors (AChRs) are divided into two classes, muscarinic and nicotinic. The aim of this project was to develop two novel SPECT AChR ligands: (R,R)[123I]I-QNB, a M1 subtype selective muscarinic acetylcholine receptor (mAChR) ligand, and 5-[123I]-A-85380, a alpha4beta2 subtype selective nicotinic receptor (nAChR) ligand, for use in human SPECT imaging studies. The calculation of the binding potential of a ligand can be used to obtain quantitative information from a SPECT scan, enabling comparisons to be made between studies. Methodological issues involved in the calculation of binding potential are therefore crucial for the accuracy of results. A particularly important parameter is the amount of authentic radioligand available to cross the blood brain barrier. This was characterised in the research performed for this thesis. The radiosynthesis of two novel neuroreceptor radioligands has been optimised for use in humans. (R, R)[123I]I-QNB has been used in human studies to provide useful information on the human mAChR function in disease. Pre-clinical evaluation of 5-[123I]-A-85380 provided useful information for in vivo human studies. Both radioligands are concluded to successfully provide novel information on the function of the acetylcholine system. Methodological issues involved in the blood metabolite analysis and measurement of plasma protein binding have been investigated and discussed, with particular reference made to the factors that must be taken into account when designing these experiments. (Abstract shortened by ProQuest.).

Structural and functional insights into 5'-ppp RNA pattern recognition by the innate immune receptor RIG-I.

PubMed

Wang, Yanli; Ludwig, Janos; Schuberth, Christine; Goldeck, Marion; Schlee, Martin; Li, Haitao; Juranek, Stefan; Sheng, Gang; Micura, Ronald; Tuschl, Thomas; Hartmann, Gunther; Patel, Dinshaw J

2010-07-01

RIG-I is a cytosolic helicase that senses 5'-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5'-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5'-ppp dsRNA 12-mer, with interactions involving 5'-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5'-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5'-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2'-OCH(3) RNA modifications on the interferon response.

MPP+ analogs acting on mitochondria and inducing neuro-degeneration.

PubMed

Kotake, Y; Ohta, S

2003-12-01

This review focuses on the mechanisms of action and the injurious effect of complex I inhibitors, of which 1-methyl-4-phenylpyridinium ion (MPP(+)) is a well studied example. These compounds can be divided into two groups, i.e. competitive inhibitors with respect to ubiquinone, such as piericidine A, and non-competitive inhibitors such as rotenone. Complex I inhibitors such as MPP(+) have been reported to induce anatomical, behavioral, and biochemical changes similar to those seen in Parkinson's disease, which is characterized by nigrostriatal dopaminergic neuro-degeneration. Spectroscopic analyses and structure-activity relationship studies have indicated that the V-shaped structure of the rotenone molecule is critical for binding to the rotenone binding site on complex I. Many isoquinoline derivatives, some of them endogenous, are also complex I inhibitors. Many lines of evidence show that complex I inhibitors elicit neuronal cell death. Recently, it was reported that chronic and systemic exposure to low-dose rotenone reproduces the features of Parkinson's disease. This work further focused attention on compounds acting on mitochondria, such as MPP(+). In Guadeloupe, the French West Indies, patients with atypical parkinsonism or progressive supranuclear palsy are frequently encountered. These diseases seem to be associated with ingestion of tropical herbal teas or tropical fruits of the Annonaceae family, which contain complex I inhibitors such as benzylisoquinoline derivatives and acetogenins. Complex I inhibitors may not simply result in reactive oxygen species generation or ATP exhaustion, but may influence complex downstream signal transduction processes. An understanding of these changes would throw light on the ways in which complex I inhibitors induce a wide range of abnormalities.

Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.

PubMed

Nakayama, Youhei; Nakajima, Yu; Kato, Naoko; Takai, Hideki; Kim, Dong-Soon; Arai, Masato; Mezawa, Masaru; Araki, Shouta; Sodek, Jaro; Ogata, Yorimasa

2006-08-01

Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.

The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

PubMed Central

Jung, Goeh; Remmert, Kirsten; Wu, Xufeng; Volosky, Joanne M.; III, John A. Hammer

2001-01-01

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker. PMID:11425877

Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera

PubMed Central

1994-01-01

Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose- dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N- formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria. PMID:8270862

Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

NASA Astrophysics Data System (ADS)

Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

2014-01-01

Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

Molecular dynamics studies on troponin (TnI-TnT-TnC) complexes: insight into the regulation of muscle contraction.

PubMed

Varughese, Jayson F; Chalovich, Joseph M; Li, Yumin

2010-10-01

Mutations of any subunit of the troponin complex may lead to serious disorders. Rational approaches to managing these disorders require knowledge of the complex interactions among the three subunits that are required for proper function. Molecular dynamics (MD) simulations were performed for both skeletal (sTn) and cardiac (cTn) troponin. The interactions and correlated motions among the three components of the troponin complex were analyzed using both Molecular Mechanics-Generalized Born Surface Area (MMGBSA) and cross-correlation techniques. The TnTH2 helix was strongly positively correlated with the two long helices of TnI. The C domain of TnC was positively correlated with TnI and TnT. The N domain of TnC was negatively correlated with TnI and TnT in cTn, but not in sTn. The two C-domain calcium-binding sites of TnC were dynamically correlated. The two regulatory N-domain calcium-binding sites of TnC were dynamically correlated, even though the calcium-binding site I is dysfunctional. The strong interaction residue pairs and the strong dynamically correlated residues pairs among the three components of troponin complexes were identified. These correlated motions are consistent with the idea that there is a high degree of cooperativity among the components of the regulatory complex in response to Ca(2+) and other effectors. This approach may give insight into the mechanism by which mutations of troponin cause disease. It is interesting that some observed disease causing mutations fall within regions of troponin that are strongly correlated or interacted.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic Organization of the Chromosome Region Surrounding mecA in Clinical Staphylococcal Strains: Role of IS431-Mediated mecI Deletion in Expression of Resistance in mecA-Carrying, Low-Level Methicillin- Resistant Staphylococcus haemolyticus

PubMed Central

Katayama, Yuki; Ito, Teruyo; Hiramatsu, Keiichi

2001-01-01

We report on the structural diversity of mecA gene complexes carried by 38 methicillin-resistant Staphylococcus aureus and 91 methicillin-resistant coagulase-negative Staphylococcus strains of seven different species with a special reference to its correlation with phenotypic expression of methicillin resistance. The most prevalent and widely disseminated mec complex had the structure mecI-mecR1-mecA-IS431R (or IS431mec), designated the class A mecA gene complex. In contrast, in S. haemolyticus, mecA was bracketed by two copies of IS431, forming the structure IS431L-mecA-IS431R. Of the 38 S. haemolyticus strains, 5 had low-level methicillin resistance (MIC, 1 to 4 mg/liter) and characteristic heterogeneous methicillin resistance as judged by population analysis. In these five strains, IS431L was located to the left of an intact mecI gene, forming the structure IS431L-class A mecA-gene complex. In other S. haemolyticus strains, IS431L was associated with the deletion of mecI and mecR1, forming the structure IS431L-ΔmecR1-mecA-IS431mec, designated the class C mecA gene complex. Mutants with the class C mecA gene complex were obtained in vitro by selecting strain SH621, containing the IS431L-class A mecA gene complex with low concentrations of methicillin (1 and 3 mg/liter). The mutants had intermediate level of methicillin resistance (MIC, 16 to 64 mg/liter). The mecA gene transcription was shown to be derepressed in a representative mutant strain, SH621-37. Our study indicated that the mecI-encoded repressor function is responsible for the low-level methicillin resistance of some S. haemolyticus clinical strains and that the IS431-mediated mecI gene deletion causes the expression of methicillin resistance through the derepression of mecA gene transcription. PMID:11408208

Structure of tropinone reductase-II complexed with NADP+ and pseudotropine at 1.9 A resolution: implication for stereospecific substrate binding and catalysis.

PubMed

Yamashita, A; Kato, H; Wakatsuki, S; Tomizaki, T; Nakatsu, T; Nakajima, K; Hashimoto, T; Yamada, Y; Oda, J

1999-06-15

Tropinone reductase-II (TR-II) catalyzes the NADPH-dependent reduction of the carbonyl group of tropinone to a beta-hydroxyl group. The crystal structure of TR-II complexed with NADP+ and pseudotropine (psi-tropine) has been determined at 1.9 A resolution. A seven-residue peptide near the active site, disordered in the unliganded structure, is fixed in the ternary complex by participation of the cofactor and substrate binding. The psi-tropine molecule is bound in an orientation which satisfies the product configuration and the stereochemical arrangement toward the cofactor. The substrate binding site displays a complementarity to the bound substrate (psi-tropine) in its correct orientation. In addition, electrostatic interactions between the substrate and Glu156 seem to specify the binding position and orientation of the substrate. A comparison between the active sites in TR-II and TR-I shows that they provide different van der Waals surfaces and electrostatic features. These differences likely contribute to the correct binding mode of the substrates, which are in opposite orientations in TR-II and TR-I, and to different reaction stereospecificities. The active site structure in the TR-II ternary complex also suggests that the arrangement of the substrate, cofactor, and catalytic residues is stereoelectronically favorable for the reaction.

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

PubMed

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

Placental Extravillous Cytotrophoblasts Persistently Express Class I Major Histocompatibility Complex Molecules after Human Cytomegalovirus Infection

PubMed Central

Terauchi, Masakazu; Koi, Hideki; Hayano, Chikako; Toyama-Sorimachi, Noriko; Karasuyama, Hajime; Yamanashi, Yuji; Aso, Takeshi; Shirakata, Masaki

2003-01-01

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules. PMID:12857887

Lactoferrin-binding proteins in Shigella flexneri.

PubMed Central

Tigyi, Z; Kishore, A R; Maeland, J A; Forsgren, A; Naidu, A S

1992-01-01

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri. Images PMID:1319403

Structural Basis of Differential Ligand Recognition by Two Classes of bis-(3-5)-cyclic Dimeric Guanosine Monophosphate-binding Riboswitches

DOE Office of Scientific and Technical Information (OSTI.GOV)

K Smith; C Shanahan; E Moore

2011-12-31

The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbonemore » is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.« less

Identification and characterization of Cu(2)O- and ZnO-binding polypeptides by Escherichia coli cell surface display: toward an understanding of metal oxide binding.

PubMed

Thai, Corrine K; Dai, Haixia; Sastry, M S R; Sarikaya, Mehmet; Schwartz, Daniel T; Baneyx, François

2004-07-20

We have used the FliTrx cell surface display system to identify disulfide-constrained dodecapeptides binding to the semiconducting metal oxides Cu(2)O and ZnO. Sequence analysis of the inserts revealed that the two populations exhibit similar, yet subtly different patterns of amino acid usage. Both sets of binders were enriched in arginine, tryptophan, and glycine with a higher degree of positional preference in the case of Cu(2)O binders. Tyrosine, proline, and serine were underrepresented in both populations. Peptides binding electrodeposited Cu(2)O or ZnO with high avidity could be subdivided into two classes based on pI and hydrophilicity. In the hydrophilic and positively charged Class I binders, the Arg-X-X-Arg tetrapeptide appears to be implicated in metal oxide binding, whereas Arg-Arg and Arg-Lys pairs allow for discrimination between Cu(2)O and ZnO. Molecular dynamics simulations of the disulfide-constrained peptides suggest that the aforementioned motifs are important to properly orient two basic residues that are likely to contact the metal oxides. The implications of our results in understanding the rules governing the interaction between peptides and inorganic compounds and in their use for the design of hybrid nanoarchitectures are discussed. Copyright 2004 Wiley Periodicals, Inc.

The major histocompatibility complex of tassel-eared squirrels. II. Genetic diversity associated with Abert squirrels.

PubMed

Wettstein, P J; States, J S

1986-01-01

The extent of polymorphism and the rate of divergence of class I and class II sequences mapping to the mammalian major histocompatibility complex (MHC) have been the subject of experimentation and speculation. To provide further insight into the evolution of the MHC we have initiated the analysis of two geographically isolated subspecies of tassel-eared squirrels. In the preceding communication we described the number and polymorphism of TSLA class I and class II sequences in Kaibab squirrels (S. aberti kaibabensis), which live north of the Grand Canyon. In this report we present a parallel analysis of Abert squirrels (S. aberti aberti), which live south of the Grand Canyon in northern Arizona. Genomic DNA from 12 Abert squirrels was digested with restriction enzymes, electrophoresed, blotted, and hybridized with DR alpha, DR beta, DQ alpha, DQ beta, and HLA-B7 probes. The results of these hybridizations were remarkably similar to those obtained in Kaibab squirrels. The majority of class I and class II bands were identical in size and number, suggesting that Abert and Kaibab squirrels have not significantly diverged in the TSLA complex despite their geographical separation. Relative polymorphism of class II sequences was similar to that observed with Kaibab squirrels: beta sequences exhibited higher polymorphism than alpha sequences. As in Kaibab squirrels, a number of alpha and beta sequences were apparently carried on the same fragments. In comparison to class II beta sequences, there was limited polymorphism in class I sequences, although a diverse number of class I genotypes were observed. Attempts to identify segregating TSLA haplotypes were futile in that the only families of sequences with concordant distributions were DQ alpha and DQ beta. These observations and those obtained with Kaibab squirrels suggest that the present-day TSLA haplotypes of both subspecies are derived from a limited number of common, progenitor haplotypes through repeated intra-TSLA recombination.

Conformational Entropy of FK506 Binding to FKBP12 Determined by Nuclear Magnetic Resonance Relaxation and Molecular Dynamics Simulations.

PubMed

Solomentsev, Gleb; Diehl, Carl; Akke, Mikael

2018-03-06

FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15 N and 2 H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔ S = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔ S = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM ( TΔ S = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.

Use of 2-(/sup 125/I)iodomelatonin to characterize melatonin binding sites in chicken retina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dubocovich, M.L.; Takahashi, J.S.

2-(/sup 125/I)Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-(/sup 125/I)iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-(/sup 125/I)iodomelatonin labeled a single class of sites with an affinity constant (Kd) of 434 +/- 56 pM and a total number of binding sites (Bmax) of 74.0 +/- 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-(/sup 125/I)iodomelatonin binding with a pharmacological ordermore » of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin much greater than N-acetyltryptamine greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-(/sup 125/I)iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of (3H)dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-(/sup 125/I)iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.« less

Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs.

PubMed

Sørensen, Maria Rathmann; Ilsøe, Mette; Strube, Mikael Lenz; Bishop, Richard; Erbs, Gitte; Hartmann, Sofie Bruun; Jungersen, Gregers

2017-01-01

The need for typing of the swine leukocyte antigen (SLA) is increasing with the expanded use of pigs as models for human diseases and organ-transplantation experiments, their use in infection studies, and for design of veterinary vaccines. Knowledge of SLA sequences is furthermore a prerequisite for the prediction of epitope binding in pigs. The low number of known SLA class I alleles and the limited knowledge of their prevalence in different pig breeds emphasizes the need for efficient SLA typing methods. This study utilizes an SLA class I-typing method based on next-generation sequencing of barcoded PCR amplicons. The amplicons were generated with universal primers and predicted to resolve 68-88% of all known SLA class I alleles dependent on amplicon size. We analyzed the SLA profiles of 72 pigs from four different pig populations; Göttingen minipigs and Belgian, Kenyan, and Danish fattening pigs. We identified 67 alleles, nine previously described haplotypes and 15 novel haplotypes. The highest variation in SLA class I profiles was observed in the Danish pigs and the lowest among the Göttingen minipig population, which also have the highest percentage of homozygote individuals. Highlighting the fact that there are still numerous unknown SLA class I alleles to be discovered, a total of 12 novel SLA class I alleles were identified. Overall, we present new information about known and novel alleles and haplotypes and their prevalence in the tested pig populations.

Signature Patterns of MHC Diversity in Three Gombe Communities of Wild Chimpanzees Reflect Fitness in Reproduction and Immune Defense against SIVcpz.

PubMed

Wroblewski, Emily E; Norman, Paul J; Guethlein, Lisbeth A; Rudicell, Rebecca S; Ramirez, Miguel A; Li, Yingying; Hahn, Beatrice H; Pusey, Anne E; Parham, Peter

2015-05-01

Major histocompatibility complex (MHC) class I molecules determine immune responses to viral infections. These polymorphic cell-surface glycoproteins bind peptide antigens, forming ligands for cytotoxic T and natural killer cell receptors. Under pressure from rapidly evolving viruses, hominoid MHC class I molecules also evolve rapidly, becoming diverse and species-specific. Little is known of the impact of infectious disease epidemics on MHC class I variant distributions in human populations, a context in which the chimpanzee is the superior animal model. Population dynamics of the chimpanzees inhabiting Gombe National Park, Tanzania have been studied for over 50 years. This population is infected with SIVcpz, the precursor of human HIV-1. Because HLA-B is the most polymorphic human MHC class I molecule and correlates strongly with HIV-1 progression, we determined sequences for its ortholog, Patr-B, in 125 Gombe chimpanzees. Eleven Patr-B variants were defined, as were their frequencies in Gombe's three communities, changes in frequency with time, and effect of SIVcpz infection. The growing populations of the northern and central communities, where SIVcpz is less prevalent, have stable distributions comprising a majority of low-frequency Patr-B variants and a few high-frequency variants. Driving the latter to high frequency has been the fecundity of immigrants to the northern community, whereas in the central community, it has been the fecundity of socially dominant individuals. In the declining population of the southern community, where greater SIVcpz prevalence is associated with mortality and emigration, Patr-B variant distributions have been changing. Enriched in this community are Patr-B variants that engage with natural killer cell receptors. Elevated among SIVcpz-infected chimpanzees, the Patr-B*06:03 variant has striking structural and functional similarities to HLA-B*57, the human allotype most strongly associated with delayed HIV-1 progression. Like HLA-B*57, Patr-B*06:03 correlates with reduced viral load, as assessed by detection of SIVcpz RNA in feces.

Detection of novel cancer-testis antigen-specific T-cell responses in TIL, regional lymph nodes, and PBL in patients with esophageal squamous cell carcinoma.

PubMed

Mizukami, Yoshiki; Kono, Koji; Daigo, Yataro; Takano, Atsushi; Tsunoda, Takuya; Kawaguchi, Yoshihiko; Nakamura, Yusuke; Fujii, Hideki

2008-07-01

We recently identified three HLA-A2402-restricted epitope peptides derived from cancer-testis antigens (CTA), TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K), and insulin-like growth factor (IGF)-II mRNA binding protein 3 (IMP-3) for the development of immunotherapies against esophageal squamous cell carcinoma (ESCC). In order to evaluate their immunotherapeutic potential in ESCC patients, we estimated by ELISPOT assay the TTK-, LY6K-, or IMP-3-specific T-cell immune responses in tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) expanded from 20HLA-A2402 (+) ESCC patients, and correlated their immune activity with the expression levels of TTK, LY6K, and IMP-3, and MHC class I in the tumors. Induction of TTK-antigen specific T-cell response in TIL to the peptide-pulsed target cells was detected in 14 out of 20 (70%) cases, while LY6K or IMP-3 specific T-cell activity was observed in 11 of 20 (55%) or in eight of 20 (40%) cases, respectively. Furthermore, T-cell activity in RLNL and PBL was detectable in the similar proportion of the 20 ESCC patients. Interestingly, CTA-specific T-cell immune response was found in 13 of 14 (93%) TIL obtained from ESCC tumors with strong MHC class I expression, while it could be observed only in two of six (33%) TIL from ESCC tumors with weak MHC class I expression. These results strongly suggest the pre-existence of specific T-cell responses to HLA-A24-restricted epitope peptides from TTK, LY6K, and IMP-3 in ESCC patients. Monitoring antigen-specific T-cell responses, as well as the expression levels of MHC class I and epitope CTA in tumors, should be a selection index for application of cancer vaccine therapies to the patients who are likely to show good immune response.

Structure of the Deactive State of Mammalian Respiratory Complex I.

PubMed

Blaza, James N; Vinothkumar, Kutti R; Hirst, Judy

2018-02-06

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly active preparation of Bos taurus complex I into the biochemically defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. We show that the deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding to the NADH-reduced enzyme templates their reordering. Our structure both rationalizes biochemical data on the deactive state and offers new insights into its physiological and cellular roles. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Theory and Normal Mode Analysis of Change in Protein Vibrational Dynamics on Ligand Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mortisugu, Kei; Njunda, Brigitte; Smith, Jeremy C

2009-12-01

The change of protein vibrations on ligand binding is of functional and thermodynamic importance. Here, this process is characterized using a simple analytical 'ball-and-spring' model and all-atom normal-mode analysis (NMA) of the binding of the cancer drug, methotrexate (MTX) to its target, dihydrofolate reductase (DHFR). The analytical model predicts that the coupling between protein vibrations and ligand external motion generates entropy-rich, low-frequency vibrations in the complex. This is consistent with the atomistic NMA which reveals vibrational softening in forming the DHFR-MTX complex, a result also in qualitative agreement with neutron-scattering experiments. Energy minimization of the atomistic bound-state (B) structure whilemore » gradually decreasing the ligand interaction to zero allows the generation of a hypothetical 'intermediate' (I) state, without the ligand force field but with a structure similar to that of B. In going from I to B, it is found that the vibrational entropies of both the protein and MTX decrease while the complex structure becomes enthalpically stabilized. However, the relatively weak DHFR:MTX interaction energy results in the net entropy gain arising from coupling between the protein and MTX external motion being larger than the loss of vibrational entropy on complex formation. This, together with the I structure being more flexible than the unbound structure, results in the observed vibrational softening on ligand binding.« less

Evidence for a non-canonical role of HDAC5 in regulation of the cardiac Ncx1 and Bnp genes.

PubMed

Harris, Lillianne G; Wang, Sabina H; Mani, Santhosh K; Kasiganesan, Harinath; Chou, C James; Menick, Donald R

2016-05-05

Class IIa histone deacetylases (HDACs) are very important for tissue specific gene regulation in development and pathology. Because class IIa HDAC catalytic activity is low, their exact molecular roles have not been fully elucidated. Studies have suggested that class IIa HDACs may serve as a scaffold to recruit the catalytically active class I HDAC complexes to their substrate. Here we directly address whether the class IIa HDAC, HDAC5 may function as a scaffold to recruit co-repressor complexes to promoters. We examined two well-characterized cardiac promoters, the sodium calcium exchanger (Ncx1) and the brain natriuretic peptide (Bnp) whose hypertrophic upregulation is mediated by both class I and IIa HDACs. Selective inhibition of class IIa HDACs did not prevent adrenergic stimulated Ncx1 upregulation, however HDAC5 knockout prevented pressure overload induced Ncx1 upregulation. Using the HDAC5((-/-)) mouse we show that HDAC5 is required for the interaction of the HDAC1/2/Sin3a co-repressor complexes with the Nkx2.5 and YY1 transcription factors and critical for recruitment of the HDAC1/Sin3a co-repressor complex to either the Ncx1 or Bnp promoter. Our novel findings support a non-canonical role of class IIa HDACs in the scaffolding of transcriptional regulatory complexes, which may be relevant for therapeutic intervention for pathologies. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of MHC class I genes across horse MHC haplotypes

PubMed Central

Tallmadge, Rebecca L.; Campbell, Julie A.; Miller, Donald C.; Antczak, Douglas F.

2010-01-01

The genomic sequences of 15 horse Major Histocompatibility Complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and non-classical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal, and two to three non-classical sequences. Phylogenetic analysis was applied to these sequences and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The non-classical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine Major Histocompatibility Complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci. PMID:20099063

The role of aromatic phenylalanine residues in binding carotenoid to light-harvesting model and wild-type complexes.

PubMed

García-Martín, A; Pazur, A; Wilhelm, B; Silber, M; Robert, B; Braun, P

2008-09-26

The mode of carotenoid (Crt) binding to polypeptide and specifying its function is as yet largely unknown. Statistical analysis of major photosystems I and II suggests that aromatic residues make up a significant part of the Crt binding pockets. Phenylalanine residues ensure approximately 25%--at some carbon atoms even up to 40%--of the total contacts with Crts. By use of an alanine-leucine model transmembrane helix that replaces the native helix of the bacterial light-harvesting complex 2 (LH2) alpha-subunit, we study the effects of polypeptide residues on cofactor binding in a model sequence context. Here, it is shown that phenylalanine residues located in the close vicinity of the Crts' polyene backbone significantly contribute to the binding of the Crt to the model protein. The replacement of a phenylalanine with leucine in the model helix results in significant reduction in the complexes' Crt content. This effect is strongly enhanced by the removal of a second phenylalanine in close vicinity to the Crt, i.e., of the wild-type (WT) beta-subunit. Remarkably, the mutation of only two phenylalanine residues in the LH2 WT sequence, alpha-Phe at position -12 and beta-Phe at -8, results in the loss of nearly 50% of functional Crt. Resonance Raman spectra indicate that the Crt conformation is fundamentally altered by the absence of the phenylalanines' aromatic side chains, suggesting that they lock the Crt into a precise, well-defined configuration. Thus, binding and specific functionalisation of Crt in the model and WT light-harvesting complex is reliant on the aromatic residue phenylalanine. The use of the light-harvesting complex as a model system thus substantiates the notion that the aromatic residue phenylalanine is a key factor for the binding of Crt to transmembrane proteins.

IFN-gamma priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFNs.

PubMed

Wong, L H; Hatzinisiriou, I; Devenish, R J; Ralph, S J

1998-06-01

IFN-stimulated gene factor 3 (ISGF3) mediates transcriptional activation of IFN-sensitive genes (ISGs). The component subunits of ISGF3, STAT1alphabeta, STAT2, and p48-ISGF3gamma, are tyrosine phosphorylated before their assembly into a complex. Subsequently, the ISGF3 complex is translocated to the nucleus. We have recently established that the responsiveness of human melanoma cell lines to type I IFNs correlates directly with their intracellular levels of ISGF3 components, particularly STAT1. In the present study, we show that pretreating IFN-resistant melanoma cell lines with IFN-gamma (IFN-gamma priming) before stimulation with type I IFN also results in increased levels of ISGF3 components and enhanced DNA-binding activation of ISGF3. In addition, IFN-gamma priming of IFN-resistant melanoma cell lines increased expression of type I IFN-induced ISG products, including ISG54, 2'-5'-oligoadenylate synthase, HLA class I, B7-1, and ICAM-1 Ags. Furthermore, IFN-gamma priming enhanced the antiviral effect of IFN-beta on the IFN-resistant melanoma cell line, MM96. These results support a role for IFN-gamma priming in up-regulating ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma cell lines to type I IFN and providing a molecular basis and justification for using sequential IFN therapy, as proposed by others, to enhance the use of IFNs in the treatment of melanoma.

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model.

PubMed

Balreira, Andrea; Lacerda, Lúcia; Miranda, Clara Sá; Arosa, Fernando A

2005-06-01

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase-deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol-B-epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid-binding molecule CD1d. GluCerase-deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)-class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC-class I. However, CD1d and MHC-class II mRNA levels were not increased. GluCerase-deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC-class II and imbalances in the percentage of CD4+, CD8+, and Valpha24+ T cells. Interestingly, follow-up studies revealed that enzyme replacement therapy induced a decrease in MHC-class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.

Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krantic, S.; Petrovic, V.M.; Quirion, R.

1989-01-01

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using /sup 125/I-(Tyr0-D-Trp8)-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of /sup 125/I-(Tyr0-D-Trp8)-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was notmore » dependent on the animal's life-cycle phase.« less

Androgen Receptor (AR) Suppresses Normal Human Prostate Epithelial Cell Proliferation via AR/β-catenin/TCF-4 Complex Inhibition of c-MYC Transcription

PubMed Central

Antony, Lizamma; van der Schoor, Freek; Dalrymple, Susan L.; Isaacs, John T.

2016-01-01

INTRODUCTION Physiologic testosterone continuously stimulates prostate stromal cell secretion of paracrine growth factors (PGFs), which if unopposed would induce hyperplastic overgrowth of normal prostate epithelial cells (PrECs). METHODS Lentiviral shRNA stable knock down of c-MYC, β-catenin, or TCF-4 completely inhibits normal (i.e., non-transformed) human PrECs growth. c-MYC enhancer driven reporter expression and growth is inhibited by two chemically distinct molecules, which prevent β-catenin signaling either by blocking TCF-4 binding (i.e., toxoflavin) or by stimulating degradation (i.e., AVX939). Recombinant DKK1 protein at a dose, which inhibits activation of canonical Wnt signaling does not inhibit PrEC growth. Nuclear β-catenin translocation and PrEC growth is prevented by both lack of PGFs or Akt inhibitor-I. Growth inhibition induced by lack of PGFs, toxoflavin, or Akt inhibitor-I is overcome by constitutive c-MYC transcription. RESULTS In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen binding to AR suppressing c-MYC transcription, resulting in G0 arrest/terminal differentiation independent of Rb, p21, p27, FoxP3, or down regulation of growth factors receptors and instead involves androgen-induced formation of AR/β-catenin/TCF-4 complexes, which suppress c-MYC transcription. Such suppression does not occur when AR is mutated in its zinc-finger binding domain. DISCUSSION Proliferation of non-transformed human PrECs is dependent upon c-MYC transcription via formation/binding of β-catenin/TCF-4 complexes at both 5′ and 3′ c-MYC enhancers stimulated by Wnt-independent, PGF induced Akt signaling. In the presence of continuous PGF signaling, PrEC hyperplasia is prevented by androgen-induced formation of AR/β-catenin/TCF-4 complexes, which retains binding to 3′ c-MYC enhancer, but now suppresses c-MYC transcription. PMID:24913829

Protein-protein interfaces are vdW dominant with selective H-bonds and (or) electrostatics towards broad functional specificity.

PubMed

Nilofer, Christina; Sukhwal, Anshul; Mohanapriya, Arumugam; Kangueane, Pandjassarame

2017-01-01

Several catalysis, cellular regulation, immune function, cell wall assembly, transport, signaling and inhibition occur through Protein- Protein Interactions (PPI). This is possible with the formation of specific yet stable protein-protein interfaces. Therefore, it is of interest to understand its molecular principles using structural data in relation to known function. Several interface features have been documented using known X-ray structures of protein complexes since 1975. This has improved our understanding of the interface using structural features such as interface area, binding energy, hydrophobicity, relative hydrophobicity, salt bridges and hydrogen bonds. The strength of binding between two proteins is dependent on interface size (number of residues at the interface) and thus its corresponding interface area. It is known that large interfaces have high binding energy (sum of (van der Waals) vdW, H-bonds, electrostatics). However, the selective role played by each of these energy components and more especially that of vdW is not explicitly known. Therefore, it is important to document their individual role in known protein-protein structural complexes. It is of interest to relate interface size with vdW, H-bonds and electrostatic interactions at the interfaces of protein structural complexes with known function using statistical and multiple linear regression analysis methods to identify the prominent force. We used the manually curated non-redundant dataset of 278 hetero-dimeric protein structural complexes grouped using known functions by Sowmya et al. (2015) to gain additional insight to this phenomenon using a robust inter-atomic non-covalent interaction analyzing tool PPCheck (Anshul and Sowdhamini, 2015). This dataset consists of obligatory (enzymes, regulator, biological assembly), immune and nonobligatory (enzyme and regulator inhibitors) complexes. Results show that the total binding energy is more for large interfaces. However, this is not true for its individual energy factors. Analysis shows that vdW energies contribute to about 75% ± 11% on average among all complexes and it also increases with interface size (r2 ranging from 0.67 to 0.89 with p<0.01) at 95% confidence limit irrespective of molecular function. Thus, vdW is both dominant and proportional at the interface independent of molecular function. Nevertheless, H bond energy contributes to 15% ± 6.5% on average in these complexes. It also moderately increases with interface size (r2 ranging from 0.43 to 0.61 with p<0.01) only among obligatory and immune complexes. Moreover, there is about 11.3% ± 8.7% contribution by electrostatic energy. It increases with interface size specifically among non-obligatory regulator-inhibitors (r2 = 0.44). It is implied that both H-bonds and electrostatics are neither dominant nor proportional at the interface. Nonetheless, their presence cannot be ignored in binding. Therefore, H-bonds and (or) electrostatic energy having specific role for improved stability in complexes is implied. Thus, vdW is common at the interface stabilized further with selective H-bonds and (or) electrostatic interactions at an atomic level in almost all complexes. Comparison of this observation with residue level analysis of the interface is compelling. The role by H-bonds (14.83% ± 6.5% and r2 = 0.61 with p<0.01) among obligatory and electrostatic energy (8.8% ± 4.77% and r2 = 0.63 with p <0.01) among non-obligatory complexes within interfaces (class A) having more non-polar residues than surface is influencing our inference. However, interfaces (class B) having less non-polar residues than surface show 1.5 fold more electrostatic energy on average. The interpretation of the interface using inter-atomic (vdW, H-bonds, electrostatic) interactions combined with inter-residue predominance (class A and class B) in relation to known function is the key to reveal its molecular principles with new challenges.

The major histocompatibility complex class Ib molecule HLA-E at the interface between innate and adaptive immunity.

PubMed

Sullivan, L C; Clements, C S; Rossjohn, J; Brooks, A G

2008-11-01

The non-classical major histocompatibility complex (MHC) class I molecule human leucocyte antigen (HLA)-E is the least polymorphic of all the MHC class I molecules and acts as a ligand for receptors of both the innate and the adaptive immune systems. The recognition of self-peptides complexed to HLA-E by the CD94-NKG2A receptor expressed by natural killer (NK) cells represents a crucial checkpoint for immune surveillance by NK cells. However, HLA-E can also be recognised by the T-cell receptor expressed by alphabeta CD8 T cells and therefore can play a role in the adaptive immune response to invading pathogens. The recent resolution of HLA-E in complex with both innate and adaptive ligands has provided insight into the dual role of this molecule in immunity.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes

PubMed Central

2017-01-01

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process. PMID:29290109

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

NASA Astrophysics Data System (ADS)

Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-02-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

PubMed Central

Man-Kupisinska, Aleksandra; Michalski, Mateusz; Maciejewska, Anna; Swierzko, Anna S.; Cedzynski, Maciej; Lugowski, Czeslaw; Lukasiewicz, Jolanta

2016-01-01

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3. PMID:27232184

Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2‧-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity

NASA Astrophysics Data System (ADS)

Hasan, Md. Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

2016-01-01

Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2 = 3-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2 = 4-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy = 2,2‧-bipyridine, Phen = 1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH ∼ 7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4 > 3 > 1 > 2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4‧,6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87 μM respectively.

Demonstration of a specific C3a receptor on guinea pig platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuoka, Y.; Hugli, T.E.

1988-05-15

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 xmore » 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin.« less

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

PubMed

Wentsch, Heike K; Walter, Niklas M; Bührmann, Mike; Mayer-Wrangowski, Svenja; Rauh, Daniel; Zaman, Guido J R; Willemsen-Seegers, Nicole; Buijsman, Rogier C; Henning, Melanie; Dauch, Daniel; Zender, Lars; Laufer, Stefan

2017-05-02

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Parkin-phosphoubiquitin complex reveals a cryptic ubiquitin binding site required for RBR ligase activity

PubMed Central

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-01-01

RING-BETWEENRING-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR is Parkin, mutations in which lead to early onset hereditary Parkinsonism. Parkin and other RBRs share a catalytic RBR module, but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during activation of Parkin. However, current data on active RBRs are in the absence of regulatory domains. Therefore, how individual RBRs are activated, and whether they share a common mechanism remains unclear. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces a movement in the IBR domain to reveal a cryptic ubiquitin binding site. Mutation of this site negatively impacts on Parkin’s activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, suggesting a role for interdomain association in RBR ligase mechanism. PMID:28414322

Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

PubMed

Kumar, Atul; Chaugule, Viduth K; Condos, Tara E C; Barber, Kathryn R; Johnson, Clare; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Knebel, Axel; Shaw, Gary S; Walden, Helen

2017-05-01

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism.

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

PubMed

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Booe, Jason M.; Walker, Christopher S.; Barwell, James

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

DOE PAGES

Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...

2015-05-14

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases.

PubMed

Triki, Dhoha; Billot, Telli; Visseaux, Benoit; Descamps, Diane; Flatters, Delphine; Camproux, Anne-Claude; Regad, Leslie

2018-04-10

HIV-2 protease (PR2) is naturally resistant to most FDA (Food and Drug Administration)-approved HIV-1 protease inhibitors (PIs), a major antiretroviral class. In this study, we compared the PR1 and PR2 binding pockets extracted from structures complexed with 12 ligands. The comparison of PR1 and PR2 pocket properties showed that bound PR2 pockets were more hydrophobic with more oxygen atoms and fewer nitrogen atoms than PR1 pockets. The structural comparison of PR1 and PR2 pockets highlighted structural changes induced by their sequence variations and that were consistent with these property changes. Specifically, substitutions at residues 31, 46, and 82 induced structural changes in their main-chain atoms that could affect PI binding in PR2. In addition, the modelling of PR1 mutant structures containing V32I and L76M substitutions revealed a cooperative mechanism leading to structural deformation of flap-residue 45 that could modify PR2 flexibility. Our results suggest that substitutions in the PR1 and PR2 pockets can modify PI binding and flap flexibility, which could underlie PR2 resistance against PIs. These results provide new insights concerning the structural changes induced by PR1 and PR2 pocket variation changes, improving the understanding of the atomic mechanism of PR2 resistance to PIs.

The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase.

PubMed Central

Thomson, G J; Howlett, G J; Ashcroft, A E; Berry, A

1998-01-01

The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies. PMID:9531482

Quantitative HDL Proteomics Identifies Peroxiredoxin-6 as a Biomarker of Human Abdominal Aortic Aneurysm

PubMed Central

Burillo, Elena; Jorge, Inmaculada; Martínez-López, Diego; Camafeita, Emilio; Blanco-Colio, Luis Miguel; Trevisan-Herraz, Marco; Ezkurdia, Iakes; Egido, Jesús; Michel, Jean-Baptiste; Meilhac, Olivier; Vázquez, Jesús; Martin-Ventura, Jose Luis

2016-01-01

High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas α-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA. PMID:27934969

Structural basis of semaphorin–plexin signalling

PubMed Central

Janssen, Bert J. C.; Robinson, Ross A.; Pérez-Brangulí, Francesc; Bell, Christian H.; Mitchell, Kevin J.; Siebold, Christian; Jones, E. Yvonne

2013-01-01

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses1. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively2,3. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB11–2–SEMA4Decto and murine PlxnA21–4–Sema6Aecto), plus unliganded structures of PlxnA21–4 and Sema6Aecto. These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed β-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin–plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity. PMID:20877282

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, M.; Allison, W.S.

1986-05-05

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with (/sup 3/H)ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. (/sup 3/H)ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with (/sup 3/H)ADP in 30more » min with a Kd of 30 microM. (/sup 3/H)ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of (/sup 3/H)ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. (/sup 3/H)ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits.« less

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PubMed

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Nutrient-dependent increased dendritic arborization of somatosensory neurons.

PubMed

Watanabe, Kaori; Furumizo, Yuki; Usui, Tadao; Hattori, Yukako; Uemura, Tadashi

2017-01-01

Suboptimal nutrition imposes developmental constraints on infant animals, which marshal adaptive responses to eventually become mature adults. Such responses are mounted at multiple levels from systemic to cellular. At the cellular level, the underlying mechanisms of cell proliferation control have been intensively studied. However, less is known about how growth of postmitotic and morphologically complex cells, such as neurons, is controlled by nutritional status. We address this question using Class I and Class IV dendritic arborization neurons in Drosophila larvae. Class IV neurons have been shown to sense nociceptive thermal, mechanical and light stimuli, whereas Class I neurons are proprioceptors. We reared larvae on diets with different protein and carbohydrate content throughout larval stages and examined how morphologies of Class I or Class IV neurons were affected. Dendritic arbors of Class IV neurons became more complex when larvae were reared on a low-yeast diet, which contains lower amounts of amino acids and other ingredients, compared to a high-yeast diet. In contrast, such low-yeast-dependent hyperarborization was not seen in Class I neurons. The physiological and metabolic implications of the hyperarborization phenotype are discussed in relation to a recent hypothesis that Class IV neurons sense protein-deficient stress and to our characterization of how the dietary yeast contents impacted larval metabolism. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Pharmacological evaluation of an [(123)I] labelled imidazopyridine-3-acetamide for the study of benzodiazepine receptors.

PubMed

Mattner, Filomena; Mardon, Karine; Loc'h, Christian; Katsifis, Andrew

2006-06-13

In vitro binding of the iodinated imidazopyridine, N',N'-dimethyl-6-methyl-(4'-[(123)I]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide [(123)I]IZOL to benzodiazepine binding sites on brain cortex, adrenal and kidney membranes is reported. Saturation experiments showed that [(123)I]IZOL, bound to a single class of binding site (n(H)=0.99) on adrenal and kidney mitochondrial membranes with a moderate affinity (K(d)=30 nM). The density of binding sites was 22+/-6 and 1.2+/-0.4 pmol/mg protein on adrenal and kidney membranes, respectively. No specific binding was observed in mitochondrial-synaptosomal membranes of brain cortex. In biodistribution studies in rats, the highest uptake of [(123)I]IZOL was found 30 min post injection in adrenals (7.5% ID/g), followed by heart, kidney, lung (1% ID/g) and brain (0.12% ID/g), consistent with the distribution of peripheral benzodiazepine binding sites. Pre-administration of unlabelled IZOL and the specific PBBS drugs, PK 11195 and Ro 5-4864 significantly reduced the uptake of [(123)I]IZOL by 30% (p<0.05) in olfactory bulbs and by 51-86% (p<0.01) in kidney, lungs, heart and adrenals, while it increased by 30% to 50% (p<0.01) in the rest of the brain and the blood. Diazepam, a mixed CBR-PBBS drug, inhibited the uptake in kidney, lungs, heart, adrenals and olfactory bulbs by 32% to 44% (p<0.01) but with no effect on brain uptake and in blood concentration. Flumazenil, a central benzodiazepine drug and haloperidol (dopamine antagonist/sigma receptor drug) displayed no effect in [(123)I]IZOL in peripheral organs and in the brain. [(123)I]IZOL may deserve further development for imaging selectively peripheral benzodiazepine binding sites.

Structural Basis of Chemokine Sequestration by CrmD, a Poxvirus-Encoded Tumor Necrosis Factor Receptor

PubMed Central

Wang, Dongli; Chen, Dongwei; He, Guangjun; Huang, Li; Wang, Hanzhong; Wang, Xinquan

2011-01-01

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions. PMID:21829356

Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module.

PubMed

Wojciechowski, Michał; Różycki, Bartosz; Huy, Pham Dinh Quoc; Li, Mai Suan; Bayer, Edward A; Cieplak, Marek

2018-03-22

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

Selector function of MHC I molecules is determined by protein plasticity

NASA Astrophysics Data System (ADS)

Bailey, Alistair; Dalchau, Neil; Carter, Rachel; Emmott, Stephen; Phillips, Andrew; Werner, Jörn M.; Elliott, Tim

2015-10-01

The selection of peptides for presentation at the surface of most nucleated cells by major histocompatibility complex class I molecules (MHC I) is crucial to the immune response in vertebrates. However, the mechanisms of the rapid selection of high affinity peptides by MHC I from amongst thousands of mostly low affinity peptides are not well understood. We developed computational systems models encoding distinct mechanistic hypotheses for two molecules, HLA-B*44:02 (B*4402) and HLA-B*44:05 (B*4405), which differ by a single residue yet lie at opposite ends of the spectrum in their intrinsic ability to select high affinity peptides. We used in vivo biochemical data to infer that a conformational intermediate of MHC I is significant for peptide selection. We used molecular dynamics simulations to show that peptide selector function correlates with protein plasticity, and confirmed this experimentally by altering the plasticity of MHC I with a single point mutation, which altered in vivo selector function in a predictable way. Finally, we investigated the mechanisms by which the co-factor tapasin influences MHC I plasticity. We propose that tapasin modulates MHC I plasticity by dynamically coupling the peptide binding region and α3 domain of MHC I allosterically, resulting in enhanced peptide selector function.

Fine-mapping classical HLA variation associated with durable host control of HIV-1 infection in African Americans.

PubMed

McLaren, Paul J; Ripke, Stephan; Pelak, Kimberly; Weintrob, Amy C; Patsopoulos, Nikolaos A; Jia, Xiaoming; Erlich, Rachel L; Lennon, Niall J; Kadie, Carl M; Heckerman, David; Gupta, Namrata; Haas, David W; Deeks, Steven G; Pereyra, Florencia; Walker, Bruce D; de Bakker, Paul I W

2012-10-01

A small proportion of human immunodeficiency virus-1 (HIV-1) infected individuals, termed HIV-1 controllers, suppress viral replication to very low levels in the absence of therapy. Genetic investigations of this phenotype have strongly implicated variation in the class I major histocompatibility complex (MHC) region as key to HIV-1 control. We collected sequence-based classical class I HLA genotypes at 4-digit resolution in HIV-1-infected African American controllers and progressors (n = 1107), and tested them for association with host control using genome-wide single nucleotide polymorphism data to account for population structure. Several classical alleles at HLA-B were associated with host control, including B*57:03 [odds ratio (OR) = 5.1; P= 3.4 × 10(-18)] and B*81:01 (OR = 4.8; P= 1.3 × 10(-9)). Analysis of variable amino acid positions demonstrates that HLA-B position 97 is the most significant association with host control in African Americans (omnibus P = 1.2 × 10(-21)) and explains the signal of several HLA-B alleles, including B*57:03. Within HLA-B, we also identified independent effects at position 116 (omnibus P= 2.8 × 10(-15)) in the canonical F pocket, position 63 in the B pocket (P= 1.5 × 10(-3)) and the non-pocket position 245 (P= 8.8 × 10(-10)), which is thought to influence CD8-binding kinetics. Adjusting for these HLA-B effects, there is evidence for residual association in the MHC region. These results underscore the key role of HLA-B in affecting HIV-1 replication, likely through the molecular interaction between HLA-B and viral peptides presented by infected cells, and suggest that sites outside the peptide-binding pocket also influence HIV-1 control.

TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

PubMed Central

Neerincx, Andreas; Hermann, Clemens; Antrobus, Robin; van Hateren, Andy; Cao, Huan; Trautwein, Nico; Stevanović, Stefan; Elliott, Tim; Deane, Janet E; Boyle, Louise H

2017-01-01

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex. DOI: http://dx.doi.org/10.7554/eLife.23049.001 PMID:28425917

Class I TCP-DELLA interactions in inflorescence shoot apex determine plant height.

PubMed

Davière, Jean-Michel; Wild, Michael; Regnault, Thomas; Baumberger, Nicolas; Eisler, Herfried; Genschik, Pascal; Achard, Patrick

2014-08-18

Regulation of plant height, one of the most important agronomic traits, is the focus of intensive research for improving crop performance. Stem elongation takes place as a result of repeated cell divisions and subsequent elongation of cells produced by apical and intercalary meristems. The gibberellin (GA) phytohormones have long been known to control stem and internodal elongation by stimulating the degradation of nuclear growth-repressing DELLA proteins; however, the mechanism allowing GA-responsive growth is only slowly emerging. Here, we show that DELLAs directly regulate the activity of the plant-specific class I TCP transcription factor family, key regulators of cell proliferation. Our results demonstrate that class I TCP factors directly bind the promoters of core cell-cycle genes in Arabidopsis inflorescence shoot apices while DELLAs block TCP function by binding to their DNA-recognition domain. GAs antagonize such repression by promoting DELLA destruction and therefore cause a concomitant accumulation of TCP factors on promoters of cell-cycle genes. Consistent with this model, the quadruple mutant tcp8 tcp14 tcp15 tcp22 exhibits severe dwarfism and reduced responsiveness to GA action. Altogether, we conclude that GA-regulated DELLA-TCP interactions in inflorescence shoot apex provide a novel mechanism to control plant height. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure based design of 11β-HSD1 inhibitors.

PubMed

Singh, Suresh; Tice, Colin

2010-11-01

Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling.

PubMed

Wu, Jia Wei; Krawitz, Ariel R; Chai, Jijie; Li, Wenyu; Zhang, Fangjiu; Luo, Kunxin; Shi, Yigong

2002-11-01

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.

Rhenium(i) complexes of N-heterocyclic carbene ligands that bind to amyloid plaques of Alzheimer's disease.

PubMed

Chan, Chung Ying; Noor, Asif; McLean, Catriona A; Donnelly, Paul S; Barnard, Peter J

2017-02-16

A series of [Re(i)L(CO) 3 ] + complexes (where L is a bifunctional bis(NHC)-amine ligand) that are analogues of potential Tc-99m diagnostic imaging agents for Alzheimer's disease have been synthesised. One of the complexes bound to amyloid plaques in human frontal cortex brain tissue from subjects with Alzheimer's disease.

CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode.

PubMed

Taneva, Svetla G; Patty, Philipus J; Frisken, Barbara J; Cornell, Rosemary B

2005-07-05

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.

The EED protein–protein interaction inhibitor A-395 inactivates the PRC2 complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Yupeng; Selvaraju, Sujatha; Curtin, Michael L.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed inmore » vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein–protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.« less