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Sample records for binding domain protein

  1. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  2. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  3. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  4. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  5. Fused protein domains inhibit DNA binding by LexA.

    PubMed Central

    Golemis, E A; Brent, R

    1992-01-01

    Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions. Images PMID:1620111

  6. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  7. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  8. Targeting the inhibitor of Apoptosis Protein BIR3 binding domains.

    PubMed

    Jaquith, James B

    2014-05-01

    The Inhibitor of Apoptosis Proteins (IAPs) play a critical role in the regulation of cellular apoptosis and cytokine signaling. IAP family members include XIAP, cIAP1, cIAP2, NAIP, survivin, Apollon/Bruce, ML-IAP/livin and TIAP. The IAPs have been targeted using both antisense oligonucleotides and small molecule inhibitors. Several research teams have advanced compounds that bind the highly conserved BIR3 domains of the IAPs into clinical trials, as single agents and in combination with standard of care. This patent review highlights the medicinal chemistry strategies that have been applied to the development of clinical compounds. PMID:24998289

  9. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    PubMed

    Khan, Waqasuddin; Duffy, Fergal; Pollastri, Gianluca; Shields, Denis C; Mooney, Catherine

    2013-01-01

    Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif) containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58).Next, we trained a bidirectional recurrent neural network (BRNN) using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72) showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods) clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors. PMID:24019881

  10. Control of domain swapping in bovine odorant-binding protein.

    PubMed Central

    Ramoni, Roberto; Vincent, Florence; Ashcroft, Alison E; Accornero, Paolo; Grolli, Stefano; Valencia, Christel; Tegoni, Mariella; Cambillau, Christian

    2002-01-01

    As revealed by the X-ray structure, bovine odorant-binding protein (OBPb) is a domain swapped dimer [Tegoni, Ramoni, Bignetti, Spinelli and Cambillau (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, Bains, Petosi, Pevsner, Snyder, Monaco and Amzel (1996) Nat. Struct. Biol. 3, 934-939]. This contrasts with all known mammalian OBPs, which are monomers, and in particular with porcine OBP (OBPp), sharing 42.3% identity with OBPb. By the mechanism of domain swapping, monomers are proposed to evolve into dimers and oligomers, as observed in human prion. Comparison of bovine and porcine OBP sequences pointed at OBPp glycine 121, in the hinge linking the beta-barrel to the alpha-helix. The absence of this residue in OBPb might explain why the normal lipocalin beta-turn is not formed. In order to decipher the domain swapping determinants we have produced a mutant of OBPb in which a glycine residue was inserted after position 121, and a mutant of OBPp in which glycine 121 was deleted. The latter mutation did not result in dimerization, while OBPb-121Gly+ became monomeric, suggesting that domain swapping was reversed. Careful structural analysis revealed that besides the presence of a glycine in the hinge, the dimer interface formed by the C-termini and by the presence of the lipocalins conserved disulphide bridge may also control domain swapping. PMID:11931632

  11. NMR Solution Structure and DNA Binding Model of the DNA Binding Domain of Competence Protein A

    PubMed Central

    Hobbs, Carey A.; Bobay, Benjamin G.; Thompson, Richele J.; Perego, Marta; Cavanagh, John

    2010-01-01

    Competence protein A (ComA) is a response regulator protein involved in the development of genetic competence in the Gram-positive spore forming bacterium Bacillus subtilis, as well as the regulation of the production of degradative enzymes and antibiotic synthesis. ComA belongs to the NarL family of proteins which are characterized by a C-terminal transcriptional activator domain that consists of a bundle of four helices, where the second and third helices (α8 and α9) form a helix-turn-helix DNA binding domain. Using NMR spectroscopy, the high resolution three-dimensional solution structure of the C-terminal DNA-binding domain of ComA (ComAC) has been determined. In addition, surface plasmon resonance and NMR protein-DNA titration experiments allowed for the analysis of the interaction of ComAC with its target DNA sequences. Combining the solution structure and biochemical data, a model of ComAC bound to the ComA recognition sequences on the srfA promoter has been developed. The model shows that for DNA binding, ComA uses the conserved helix-turn-helix motif present in other NarL family members. However, the model also reveals that ComA may use a slightly different part of the helix-turn-helix motif and there appears to be some associated domain re-orientation. These observations suggest a basis for DNA binding specificity within the NarL family. PMID:20302877

  12. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.

    PubMed

    Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H; Van Etten, Jamie; Goldstrohm, Aaron C

    2014-08-01

    PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation. PMID:24942623

  13. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  14. Ligand binding to the PDZ domains of postsynaptic density protein 95.

    PubMed

    Toto, Angelo; Pedersen, Søren W; Karlsson, O Andreas; Moran, Griffin E; Andersson, Eva; Chi, Celestine N; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2016-05-01

    Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i)N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain. PMID:26941280

  15. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

    PubMed

    Pearson, M A; Reczek, D; Bretscher, A; Karplus, P A

    2000-04-28

    The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin. PMID:10847681

  16. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

    PubMed Central

    Leberer, E; Wu, C; Leeuw, T; Fourest-Lieuvin, A; Segall, J E; Thomas, D Y

    1997-01-01

    Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins. PMID:9009270

  17. Calmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis.

    PubMed

    O'Day, Danton H; Myre, Michael A

    2004-08-01

    The calcium hypothesis of Alzheimer's disease (AD) invokes the disruption of calcium signaling as the underlying cause of neuronal dysfunction and ultimately apoptosis. As a primary calcium signal transducer, calmodulin (CaM) responds to cytosolic calcium fluxes by binding to and regulating the activity of target CaM-binding proteins (CaMBPs). Ca(2+)-dependent CaMBPs primarily contain domains (CaMBDs) that can be classified into motifs based upon variations on the basic amphiphilic alpha-helix domain involving conserved hydrophobic residues at positions 1-10, 1-14 or 1-16. In contrast, an IQ or IQ-like domain often mediates Ca(2+)-independent CaM-binding. Based on these attributes, a search for CaMBDs reveals that many of the proteins intimately linked to AD may be calmodulin-binding proteins, opening new avenues for research on this devastating disease. PMID:15249195

  18. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    SciTech Connect

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  19. Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

    PubMed

    Pessler, Frank; Hernandez, Nouria

    2003-08-01

    POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat. PMID:12750370

  20. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

    SciTech Connect

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M.F.; Downes, C. Peter; Batty, Ian H.

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

  1. IQGAP proteins reveal an atypical phosphoinositide (aPI) binding domain with a pseudo C2 domain fold.

    PubMed

    Dixon, Miles J; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R; van Aalten, Daan M F; Downes, C Peter; Batty, Ian H

    2012-06-29

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

  2. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.

    PubMed Central

    Dowler, S; Currie , R A; Campbell , D G; Deak, M; Kular, G; Downes, C P; Alessi, D R

    2000-01-01

    The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s). PMID:11001876

  3. Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

    PubMed Central

    Lobingier, Braden T.; Merz, Alexey J.

    2012-01-01

    Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal Habc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function. PMID:23051737

  4. Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

    PubMed Central

    Mi, Michael Y; Zhang, Jiying; He, Yukai

    2005-01-01

    Background A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells. Results We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays. Conclusion Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs. PMID:16293193

  5. A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

    PubMed Central

    Glanzer, Jason G.; Carnes, Katie A.; Soto, Patricia; Liu, Shengqin; Parkhurst, Lawrence J.; Oakley, Gregory G.

    2013-01-01

    Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53–RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome. PMID:23267009

  6. ADAR Proteins: Double-stranded RNA and Z-DNA Binding Domains

    PubMed Central

    Barraud, Pierre; Allain, Frédéric H.-T

    2012-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine editing within double-stranded RNA (dsRNA) substrates. Inosine is read as a guanine by most cellular processes and therefore these changes create codons for a different amino acid, stop codons or even a new splice-site allowing protein diversity generated from a single gene. We are reviewing here the current structural and molecular knowledge on RNA editing by the ADAR family of protein. We focus especially on two types of nucleic acid binding domains present in ADARs, namely the double-stranded RNA and Z-DNA binding domains. PMID:21728134

  7. Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization

    PubMed Central

    Vinogradova, Maia V.; Malanina, Galina G.; Waitzman, Joshua S.; Rice, Sarah E.; Fletterick, Robert J.

    2013-01-01

    Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca2+-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca2+ signaling since Ca2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface. PMID:23805258

  8. Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization.

    PubMed

    Vinogradova, Maia V; Malanina, Galina G; Waitzman, Joshua S; Rice, Sarah E; Fletterick, Robert J

    2013-01-01

    Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+)-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface. PMID:23805258

  9. Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein.

    PubMed

    Krois, Alexander S; Ferreon, Josephine C; Martinez-Yamout, Maria A; Dyson, H Jane; Wright, Peter E

    2016-03-29

    An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2-p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1-p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the full-length p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stress-related posttranslational modification. PMID:26976603

  10. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    SciTech Connect

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N.

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  11. Engineered staphylococcal protein A's IgG-binding domain with cathepsin L inhibitory activity

    SciTech Connect

    Bratkovic, Tomaz . E-mail: tomaz.bratkovic@ffa.uni-lj.si; Berlec, Ales; Popovic, Tatjana; Lunder, Mojca; Kreft, Samo; Urleb, Uros; Strukelj, Borut

    2006-10-13

    Inhibitory peptide of papain-like cysteine proteases, affinity selected from a random disulfide constrained phage-displayed peptide library, was grafted to staphylococcal protein A's B domain. Scaffold protein was additionally modified in order to allow solvent exposed display of peptide loop. Correct folding of fusion proteins was confirmed by CD-spectroscopy and by the ability to bind the Fc-region of rabbit IgG, a characteristic of parent domain. The recombinant constructs inhibited cathepsin L with inhibitory constants in the low-micromolar range.

  12. Kits and methods of detection using cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.

    1998-04-14

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  13. Kits and methods of detection using cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  14. Retinol Binding Protein-Albumin Domain III Fusion Protein Deactivates Hepatic Stellate Cells

    PubMed Central

    Park, Sangeun; Choi, Soyoung; Lee, Min-Goo; Lim, Chaeseung; Oh, Junseo

    2012-01-01

    Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis. PMID:23161170

  15. A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein.

    PubMed

    Watanabe, T; Sukegawa, J; Sukegawa, I; Tomita, S; Iijima, K; Oguchi, S; Suzuki, T; Nairn, A C; Greengard, P

    1999-02-01

    Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP. PMID:9930726

  16. Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction.

    PubMed

    Low, Diana Hooi Ping; Frecer, Vladimir; Le Saux, Agnès; Srinivasan, Ganesh Anand; Ho, Bow; Chen, Jianzhu; Ding, Jeak Ling

    2010-03-26

    Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human. PMID:20118243

  17. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    NASA Technical Reports Server (NTRS)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  18. LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

    SciTech Connect

    Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike; Schwartz, Thomas U.

    2012-08-31

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

  19. Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus.

    PubMed

    Enemark, E J; Chen, G; Vaughn, D E; Stenlund, A; Joshua-Tor, L

    2000-07-01

    Papillomaviral infection causes both benign and malignant lesions and is a necessary cause of cervical carcinoma. Replication of this virus requires the replication initiation proteins E1 and E2, which bind cooperatively at the origin of replication (ori) as an (E1)2-(E2)2-DNA complex. This is a precursor to larger E1 complexes that distort and unwind the ori. We present the crystal structure of the E1 DNA binding domain refined to 1.9 A resolution. Residues critical for DNA binding are located on an extended loop and an alpha helix. We identify the E1 dimerization surface by selective mutations at an E1/E1 interface observed in the crystal and propose a model for the (E1)2-DNA complex. These and other observations suggest how the E1 DNA binding domain orchestrates assembly of the hexameric helicase on the ori. PMID:10949036

  20. Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins.

    PubMed Central

    Carson, M R; Welsh, M J

    1995-01-01

    The opening and closing of the CFTR Cl- channel are regulated by ATP hydrolysis at its two nucleotide binding domains (NBDs). However, the mechanism and functional significance of ATP hydrolysis are unknown. Sequence similarity between the NBDs of CFTR and GTP-binding proteins suggested the NBDs might have a structure and perhaps a function like that of GTP-binding proteins. Based on this similarity, we predicted that the terminal residue of the LSGGQ motif in the NBDs of CFTR corresponds to a highly conserved glutamine residue in GTP-binding proteins that directly catalyzes the GTPase reaction. Mutations of this residue in NBD1 or NBD2, which were predicted to increase or decrease the rate of hydrolysis, altered the duration of channel closed and open times in a specific manner without altering ion conduction properties or ADP-dependent inhibition. These results suggest that the NBDs of CFTR, and consequently other ABC transporters, may have a structure and a function analogous to those of GTP-binding proteins. We conclude that the rates of ATP hydrolysis at NBD1 and at NBD2 determine the duration of the two states of the channel, closed and open, much as the rate of GTP hydrolysis by GTP-binding proteins determines the duration of their active state. Images FIGURE 3 FIGURE 4 PMID:8599650

  1. The calmodulin-binding domain of the mouse 90-kDa heat shock protein.

    PubMed

    Minami, Y; Kawasaki, H; Suzuki, K; Yahara, I

    1993-05-01

    The mouse 90-kDa heat shock protein (HSP90) and Ca(2+)-calmodulin were cross-linked at an equimolar ratio using a carbodiimide zero-length cross-linker. To identify the calmodulin-binding domain(s) of HSP90, CNBr-cleaved peptide fragments of HSP90 were mixed with Ca(2+)-calmodulin and cross-linked. Amino acid sequence determination revealed that an HSP90 alpha-derived peptide starting at the 486th amino acid residue was contained in the cross-linked products, which contains a calmodulin-binding motif (from Lys500 to Ile520). A similar motif is present also in HSP90 beta (from Lys491 to Val511). The synthetic peptides corresponding to these putative calmodulin-binding sequences were found to be cross-linked with Ca(2+)-calmodulin and to prevent the cross-linking of HSP90 and Ca(2+)-calmodulin. Both HSP90 alpha and HSP90 beta bind Ca2+. The HSP90 peptides bind HSP90 and thereby inhibit the binding of Ca2+. In addition, the HSP90 peptides augment the self-oligomerization of HSP90 induced at elevated temperatures. These results suggest that the calmodulin-binding domain of HSP90 might interact with another part of the same molecule and that Ca(2+)-calmodulin might modulate the structure and function of HSP90 through abolishing the intramolecular interaction. PMID:8486648

  2. PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL.

    PubMed

    Cuppen, E; Gerrits, H; Pepers, B; Wieringa, B; Hendriks, W

    1998-03-01

    The specificity of protein-protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells. PMID:9487134

  3. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    PubMed Central

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  4. Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins.

    PubMed

    Gimm, J Aura; An, Xiuli; Nunomura, Wataru; Mohandas, Narla

    2002-06-11

    Protein 4.1R is the prototypical member of a protein family that includes 4.1G, 4.1B, and 4.1N. 4.1R plays a crucial role in maintaining membrane mechanical integrity by binding cooperatively to spectrin and actin through its spectrin-actin-binding (SAB) domain. While the binary interaction between 4.1R and spectrin has been well characterized, the actin binding site in 4.1R remains unidentified. Moreover, little is known about the interaction of 4.1R homologues with spectrin and actin. In the present study, we showed that the 8 aa motif (LKKNFMES) within the 10 kDa spectrin-actin-binding domain of 4.1R plays a critical role in binding of 4.1R to actin. Recombinant 4.1R SAB domain peptides with mutations in this motif showed a marked decrease in their ability to form ternary complexes with spectrin and actin. Binary protein-protein interaction studies revealed that this decrease resulted from the inability of mutant SAB peptides to bind to actin filaments while affinity for spectrin was unchanged. We also documented that the 14 C-terminal residues of the 21 amino acid cassette encoded by exon 16 in conjunction with residues 27-43 encoded by exon 17 constituted a fully functional minimal spectrin-binding motif. Finally, we showed that 4.1N SAB domain was unable to form a ternary complex with spectrin and actin, while 4.1G and 4.1B SAB domains were able to form such a complex but less efficiently than 4.1R SAB. This was due to a decrease in the ability of 4.1G and 4.1B SAB domain to interact with actin but not with spectrin. These data enabled us to propose a model for the 4.1R-spectrin-actin ternary complex which may serve as a general paradigm for regulation of spectrin-based cytoskeleton interaction in various cell types. PMID:12044158

  5. PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

    PubMed Central

    Cuppen, Edwin; Gerrits, Herlinde; Pepers, Barry; Wieringa, Bé; Hendriks, Wiljan

    1998-01-01

    The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells. PMID:9487134

  6. The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

    PubMed Central

    Wang, Jingang; Zhou, Dan; Prabhu, Anjali; Schlegel, Richard; Yuan, Hang

    2010-01-01

    The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb. PMID:20824099

  7. Ligand-Binding Properties of the Carboxyl-Terminal Repeat Domain of Streptococcus mutans Glucan-Binding Protein A

    PubMed Central

    Haas, Wolfgang; Banas, Jeffrey A.

    2000-01-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two S. mutans GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan. PMID:10633107

  8. Ligand-binding properties of the carboxyl-terminal repeat domain of Streptococcus mutans glucan-binding protein A.

    PubMed

    Haas, W; Banas, J A

    2000-02-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two S. mutans GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan. PMID:10633107

  9. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis

    PubMed Central

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-01-01

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein–albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug. PMID:25864124

  10. Mutation analysis of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

    PubMed Central

    Goldstein, M A; Doi, R H

    1994-01-01

    Cellulose-binding protein A (CbpA) has been previously shown to mediate the interaction between crystalline cellulose substrates and the cellulase enzyme complex of Clostridium cellulovorans. CbpA contains a family III cellulose-binding domain (CBD) which, when expressed independently, binds specifically to crystalline cellulose. A series of N- and C-terminal deletions and a series of small internal deletions of the CBD were created to determine whether the entire region previously described as a CBD is required for the cellulose-binding function. The N- and C-terminal deletions reduced binding affinity by 10- to 100-fold. Small internal deletions of the CBD resulted in substantial reduction of CBD function. Some, but not all, point mutations throughout the sequence had significant disruptive effects on the binding ability of the CBD. Thus, mutations in any region of the CBD had effects on the binding of the fragment to cellulose. The results indicate that the entire 163-amino-acid region of the CBD is required for maximal binding to crystalline cellulose. Images PMID:7961505

  11. RNA-binding proteins with prion-like domains in ALS and FTLD-U.

    PubMed

    Gitler, Aaron D; Shorter, James

    2011-01-01

    Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a debilitating, and universally fatal, neurodegenerative disease that devastates upper and lower motor neurons. The causes of ALS are poorly understood. A central role for RNA-binding proteins and RNA metabolism in ALS has recently emerged. The RNA-binding proteins, TDP-43 and FUS, are principal components of cytoplasmic inclusions found in motor neurons of ALS patients and mutations in TDP-43 and FUS are linked to familial and sporadic ALS. Pathology and genetics also connect TDP-43 and FUS with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). It was unknown whether mechanisms of FUS aggregation and toxicity were similar or different to those of TDP-43. To address this issue, we have employed yeast models and pure protein biochemistry to define mechanisms underlying TDP-43 and FUS aggregation and toxicity, and to identify genetic modifiers relevant for human disease. We have identified prion-like domains in FUS and TDP-43 and provide evidence that these domains are required for aggregation. Our studies have defined key similarities as well as important differences between the two proteins. Collectively, however, our findings lead us to suggest that FUS and TDP-43, though similar RNA-binding proteins, likely aggregate and confer disease phenotypes via distinct mechanisms. PMID:21847013

  12. DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the Saccharomyces cervisiae TATA Binding Protein

    SciTech Connect

    Gupta,S.; Cheng, H.; Mollah, A.; Jamison, E.; Morris, S.; Chance, M.; Khrapunov, S.; Brenowitz, M.

    2007-01-01

    Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by 'protein footprinting' with hydroxyl radical ({center_dot}OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

  13. Structure of Alzheimer’s disease amyloid precursor protein copper-binding domain at atomic resolution

    SciTech Connect

    Kong, Geoffrey Kwai-Wai; Adams, Julian J.; Cappai, Roberto; Parker, Michael W.

    2007-10-01

    An atomic resolution structure of the copper-binding domain of the Alzheimer’s disease amyloid precursor protein is presented. Amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer’s disease, as its cleavage generates the Aβ peptide that is toxic to cells. APP is able to bind Cu{sup 2+} and reduce it to Cu{sup +} through its copper-binding domain (CuBD). The interaction between Cu{sup 2+} and APP leads to a decrease in Aβ production and to alleviation of the symptoms of the disease in mouse models. Structural studies of CuBD have been undertaken in order to better understand the mechanism behind the process. Here, the crystal structure of CuBD in the metal-free form determined to ultrahigh resolution (0.85 Å) is reported. The structure shows that the copper-binding residues of CuBD are rather rigid but that Met170, which is thought to be the electron source for Cu{sup 2+} reduction, adopts two different side-chain conformations. These observations shed light on the copper-binding and redox mechanisms of CuBD. The structure of CuBD at atomic resolution provides an accurate framework for structure-based design of molecules that will deplete Aβ production.

  14. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein.

    PubMed

    Leong, J M; Fournier, R S; Isberg, R R

    1990-06-01

    The invasin protein of the pathogenic Yersinia pseudotuberculosis mediates entry of the bacterium into cultured mammalian cells by binding several beta 1 chain integrins. In this study, we identified the region of invasin responsible for cell recognition. Thirty-two monoclonal antibodies directed against invasin were isolated, and of those, six blocked cell attachment to invasin. These six antibodies recognized epitopes within the last 192 amino acids of invasin. Deletion mutants of invasin and maltose-binding protein (MBP)--invasin fusion proteins were generated and tested for cell attachment. All of the invasin derivatives that carried the carboxyl-terminal 192 amino acids retained cell binding activity. One carboxyl-terminal invasin fragment and seven MBP--invasin fusion proteins were purified. The purified derivatives that retained binding activity inhibited bacterial entry into cultured mammalian cells. These results indicated that the carboxyl-terminal 192 amino acids of invasin contains the integrin-binding domain, even though this region does not contain the tripeptide sequence Arg-Gly-Asp. PMID:1693333

  15. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

    PubMed Central

    Weng, Z; Thomas, S M; Rickles, R J; Taylor, J A; Brauer, A W; Seidel-Dugan, C; Michael, W M; Dreyfuss, G; Brugge, J S

    1994-01-01

    Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through

  16. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

    PubMed Central

    Cooper, J A; Kashishian, A

    1993-01-01

    We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

  17. Identification of the Receptor Binding Domain of the Mouse Mammary Tumor Virus Envelope Protein

    PubMed Central

    Zhang, Yuanming; Rassa, John C.; deObaldia, Maria Elena; Albritton, Lorraine M.; Ross, Susan R.

    2003-01-01

    Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe40 to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe40 residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation. PMID:12970432

  18. Structure-Based Design of a Periplasmic Binding Protein Antagonist that Prevents Domain Closure

    SciTech Connect

    Borrok, M. Jack; Zhu, Yimin; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-D-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

  19. Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences.

    PubMed

    Choudhury, Susobhan; Ghosh, Basusree; Singh, Priya; Ghosh, Raka; Roy, Siddhartha; Pal, Samir Kumar

    2016-07-21

    The nature of the interface of specific protein-DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3-Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DNA. PMID:27326896

  20. Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

    PubMed Central

    Kuehnert, Julia; Sommer, Gunhild; Zierk, Avery W.; Fedarovich, Alena; Brock, Alexander; Fedarovich, Dzmitry; Heise, Tilman

    2015-01-01

    The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation. PMID:25520193

  1. Nuclear protein LEDGF/p75 recognizes supercoiled DNA by a novel DNA-binding domain

    PubMed Central

    Tsutsui, Kimiko M.; Sano, Kuniaki; Hosoya, Osamu; Miyamoto, Tadashi; Tsutsui, Ken

    2011-01-01

    Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it ‘supercoiled DNA-recognition domain’ (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K9E9K9) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it. PMID:21345933

  2. Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

    PubMed

    Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

    2011-11-01

    The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

  3. Phylogenetic Distribution and Evolution of the Linked RNA-Binding and NOT1-Binding Domains in the Tristetraprolin Family of Tandem CCCH Zinc Finger Proteins

    PubMed Central

    Perera, Lalith

    2014-01-01

    In humans, the tristetraprolin or TTP family of CCCH tandem zinc finger (TZF) proteins comprises 3 members, encoded by the genes ZFP36, ZFP36L1, and ZFP36L2. These proteins have direct orthologues in essentially all vertebrates studied, with the exception of birds, which appear to lack a version of ZFP36. Additional family members are found in rodents, amphibians, and fish. In general, the encoded proteins contain 2 critical macromolecular interaction domains: the CCCH TZF domain, which is necessary for high-affinity binding to AU-rich elements in mRNA; and an extreme C-terminal domain that, in the case of TTP, interacts with NOT1, the scaffold of a large multi-protein complex that contains deadenylases. TTP and its related proteins act by first binding to AU-rich elements in mRNA, and then recruiting deadenylases to the mRNA, where they can processively remove the adenosine residues from the poly(A) tail. Highly conserved TZF domains have been found in unicellular eukaryotes such as yeasts, and these domains can bind AU-rich elements that resemble those bound by the mammalian proteins. However, certain fungi appear to lack proteins with intact TZF domains, and the TTP family proteins that are expressed in other fungi often lack the characteristic C-terminal NOT1 binding domain found in the mammalian proteins. For these reasons, we investigated the phylogenetic distribution of the relevant sequences in available databases. Both domains are present in family member proteins from most lineages of eukaryotes, suggesting their mutual presence in a common ancestor. However, the vertebrate type of NOT1-binding domain is missing in most fungi, and the TZF domain itself has disappeared or degenerated in recently evolved fungi. Nonetheless, both domains are present together in the proteins from several unicellular eukaryotes, including at least 1 fungus, and they seem to have remained together during the evolution of metazoans. PMID:24697206

  4. Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs).

    PubMed

    Gao, Yuan; Li, Yanchang; Zhang, Chengpu; Zhao, Mingzhi; Deng, Chen; Lan, Qiuyan; Liu, Zexian; Su, Na; Wang, Jingwei; Xu, Feng; Xu, Yongru; Ping, Lingyan; Chang, Lei; Gao, Huiying; Wu, Junzhu; Xue, Yu; Deng, Zixin; Peng, Junmin; Xu, Ping

    2016-04-01

    Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community. PMID:27037361

  5. The anti-trp RNA-binding attenuation protein (Anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein.

    PubMed

    Valbuzzi, Angela; Gollnick, Paul; Babitzke, Paul; Yanofsky, Charles

    2002-03-22

    In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of l-tryptophan. Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation. Conversely, the accumulation of uncharged tRNA(Trp) induces synthesis of an anti-TRAP protein (AT), which forms a complex with TRAP and inhibits its activity. In this report, we investigate the structural features of TRAP required for AT recognition. A collection of TRAP mutant proteins was examined that were known to be partially or completely defective in tryptophan binding and/or RNA binding. Analyses of AT interactions with these proteins were performed using in vitro transcription termination assays and cross-linking experiments. We observed that TRAP mutant proteins that had lost the ability to bind RNA were no longer recognized by AT. Our findings suggest that AT acts by competing with messenger RNA for the RNA binding domain of TRAP. B. subtilis AT was also shown to interact with TRAP proteins from Bacillus halodurans and Bacillus stearothermophilus, implying that the structural elements required for AT recognition are conserved in the TRAP proteins of these species. Analyses of AT interaction with B. stearothermophilus TRAP at 60 degrees C demonstrated that AT is active at this elevated temperature. PMID:11786553

  6. Fast folding of a prototypic polypeptide: the immunoglobulin binding domain of streptococcal protein G.

    PubMed

    Kuszewski, J; Clore, G M; Gronenborn, A M

    1994-11-01

    The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched-flow deuterium-hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND-NH exchange up to 10-fold higher than that expected for a random coil, occurs within the dead time (< or = 1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half-life of 5.2 ms at 5 degrees C. PMID:7703841

  7. De novo design and engineering of functional metal and porphyrin-binding protein domains

    NASA Astrophysics Data System (ADS)

    Everson, Bernard H.

    In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron transfer require only a very small number of residue positions in a protein sequence to be fixed. Net charge and hydrophobic content are important in determining protein solubility and stability. Accordingly, rational modifications were made to the aforementioned design procedure in order to improve its overall success rate. The effects of these modifications are explored using two `next-generation' sequence libraries, which were separately expressed and evaluated. Particular modifications to these design parameters are demonstrated to effectively double the purification success rate of the procedure. Finally, I describe the redesign of the artificial di-iron protein DF2 into CDM13, a single chain di-Manganese four-helix bundle. CDM13 acts as a functional model of natural manganese catalase, exhibiting a kcat of 0.08s-1 under steady-state conditions. The bound manganese cofactors have a reduction potential of +805 mV vs NHE, which is too high for efficient dismutation of hydrogen peroxide. These results indicate that as a high-potential manganese complex, CDM13 may represent a promising first step toward a polypeptide model of the Oxygen Evolving Complex of the photosynthetic enzyme Photosystem II.

  8. Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA

    SciTech Connect

    Sharma A.; Heroux A.; Jenkins K. R.; Bowman G. D.

    2011-12-09

    Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves.

  9. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    SciTech Connect

    Lechner, Mark S. . E-mail: msl27@drexel.edu; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-06-17

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

  10. Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number.

    PubMed

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number. PMID:15325281

  11. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    NASA Technical Reports Server (NTRS)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  12. Structural Studies of the Alzheimer's Amyloid Precursor Protein Copper-Binding Domain Reveal How It Binds Copper Ions

    SciTech Connect

    Kong, G.K.-W.; Adams, J.J.; Harris, H.H.; Boas, J.F.; Curtain, C.C.; Galatis, D.; Master, C.L.; Barnham, K.J.; McKinstry, W.J.; Cappai, R.; Parker, M.W.; /Sydney U. /Monash U. /Melbourne U.

    2007-07-09

    Alzheimer's disease (AD) is the major cause of dementia. Amyloid {beta} peptide (A {beta}), generated by proteolytic cleavage of the amyloid precursor protein (APP), is central to AD pathogenesis. APP can function as a metalloprotein and modulate copper (Cu) transport, presumably via its extracellular Cu-binding domain (CuBD). Cu binding to the CuBD reduces A{beta} levels, suggesting that a Cu mimetic may have therapeutic potential. We describe here the atomic structures of apo CuBD from three crystal forms and found they have identical Cu-binding sites despite the different crystal lattices. The structure of Cu[2+]-bound CuBD reveals that the metal ligands are His147, His151, Tyrl68 and two water molecules, which are arranged in a square pyramidal geometry. The site resembles a Type 2 non-blue Cu center and is supported by electron paramagnetic resonance and extended X-ray absorption fine structure studies. A previous study suggested that Met170 might be a ligand but we suggest that this residue plays a critical role as an electron donor in CuBDs ability to reduce Cu ions. The structure of Cu[+]-bound CuBD is almost identical to the Cu[2+]-bound structure except for the loss of one of the water ligands. The geometry of the site is unfavorable for Cu[+], thus providing a mechanism by which CuBD could readily transfer Cu ions to other proteins.

  13. Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.

    PubMed

    Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D

    2014-01-01

    Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

  14. Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1

    PubMed Central

    Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D.

    2014-01-01

    Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquiti-nation and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

  15. Direct Correlation of DNA Binding and Single Protein Domain Motion via Dual Illumination Fluorescence Microscopy

    PubMed Central

    2015-01-01

    We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes. PMID:25204359

  16. Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2016-05-20

    Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

  17. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA.

    PubMed

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder; Nyengaard, Jens R; Hermey, Guido; Bakke, Oddmund; Mari, Muriel; Schu, Peter; Pohlmann, Regina; Dennes, André; Petersen, Claus M

    2007-10-01

    SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 mu1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes. PMID:17646382

  18. Crystal structure of the trithorax group protein ASH2L reveals a forkhead-like DNA binding domain

    SciTech Connect

    Sarvan, Sabina; Avdic, Vanja; Tremblay, Véronique; Chaturvedi, Chandra-Prakash; Zhang, Pamela; Lanouette, Sylvain; Blais, Alexandre; Brunzelle, Joseph S; Brand, Marjorie; Couture, Jean-François

    2012-05-02

    Absent, small or homeotic discs-like 2 (ASH2L) is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. Here we report that ASH2L binds DNA using a forkhead-like helix-wing-helix (HWH) domain. In vivo, the ASH2L HWH domain is required for binding to the {beta}-globin locus control region, histone H3 Lys4 (H3K4) trimethylation and maximal expression of the {beta}-globin gene (Hbb-1), validating the functional importance of the ASH2L DNA binding domain.

  19. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

    NASA Astrophysics Data System (ADS)

    Rustgi, Anil K.; Dyson, Nicholas; Bernards, Rene

    1991-08-01

    THE proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms1. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization2-5. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation6,7. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.

  20. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham.

    PubMed

    Gallego, Marta; Mora, Leticia; Fraser, Paul D; Aristoy, María-Concepción; Toldrá, Fidel

    2014-04-15

    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered. PMID:24295685

  1. Asymmetry adjacent to the collagen-like domain in rat liver mannose-binding protein.

    PubMed Central

    Wallis, R; Drickamer, K

    1997-01-01

    Rat liver mannose-binding protein (MBP-C) is the smallest known member of the collectin family of animal lectins, many of which are involved in defence against microbial pathogens. It consists of an N-terminal collagen-like domain linked to C-terminal carbohydrate-recognition domains. MBP-C, overproduced in Chinese-hamster ovary cells, is post-translationally modified and processed in a manner similar to the native lectin. Analytical ultracentrifugation experiments indicate that MBP-C is trimeric, with a weight-averaged molecular mass of approx. 77 kDa. The rate of sedimentation of MBP-C and its mobility on gel filtration suggest a highly elongated molecule. Anomalous behaviour on gel filtration due to this extended conformation may explain previous suggestions that MBP-C forms a higher oligomer. The polypeptide chains of the MBP-C trimer are linked by disulphide bonds between two cysteine residues at the N-terminal junction of the collagen-like domain. Analysis of an N-terminal tryptic fragment reveals that the disulphide bonding in MBP-C is heterogeneous and asymmetrical. These results indicate that assembly of MBP-C oligomers probably proceeds in a C- to N-terminal direction: trimerization at the C-terminus is followed by assembly of the collagenous domain and finally formation of N-terminal disulphide bonds. The relatively simple organization of MBP-C provides a template for understanding larger, more complex collectins. PMID:9230118

  2. Structural architecture and interplay of the nucleotide- and erythrocyte binding domain of the reticulocyte binding protein Py235 from Plasmodium yoelii.

    PubMed

    Grüber, Ardina; Manimekalai, Malathy S S; Preiser, Peter R; Grüber, Gerhard

    2012-11-01

    Human malaria is caused by the cyclical invasion of the host's red blood cells (RBCs) by the invasive form of the parasite, the merozoite. The invasion of the RBC involves a range of parasite ligand receptor interactions, a process which is under intensive investigation. Two protein families are known to be important in the recognition and invasion of the human erythrocyte, the erythrocyte-binding like (EBL) proteins and the reticulocyte binding like proteins, of which the Py235 family in Plasmodium yoelii is a member. Recently the nucleotide binding domain (NBD94), that plays a role in ATP sensing, and the erythrocyte binding domain (EBD) of Py235, called EBD(1-194), have been identified. Binding of ATP leads to conformational changes within Py235 from P. yoelli and results in enhanced binding of the protein to the RBC. Structural features of these domains have been obtained, providing the platform to discuss how the structural architecture creates the basis for an interplay of the sensing NBD and the EBD domain in Py235. In analogy to the receptor-mediated ligand-dimerization model of the EBL proteins PvDBP and PfEBA-175 from Plasmodium vivax and Plasmodium falciparum, respectively, we hypothesise that Py235 of P. yoelii binds via its EBD(1-194) domain to the RBC receptor, thereby inducing dimerization of the Py235-receptor complex. PMID:22878128

  3. DNA Bending is Induced in an Enhancer by the DNA-Binding Domain of the Bovine Papillomavirus E2 Protein

    NASA Astrophysics Data System (ADS)

    Moskaluk, Christopher; Bastia, Deepak

    1988-03-01

    The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I ``footprinting'' on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucleotide) as the E2 binding site. Using electrophoretic methods we have shown that the DNA-binding domain changes conformation of the enhancer by inducing significant DNA bending.

  4. ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.; DeRisi, Joseph L.

    2013-01-01

    ABSTRACT Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIβ/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism. PMID:23572552

  5. Domain rearrangement of SRP protein Ffh upon binding 4.5S RNA and the SRP receptor FtsY.

    PubMed

    Buskiewicz, Iwona; Kubarenko, Andriy; Peske, Frank; Rodnina, Marina V; Wintermeyer, Wolfgang

    2005-06-01

    The signal recognition particle (SRP) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. In Escherichia coli, SRP consists of 4.5S RNA and a protein, Ffh, that recognizes the signal peptide emerging from the ribosome and the SRP receptor at the membrane, FtsY. In the present work, we studied the interactions between the NG and M domains in Ffh and their rearrangements upon complex formation with 4.5S RNA and/or FtsY. In free Ffh, the NG and M domains are facing one another in an orientation that allows cross-linking between positions 231 in the G domain and 377 in the M domain. There are binding interactions between the two domains, as the isolated domains form a strong complex. The interdomain contacts are disrupted upon binding of Ffh to 4.5S RNA, consuming a part of the total binding energy of 4.5S RNA-Ffh association that is roughly equivalent to the free energy of domain binding to each other. In the SRP particle, the NG domain binds to 4.5S RNA in a region adjacent to the binding site of the M domain. Ffh binding to FtsY also requires a reorientation of NG and M domains. These results suggest that in free Ffh, the binding sites for 4.5S RNA and FtsY are occluded by strong domain-domain interactions which must be disrupted for the formation of SRP or the Ffh-FtsY complex. PMID:15923378

  6. Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase.

    PubMed

    Miyao, Hiroki; Ikeda, Yusuke; Shiraishi, Arata; Kawakami, Yuji; Sueda, Shinji

    2015-09-01

    Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability. PMID:25998102

  7. Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

    PubMed Central

    Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J.; Iyer, K. Swaminathan; Ho, Diwei; Newcombe, Estella A.; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S.

    2015-01-01

    Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration. PMID:26283796

  8. Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola

    PubMed Central

    Bamford, C.V.; Francescutti, T.; Cameron, C.E.; Jenkinson, H.F.; Dymock, D.

    2011-01-01

    SUMMARY Interactions with fibronectin are important in the virulence strategies of a range of disease-related bacteria. The periodontitis-associated oral spirochaete Treponema denticola expresses at least two fibronectin-binding proteins, designated Msp (major surface protein) and OppA (oligopeptide-binding protein homologue). To identify other T. denticola outer membrane fibronectin-binding proteins, the amino acid sequence of the Treponema pallidum fibronectin-binding protein Tp0155 was used to survey the T. denticola genome. Seven T. denticola genes encoding orthologous proteins were identified. All but two were expressed in Escherichia coli and purified recombinant proteins bound fibronectin. Using antibodies to the N-terminal region of Tp0155, it was demonstrated that T. denticola TDE2318, with highest homology to Tp0155, was cell surface localized. Like Tp0155, the seven T. denticola proteins contained an M23 peptidase domain and four (TDE2318, TDE2753, TDE1738, TDE1297) contained one or two LysM domains. M23 peptidases can degrade peptidoglycan whereas LysM domains recognize carbohydrate polymers. In addition, TDE1738 may act as a bacteriocin based on homology with other bacterial lysins and the presence of an adjacent gene encoding a putative immunity factor. Collectively, these results suggest that T. denticola expresses fibronectin-binding proteins associated with the cell surface that may also have cell wall modifying or lytic functions. PMID:21040511

  9. Domain rearrangement of SRP protein Ffh upon binding 4.5S RNA and the SRP receptor FtsY

    PubMed Central

    BUSKIEWICZ, IWONA; KUBARENKO, ANDRIY; PESKE, FRANK; RODNINA, MARINA V.; WINTERMEYER, WOLFGANG

    2005-01-01

    The signal recognition particle (SRP) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. In Escherichia coli, SRP consists of 4.5S RNA and a protein, Ffh, that recognizes the signal peptide emerging from the ribosome and the SRP receptor at the membrane, FtsY. In the present work, we studied the interactions between the NG and M domains in Ffh and their rearrangements upon complex formation with 4.5S RNA and/or FtsY. In free Ffh, the NG and M domains are facing one another in an orientation that allows cross-linking between positions 231 in the G domain and 377 in the M domain. There are binding interactions between the two domains, as the isolated domains form a strong complex. The interdomain contacts are disrupted upon binding of Ffh to 4.5S RNA, consuming a part of the total binding energy of 4.5S RNA-Ffh association that is roughly equivalent to the free energy of domain binding to each other. In the SRP particle, the NG domain binds to 4.5S RNA in a region adjacent to the binding site of the M domain. Ffh binding to FtsY also requires a reorientation of NG and M domains. These results suggest that in free Ffh, the binding sites for 4.5S RNA and FtsY are occluded by strong domain–domain interactions which must be disrupted for the formation of SRP or the Ffh-FtsY complex. PMID:15923378

  10. Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

    PubMed Central

    Yu, Wookyung; Chung, Kwanghoon; Cheon, Mookyung; Heo, Muyoung; Han, Kyou-Hoon; Ham, Sihyun; Chang, Iksoo

    2008-01-01

    Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz–Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding–unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined. PMID:18272497

  11. Characterization of substrate binding of the WW domains in human WWP2 protein.

    PubMed

    Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

    2015-07-01

    WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. PMID:25999310

  12. Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains.

    PubMed

    Wang, Chen; Oliver, Erin E; Christner, Brent C; Luo, Bing-Hao

    2016-07-19

    Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 μM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect. PMID:27359086

  13. FMN binding and photochemical properties of plant putative photoreceptors containing two LOV domains, LOV/LOV proteins.

    PubMed

    Kasahara, Masahiro; Torii, Mayumi; Fujita, Akimitsu; Tainaka, Kengo

    2010-11-01

    LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly(66) in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins. PMID:20826774

  14. MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein

    PubMed Central

    Xia, Zhen-Biao; Anderson, Melanie; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2003-01-01

    The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo. PMID:12829790

  15. Comprehensive comparative analysis and identification of RNA-binding protein domains: multi-class classification and feature selection

    PubMed Central

    Jahandideh, Samad; Srinivasasainagendra, Vinodh; Zhi, Degui

    2012-01-01

    RNA-protein interaction plays an important role in various cellular processes, such as protein synthesis, gene regulation, post-transcriptional gene regulation, alternative splicing, and infections by RNA viruses. In this study, using Gene Ontology Annotated (GOA) and Structural Classification of Proteins (SCOP) databases an automatic procedure was designed to capture structurally solved RNA-binding protein domains in different subclasses. Subsequently, we applied tuned multi-class SVM (TMCSVM), Random Forest (RF), and multi-class ℓ1/ℓq-regularized logistic regression (MCRLR) for analysis and classifying RNA-binding protein domains based on a comprehensive set of sequence and structural features. In this study, we compared prediction accuracy of three different state-of-the-art predictor methods. From our results, TMCSVM outperforms the other methods and suggests the potential of TMCSVM as a useful tool for facilitating the multi-class prediction of RNA-binding protein domains. On the other hand, MCRLR by elucidating importance of features for their contribution in predictive accuracy of RNA-binding protein domains subclasses, helps us to provide some biological insights into the roles of sequences and structures in protein–RNA interactions. PMID:22884576

  16. The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain

    PubMed Central

    2013-01-01

    Background The regulatory mechanisms of motor protein-dependent intracellular transport are still not fully understood. The kinesin-1-binding protein, JIP1, can function as an adaptor protein that links kinesin-1 and other JIP1-binding “cargo” proteins. However, it is unknown whether these “cargo” proteins influence the JIP1–kinesin-1 binding. Results We show here that JIP1–kinesin-1 binding in Neuro2a cells was dependent on conserved amino acid residues in the JIP1-phosphotyrosine binding (PTB) domain, including F687. In addition, mutation of F687 severely affected the neurite tip localization of JIP1. Proteomic analysis revealed another kinesin-1 binding protein, JIP3, as a major JIP1 binding protein. The association between JIP1 and JIP3 was dependent on the F687 residue in JIP1, and this association induced the formation of a stable ternary complex with kinesin-1. On the other hand, the binding of JIP1 and JIP3 was independent of kinesin-1 binding. We also show that other PTB binding proteins can interrupt the formation of the ternary complex. Conclusions The formation of the JIP1–kinesin-1 complex depends on the protein binding-status of the JIP1 PTB domain. This may imply a regulatory mechanism of kinesin-1-dependent intracellular transport. PMID:23496950

  17. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    SciTech Connect

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  18. Methyl Binding Domain Protein 2 (MBD2) dependent proliferation and survival of breast cancer cells

    PubMed Central

    Mian, Omar Y.; Wang, Shou Zhen; Zhu, Sheng Zu; Gnanapragasam, Merlin N.; Graham, Laura; Bear, Harry D.; Ginder, Gordon D.

    2011-01-01

    Methyl Cytosine Binding Domain Protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where re-patterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable shRNA mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak anti-proliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in BALB/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by de-repression of tumor suppressor genes including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing demonstrate MBD2 binding directly to the hyper-methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG-island associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and re-silencing of the MBD2 dependent tumor suppressor genes. Our data suggest that uncoupling CpG-methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results demonstrate a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers. PMID:21693597

  19. Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4.

    PubMed Central

    Jolliffe, C N; Harvey, K F; Haines, B P; Parasivam, G; Kumar, S

    2000-01-01

    Nedd4 is a member of a growing family of ubiquitin-protein ligases which consist of a lipid-binding domain, two to four WW domains and a C-terminal ubiquitin-protein ligase domain. The Nedd4 mRNA levels are developmentally regulated and Nedd4 protein is highly expressed in many mouse embryonic tissues. In this study we have used a far-Western screen to identify embryonic proteins that interact with the WW domains in mouse Nedd4. We report here identification of eight Nedd4 WW-domain-interacting proteins from mouse embryonic cDNA expression libraries. Two of the proteins are novel, while two have been identified previously as ligands for a WW domain. All of these proteins contain one or more PY motifs. In seven of the eight proteins, these PY motifs are necessary for their interaction with the WW domains of Nedd4. Using site-directed mutagenesis, and by using individual WW domains of Nedd4 as probes for far-Western analysis, we show that the three WW domains in Nedd4 interact with varying affinities with the PY motifs present in various Nedd4-binding proteins. These results provide evidence that Nedd4 can potentially interact with multiple proteins, possibly simultaneously, through its WW domains. PMID:11042109

  20. FhCaBP3: a Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains.

    PubMed

    Banford, Samantha; Drysdale, Orla; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2013-04-01

    A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism. PMID:23142130

  1. Molecular modeling of the heterodimer of human CFTR's nucleotide-binding domains using a protein-protein docking approach.

    PubMed

    Huang, Sheng-You; Bolser, Diana; Liu, Hao-Yang; Hwang, Tzyh-Chang; Zou, Xiaoqin

    2009-04-01

    We have presented a new protein-protein docking approach to model heterodimeric structures based on the conformations of the monomeric units. The conventional modeling method relies on superimposing two monomeric structures onto the crystal structure of a homologous protein dimer. The resulting structure may exhibit severe backbone clashes at the dimeric interface depending on the backbone dissimilarity between the target and template proteins. Our method overcomes the backbone clashing problem and requires no a priori knowledge of the dimeric structure of a homologous protein. Here we used human Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel whose dysfunction causes cystic fibrosis, for illustration. The two intracellular nucleotide-binding domains (NBDs) of CFTR control the opening and closing of the channel. Yet, the structure of the CFTR's NBD1-NBD2 complex has not been experimentally determined. Thus, correct modeling of this heterodimeric structure is valuable for understanding CFTR functions and would have potential applications for drug design for cystic fibrosis treatment. Based on the crystal structure of human CFTR's NBD1, we constructed a model of the NBD1-NBD2 complex. The constructed model is consistent with the dimeric mode observed in the crystal structures of other ABC transporters. To verify our structural model, an ATP substrate was docked into the nucleotide-binding site. The predicted binding mode shows consistency with related crystallographic findings and CFTR functional studies. Finally, genistein, an agent that enhances CFTR activity, though the mechanism for such enhancement is unclear, was docked to the model. Our predictions agreed with genistein's bell-shaped dose-response relationship. Potential mutagenesis experiments were proposed for understanding the potentiation mechanism of genistein and for providing insightful information for drug design targeting at CFTR. The method used in this study can be

  2. The energetic cost of domain reorientation in maltose-binding protein as studied by NMR and fluorescence spectroscopy.

    PubMed

    Millet, Oscar; Hudson, Rhea P; Kay, Lewis E

    2003-10-28

    Maltose-binding protein (MBP) is a two-domain protein that undergoes a ligand-mediated conformational rearrangement from an "open" to a "closed" structure on binding to maltooligosaccharides. To characterize the energy landscape associated with this transition, we have generated five variants of MBP with mutations located in the hinge region of the molecule. Residual dipolar couplings, measured in the presence of a weak alignment medium, have been used to establish that the average structures of the mutant proteins are related to each other by domain rotation about an invariant axis, with the rotation angle varying from 5 degrees to 28 degrees. Additionally, the domain orientations observed in the wild-type apo and ligand-bound (maltose, maltotriose, etc.) structures are related through a rotation of 35 degrees about the same axis. Remarkably, the free energy of unfolding, measured by equilibrium denaturation experiments and monitored by fluorescence spectroscopy, shows a linear correlation with the rotation angle, with the stability of the (apo)protein decreasing with domain closure by 212 +/- 16 cal mol-1 per degree of rotation. The apparent binding energy for maltose also shows a similar correlation with the interdomain angle, suggesting that the mutations, as they relate to binding, affect predominantly the ligand-free structure. The linearity of the energy change is interpreted in terms of an increase in the extent of hydrophobic surface that becomes solvent accessible on closure. The combination of structural, stability, and binding data allows separation of the energetics of domain reorientation from ligand binding. This work presents a near quantitative structure-energy-binding relationship for a series of mutants of MBP, illustrating the power of combined studies involving protein engineering and solution NMR spectroscopy. PMID:14530390

  3. Localization of the equine IgG-binding domain in the fibrinogen-binding protein (FgBP) of Streptococcus equi subsp. equi.

    PubMed

    Meehan, Mary; Lewis, Melanie J; Byrne, Caroline; O'Hare, David; Woof, Jenny M; Owen, Peter

    2009-08-01

    Fibrinogen-binding protein (FgBP, also termed SeM) is a cell-wall-associated anti-phagocytic M-like protein of the equine pathogen Streptococcus equi subsp. equi, and binds fibrinogen (Fg) and IgG. FgBP binds Fg avidly through residues located at the extreme N terminus of the molecule, whereas the IgG-binding site is more centrally located between the A and B repeats. FgBP binds equine IgG4 and IgG7 subclasses through interaction with the CH2-CH3 interdomain region of IgG-Fc, and possesses overlapping Fc-binding sites with protein A and protein G. In this study, FgBP truncates containing defined internal deletions were used to identify a stretch of 14 aa (residues 335-348) critical for IgG binding. Protein chimeras consisting of the non-IgG-binding alpha-helical coiled-coil M5 protein fused to FgBP sequences were used to identify a minimal equine IgG-binding domain consisting of residues 329-360. Competition ELISA tests suggested that IgG does not compromise Fg binding and vice versa. PMID:19423628

  4. The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

    PubMed Central

    Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

    2011-01-01

    Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

  5. Solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP).

    PubMed

    Liew, Chu Kong; Crossley, Merlin; Mackay, Joel P; Nicholas, Hannah R

    2007-02-16

    The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers. PMID:17174978

  6. The structure of the Ca{sup 2+}-binding , glycosylated F-spondin domain of F-spondin- A C2-domain variant in an extracellular matrix protein.

    SciTech Connect

    Tan, K.; Lawler, J.

    2011-05-10

    F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin{_}N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin. We present the crystal structure of human F-spondin FS domain at 1.95{angstrom} resolution. The structure reveals a Ca{sup 2+}-binding C2 domain variant with an 8-stranded antiparallel {beta}-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain. The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca{sup 2+}- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

  7. A Novel Catalytic Function of Synthetic IgG-Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine.

    PubMed

    Inouye, Satoshi; Sahara-Miura, Yuiko

    2014-01-01

    The synthetic IgG-binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine-utilizing luciferase. The Z domain derivatives (ZZ-gCys, Z-gCys and Z-domain) were purified and the luminescence properties were characterized by comparing with coelenterazine-utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ-gCys (100%) > Z-gCys (36.8%) > Z-domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ-gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z-gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG-binding region of the Z domain. PMID:24138575

  8. Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

    PubMed Central

    de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

    2014-01-01

    The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

  9. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli; Michael Garavito, R.

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions, suggesting a role for the PRR-IC domain in PRR dimerization. -- Abstract: The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain ({approx}310 amino acids), a single transmembrane domain ({approx}20 amino acids) and an intracellular domain ({approx}19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain ({approx}30 amino acids), the IC domain is also involved in assembly of V{sub 0} portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 A (maltose-free) and 2.15 A (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR

  10. Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.

    PubMed

    Sheldon, Paul J; Busarow, Sara B; Hutchinson, C Richard

    2002-04-01

    Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon. PMID:11972782

  11. A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

    PubMed Central

    Sabogal, Alex; Rio, Donald C

    2010-01-01

    Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions. PMID:20842711

  12. Disorder and structure in the Rab11 binding domain of Rab11 family interacting protein 2.

    PubMed

    Wei, Jie; Liu, Yuqi; Bose, Kakoli; Henry, Gillian D; Baleja, James D

    2009-01-27

    Rab11 plays a central role in plasma membrane recycling which returns cellular receptors for reuse at the cell surface. A recently identified family of Rab11 interacting proteins (FIP) includes FIP2. The C-terminal region of FIP2 is essential for colocalization with Rab11 on early endosomes and for enabling formation of higher-order oligomers. Rab11 binding and oligomerization of FIP2 are separable. Here we have determined the three-dimensional structure of the 40-residue coiled-coil oligomerization domain of FIP2 in the absence of Rab11 using NMR methods. The N-terminal half showed strong NOE cross-peaks and well-dispersed NMR resonances, whereas the C-terminal half had fewer NOE cross-peaks and less chemical shift dispersion. The 10 C-terminal residues were mostly disordered. The final structures of the dimer had favorable Ramachandran angles and a root-mean-square deviation of 0.59 +/- 0.13 A over superimposed backbone residues. The structure allows a comparison to a structure of FIP2 in complex with Rab11 that was determined crystallographically. In complex with Rab11, the C-terminal residues are not disordered but have a helical structure that predicts residual dipolar coupling constants that are incompatible with those measured on the unbound FIP2. In both structures, a histidine residue is found at the normally hydrophobic position of the heptad repeat of the coiled coil, and here we show its ionization destabilizes the coiled-coil structure. Together, these data allow us to build a model in which the binding of FIP family proteins to Rab11 can be described in terms of conformational changes and that suggests new modes of regulation. PMID:19119858

  13. Fasciola hepatica calcium-binding protein FhCaBP2: structure of the dynein light chain-like domain.

    PubMed

    Nguyen, Thanh H; Thomas, Charlotte M; Timson, David J; van Raaij, Mark J

    2016-07-01

    The common liver fluke Fasciola hepatica causes an increasing burden on human and animal health, partly because of the spread of drug-resistant isolates. As a consequence, there is considerable interest in developing new drugs to combat liver fluke infections. A group of potential targets is a family of calcium-binding proteins which combine an N-terminal domain with two EF-hand motifs and a C-terminal domain with predicted similarity to dynein light chains (DLC-like domain). The function of these proteins is unknown, although in several species, they have been localised to the tegument, an important structure at the host-parasite interface. Here, we report the X-ray crystal structure of the DLC-like domain of F. hepatica calcium-binding protein 2 (FhCaBP2), solved using single-wavelength anomalous diffraction and refined at 2.3 Å resolution in two different crystal forms. The FhCaBP2 DLC-like domain has a structure similar to other DLC domains, with an anti-parallel β-sheet packed against an α-helical hairpin. Like other DLC domains, it dimerises through its β2-strand, which extends in an arch and forms the fifth strand in an extended β-sheet of the other monomer. The structure provides molecular details of the dimerisation of FhCaBP2, the first example from this family of parasite proteins. PMID:27083189

  14. Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization*

    PubMed Central

    Mayer, Magnus C.; Kaden, Daniela; Schauenburg, Linda; Hancock, Mark A.; Voigt, Philipp; Roeser, Dirk; Barucker, Christian; Than, Manuel E.; Schaefer, Michael; Multhaup, Gerhard

    2014-01-01

    The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438–452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level. PMID:24855651

  15. Transcription Factor Pip Can Enhance DNA Binding by E47, Leading to Transcriptional Synergy Involving Multiple Protein Domains

    PubMed Central

    Nagulapalli, Sujatha; Atchison, Michael L.

    1998-01-01

    The transcription factors E2A (E12/E47) and Pip are both required for normal B-cell development. Each protein binds to regulatory sequences within various immunoglobulin enhancer elements. Activity of E2A proteins can be regulated by interactions with other proteins which influence their DNA binding or activation potential. Similarly, Pip function can be influenced by interaction with the protein PU.1, which can recruit Pip to bind to DNA. We show here that a previously unidentified Pip binding site resides adjacent to the E2A binding site within the immunoglobulin κ 3′ enhancer. Both of these binding sites are crucial for high-level enhancer activity. We found that E47 and Pip can functionally interact to generate a very potent 100-fold transcriptional synergy. Through a series of mutagenesis experiments, we identified the Pip sequences necessary for transcriptional activation and for synergy with E47. Two synergy domains (residues 140 to 207 and 300 to 420) in addition to the Pip DNA binding domain (residues 1 to 134) are required for maximal synergy with E47. We also identified a Pip domain (residues 207 to 300) that appears to mask Pip transactivation potential. Part of the synergy mechanism between E47 and Pip appears to involve the ability of Pip to increase DNA binding by E47, perhaps by inducing a conformational change in the E47 protein. E47 may also induce a conformational change in Pip which unmasks sequences important for transcriptional activity. Based upon our results, we propose a model for E47-Pip transcriptional synergy. PMID:9671474

  16. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease.

    PubMed

    King, Oliver D; Gitler, Aaron D; Shorter, James

    2012-06-26

    Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable 'prion domain' enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer's disease and Huntington's disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion

  17. Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.

    PubMed

    Heimer, Brandon W; Tam, Brooke E; Sikes, Hadley D

    2015-12-01

    Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application. PMID:26384511

  18. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  19. Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

    PubMed Central

    Barros, Marilia; Jin, Danni; Lösche, Mathias; Vogt, Volker M.

    2015-01-01

    ABSTRACT The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCE The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with

  20. Evolutionary analysis of the global landscape of protein domain types and domain architectures associated with family 14 carbohydrate-binding modules.

    PubMed

    Chang, Ti-Cheng; Stergiopoulos, Ioannis

    2015-07-01

    Domain promiscuity is a powerful evolutionary force that promotes functional innovation in proteins, thus increasing proteome and organismal complexity. Carbohydrate-binding modules, in particular, are known to partake in complex modular architectures that play crucial roles in numerous biochemical and molecular processes. However, the extent, functional, and evolutionary significance of promiscuity is shrouded in mystery for most CBM families. Here, we analyzed the global promiscuity of family 14 carbohydrate-binding modules (CBM14s) and show that fusion, fission, and reorganization events with numerous other domain types interplayed incessantly in a lineage-dependent manner to likely facilitate species adaptation and functional innovation in the family. PMID:26067847

  1. A small cellulose binding domain protein in Phytophtora is cell wall localized

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellulose binding domains (CBD) are structurally conserved regions linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are not generally present in plant pathogenic fungi. A genome wide survey of CBDs w...

  2. Conserved DNA binding and self-association domains of the Drosophila zeste protein.

    PubMed Central

    Chen, J D; Chan, C S; Pirrotta, V

    1992-01-01

    The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures. Images PMID:1732733

  3. A Thermodynamic Ligand Binding Study of the Third PDZ Domain (PDZ3) from the Mammalian Neuronal Protein PSD-95†

    PubMed Central

    Saro, Dorina; Li, Tao; Rupasinghe, Chamila; Paredes, Azrael; Caspers, Nicole; Spaller, Mark R.

    2008-01-01

    The thermodynamic parameters associated with the binding of several series of linear peptides to the third PDZ domain (PDZ3) of the postsynaptic density 95 protein (PSD-95) have been measured using isothermal titration calorimetry (ITC). Two strategies were pursued in developing these binding ligands: (1) systematic N-terminal truncation of sequences derived from the C-terminal regions of identified PDZ3-binding proteins (CRIPT, neuroligin-1 and citron); and (2) selective mutation of specific positions within a consensus hexapeptide (KKETEV) known to bind PDZ3. Each synthetically prepared peptide was used to titrate PDZ3, which yielded the changes in Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (TΔS) for the binding event. Selected peptides were subjected to additional analysis, which entailed (1) measuring the change in heat capacity (ΔCp) upon association, to assess the character of the binding interface; and (2) constructing thermodynamic double-mutant cycles, to determine the presence of cooperative effects. From the first series, the CRIPT protein proved to be the better source for higher affinity sequences. From the second series, enhanced binding was associated with peptides that closely adhered to the established motif for class I PDZ domain C-termini, X-(T/S)-X-(V/I/L), and more specifically to a narrower motif of X-T-X-V. Further, in both series a length of six residues was necessary and sufficient to capture maximal affinity. In addition, there were significant influences upon binding by modifying the abutting ‘X’ positions. The cumulative results provide greater detail into the specific nature of ligand binding to PDZ3, and will assist in the development of selective molecular probes for the study of this and structurally homologous PDZ domains. PMID:17474715

  4. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    PubMed Central

    2011-01-01

    Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC). A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans. PMID:22122911

  5. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    SciTech Connect

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  6. In Vitro Genetic Analysis of the RNA Binding Site of Vigilin, a Multi-KH-Domain Protein

    PubMed Central

    Kanamori, Hiroshi; Dodson, Robin E.; Shapiro, David J.

    1998-01-01

    The function(s) and RNA binding properties of vigilin, a ubiquitous protein with 14 KH domains, remain largely obscure. We recently showed that vigilin is the estrogen-inducible protein in polysome extracts which binds specifically to a segment of the 3′ untranslated region (UTR) of estrogen-stabilized vitellogenin mRNA. In order to identify consensus mRNA sequences and structures important in binding of vigilin to RNA, before vigilin was purified, we developed a modified in vitro genetic selection protocol. We subsequently validated our selection procedure, which employed crude polysome extracts, by testing natural and in vitro-selected RNAs with purified recombinant vigilin. Most of the selected up-binding mutants exhibited hypermutation of G residues leading to a largely unstructured, single-stranded region containing multiple conserved (A)nCU and UC(A)n motifs. All eight of the selected down-binding mutants contained a mutation in the sequence (A)nCU. Deletion analysis indicated that approximately 75 nucleotides are required for maximal binding. Using this information, we predicted and subsequently identified a strong vigilin binding site near the 3′ end of human dystrophin mRNA. RNA sequences from the 3′ UTRs of transferrin receptor and estrogen receptor, which lack strong homology to the selected sequences, did not bind vigilin. These studies describe an aproach to identifying long RNA binding sites and describe sequence and structural requirements for interaction of vigilin with RNAs. PMID:9632784

  7. NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan.

    PubMed

    Bannantine, John P; Lingle, Cari K; Adam, Philip R; Ramyar, Kasra X; McWhorter, William J; Stabel, Judith R; Picking, William D; Geisbrecht, Brian V

    2016-04-01

    A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases. PMID:26799947

  8. Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

    PubMed Central

    Koyasu, S; Tse, A G; Moingeon, P; Hussey, R E; Mildonian, A; Hannisian, J; Clayton, L K; Reinherz, E L

    1994-01-01

    To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains. Images PMID:7517560

  9. Carbohydrate affinity for the glucose-galactose binding protein is regulated by allosteric domain motions.

    PubMed

    Ortega, Gabriel; Castaño, David; Diercks, Tammo; Millet, Oscar

    2012-12-01

    Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding. PMID:23148479

  10. Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.

    PubMed

    Sheth, Payal R; Shipps, Gerald W; Seghezzi, Wolfgang; Smith, Catherine K; Chuang, Cheng-Chi; Sanden, David; Basso, Andrea D; Vilenchik, Lev; Gray, Kimberly; Annis, D Allen; Nickbarg, Elliott; Ma, Yao; Lahue, Brian; Herbst, Ronald; Le, Hung V

    2010-09-28

    Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy. PMID:20718440

  11. Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

    PubMed

    Baumkötter, Frederik; Schmidt, Nadine; Vargas, Carolyn; Schilling, Sandra; Weber, Rebecca; Wagner, Katja; Fiedler, Sebastian; Klug, Wilfried; Radzimanowski, Jens; Nickolaus, Sebastian; Keller, Sandro; Eggert, Simone; Wild, Klemens; Kins, Stefan

    2014-08-13

    Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function. PMID:25122912

  12. Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

    PubMed Central

    Guryanov, Sergey G.; Selivanova, Olga M.; Nikulin, Alexey D.; Enin, Gennady A.; Melnik, Bogdan S.; Kretov, Dmitry A.; Serdyuk, Igor N.; Ovchinnikov, Lev P.

    2012-01-01

    YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions. PMID:22590640

  13. Preferential binding of high mobility group 1 protein to UV-damaged DNA. Role of the COOH-terminal domain.

    PubMed

    Pasheva, E A; Pashev, I G; Favre, A

    1998-09-18

    Binding of chromosomal high mobility group 1 protein (HMG1) to UV-damaged DNA has been studied with oligonucleotides containing a single dipyrimidine site for formation of UV photolesions. Irradiation of an oligonucleotide with unique TT dinucleotide resulted in generation of cyclobutane pyrimidine dimer with no evidence for induction of (6-4) photoproducts, whereas the analysis of irradiated TC-containing oligonucleotide detected (6-4) photoproducts but not cyclobutane pyrimidine dimers. Mobility shift assays have revealed that HMG1 protein binds preferentially to irradiated TT and TC oligonucleotides. Photoreversal of cyclobutane pyrimidine dimers with DNA photolyase and hydrolysis of the (6-4) photoproducts with hot alkali substantially reduced but did not eliminate binding of HMG1. The protein, therefore, appears to bind the two main types of UV damages in DNA, but some other photolesion(s) contributes to the preferential binding of HMG1 to irradiated DNA. By quantifying gel shift assays and considering the efficiencies of lesion formation, we determined dissociation constants of 1.2 +/- 0.5 and 4.0 +/- 1.5 microM for irradiated TT and TC oligonucleotides, respectively, and 70 +/- 20 microM for the control non-irradiated probes. Tryptic removal of the acidic COOH-terminal domain of HMG1 significantly affected binding of the protein to both irradiated and intact oligonucleotides. The potential role of HMG1 in recognition of the UV lesions in DNA is discussed. PMID:9733773

  14. Structural and functional studies of a large winged Z-DNA-binding domain of Danio rerio protein kinase PKZ.

    PubMed

    Subramani, Vinod Kumar; Kim, Doyoun; Yun, Kyunghee; Kim, Kyeong Kyu

    2016-07-01

    The Z-DNA-binding domain of PKZ from zebrafish (Danio rerio; drZαPKZ ) contains the largest β-wing among known Z-DNA-binding domains. To elucidate the functional implication of the β-wing, we solved the crystal structure of apo-drZαPKZ . Structural comparison with its Z-DNA-bound form revealed a large conformational change within the β-wing during Z-DNA binding. Biochemical studies of protein mutants revealed that two basic residues in the β-wing are responsible for Z-DNA recognition as well as fast B-Z transition. Therefore, the extra basic residues in the β-wing of drZαPKZ are necessary for the fast B-Z transition activity. PMID:27265117

  15. Tomato MBD5, a methyl CpG binding domain protein, physically interacting with UV-damaged DNA binding protein-1, functions in multiple processes.

    PubMed

    Li, Yuxiang; Deng, Heng; Miao, Min; Li, Huirong; Huang, Shengxiong; Wang, Songhu; Liu, Yongsheng

    2016-04-01

    In tomato (Solanum lycopersicum), high pigment mutations (hp-1 and hp-2) were mapped to genes encoding UV-damaged DNA binding protein 1 (DDB1) and de-etiolated-1 (DET1), respectively. Here we characterized a tomato methyl-CpG-binding domain protein SlMBD5 identified by yeast two-hybrid screening using SlDDB1 as a bait. Yeast two-hybrid assay demonstrated that the physical interaction of SlMBD5 with SlDDB1 is mediated by the C-termini of SlMBD5 and the β-propeller-C (BPC) of SlDDB1. Co-immunoprecipitation analyses revealed that SlMBD5 associates with SlDDB1-interacting partners including SlDET1, SlCUL4, SlRBX1a and SlRBX1b in vivo. SlMBD5 was shown to target to nucleus and dimerizes via its MBD motif. Electrophoresis mobility shift analysis suggested that the MBD of SlMBD5 specifically binds to methylated CpG dinucleotides but not to methylated CpHpG or CpHpH dinucleotides. SlMBD5 expressed in protoplast is capable of activating transcription of CG islands, whereas CUL4/DDB1 antagonizes this effect. Overexpressing SlMBD5 resulted in diverse developmental alterations including darker green fruits with increased plastid level and elevated pigmentation, as well as enhanced expression of SlGLK2, a key regulator of plastid biogenesis. Taken together, we hypothesize that the physical interaction of SlMBD5 with the CUL4-DDB1-DET1 complex component may affect its binding activity to methylated DNA and subsequently attenuate its transcription activation of downstream genes. PMID:26551231

  16. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    SciTech Connect

    Delorme, Caroline; Joshi, Monika; Allingham, John S.

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  17. Streptococcus gordonii collagen-binding domain protein CbdA may enhance bacterial survival in instrumented root canals ex vivo

    PubMed Central

    Moses, Peter J.; Power, Daniel A.; Jesionowski, Amy M.; Jenkinson, Howard F.; Pantera, Eugene A.; Vickerman, M. Margaret

    2012-01-01

    Introduction The surface-associated collagen-binding protein Ace of Enterococcus faecalis has been implicated as a virulence factor that contributes to bacterial persistence in endodontic infections. The purpose of this study was to determine if proteins with amino acid sequence similarity to Ace found in more abundant oral streptococci could play a similar role in potentially enhancing endodontic infections. Methods A Streptococcus gordonii gene similar to ace was identified by genome sequence searches in silico. An isogenic derivative of strain DL1 with a disruption in the identified gene was constructed by allelic replacement. Parent and mutant strains were characterized for their ability to bind immobilized collagen type-1 in a microtiter plate binding assay. Survival of the strains in a human tooth ex vivo instrumented root canal model was compared by inoculating canals with parental or mutant bacteria and determining the CFUs recovered at various time points over a 12-day period. Results The S. gordonii gene, encoding a protein with a conserved collagen-binding domain similar to that of Ace, was designated cbdA. The cbdA-deficient cells were less able to bind collagen type-1 than parental cells (P <0.0001). Genetic complementation of the cbdA-deficient strain restored the collagen-binding phenotype. By day 12 significantly fewer (P =0.03) cbdA-deficient than parental CFUs were recovered from instrumented canals. Conclusion A gene encoding a putative collagen-binding protein was identified in S. gordonii. Fewer S. gordonii cbdA-deficient cells survived ex vivo compared with parental cells, suggesting that collagen-binding proteins may contribute to persistence of oral streptococci in instrumented root canals. PMID:23228255

  18. Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

    NASA Technical Reports Server (NTRS)

    Huang, J. F.; Teyton, L.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

  19. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis.

    PubMed

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. PMID:26972250

  20. Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

    NASA Technical Reports Server (NTRS)

    Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

    2000-01-01

    Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

  1. The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal Domains*

    PubMed Central

    Dahlbäck, Madeleine; Jørgensen, Lars M.; Nielsen, Morten A.; Clausen, Thomas M.; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Arnot, David E.; Theander, Thor G.; Salanti, Ali

    2011-01-01

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. PMID:21398524

  2. Poly(A)-binding proteins: structure, domain organization, and activity regulation.

    PubMed

    Eliseeva, I A; Lyabin, D N; Ovchinnikov, L P

    2013-12-01

    RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA interactions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed. PMID:24490729

  3. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  4. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  5. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    PubMed Central

    2011-01-01

    Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket. PMID:21281498

  6. Dissection of the DNA binding domain of yeast Zn-finger protein Rme1p, a repressor of meiotic activator IME1.

    PubMed

    Shimizu, M; Hara, M; Murase, A; Shindo, H; Mitchell, A P

    1997-01-01

    A series of deletion mutants of the yeast Zn-finger protein Rme1p (Repressor of Meiosis) fused with maltose binding protein (MBP) were constructed, purified, and characterized to examine the DNA binding domain. It was shown by gel retardation assay that the DNA binding domain of Rme1p was attributed to C-terminal amino acid residues 171 to 300. All three Zn-fingers are involved in the DNA binding domain, but they are not sufficient for DNA binding ability. Notably, the C-terminal region (residues 285-300) is essential for DNA binding. Provided that the region folds into alpha-helix, the basic amino acid residues may form a ridge on one side of the helix, whereas the hydrophobic residues may form it on the other side. Thus, the DNA binding domain of Rme1p would be dissected two regions. The roles of C-terminal region in DNA recognition will be discussed. PMID:9586056

  7. The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator.

    PubMed

    Moser, T L; Enghild, J J; Pizzo, S V; Stack, M S

    1993-09-01

    This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix. PMID:8360181

  8. Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein, BCR, and Regulates Levels of Rac1-GTP*

    PubMed Central

    Gray, Elizabeth J.; Petsalaki, Evangelia; James, D. Andrew; Bagshaw, Richard D.; Stacey, Melissa M.; Rocks, Oliver; Gingras, Anne-Claude; Pawson, Tony

    2014-01-01

    SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein. PMID:25331951

  9. Insights into Duffy Binding-like Domains through the Crystal Structure and Function of the Merozoite Surface Protein MSPDBL2 from Plasmodium falciparum*

    PubMed Central

    Hodder, Anthony N.; Czabotar, Peter E.; Uboldi, Alessandro D.; Clarke, Oliver B.; Lin, Clara S.; Healer, Julie; Smith, Brian J.; Cowman, Alan F.

    2012-01-01

    Invasion of human red blood cells by Plasmodium falciparum involves interaction of the merozoite form through proteins on the surface coat. The erythrocyte binding-like protein family functions after initial merozoite interaction by binding via the Duffy binding-like (DBL) domain to receptors on the host red blood cell. The merozoite surface proteins DBL1 and -2 (PfMSPDBL1 and PfMSPDBL2) (PF10_0348 and PF10_0355) are extrinsically associated with the merozoite, and both have a DBL domain in each protein. We expressed and refolded recombinant DBL domains for PfMSPDBL1 and -2 and show they are functional. The red cell binding characteristics of these domains were shown to be similar to full-length forms of these proteins isolated from parasite cultures. Futhermore, metal cofactors were found to enhance the binding of both the DBL domains and the parasite-derived full-length proteins to erythrocytes, which has implications for receptor binding of other DBL-containing proteins in Plasmodium spp. We solved the structure of the erythrocyte-binding DBL domain of PfMSPDBL2 to 2.09 Å resolution and modeled that of PfMSPDBL1, revealing a canonical DBL fold consisting of a boomerang shaped α-helical core formed from three subdomains. PfMSPDBL2 is highly polymorphic, and mapping of these mutations shows they are on the surface, predominantly in the first two domains. For both PfMSPDBL proteins, polymorphic variation spares the cleft separating domains 1 and 2 from domain 3, and the groove between the two major helices of domain 3 extends beyond the cleft, indicating these regions are functionally important and are likely to be associated with the binding of a receptor on the red blood cell. PMID:22843685

  10. A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication▿

    PubMed Central

    Sklan, Ella H.; Staschke, Kirk; Oakes, Tina M.; Elazar, Menashe; Winters, Mark; Aroeti, Benjamin; Danieli, Tsafi; Glenn, Jeffrey S.

    2007-01-01

    Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population. PMID:17686842

  11. Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain

    PubMed Central

    Buczek, Pawel; Horvath, Martin P.

    2010-01-01

    The Oxytricha nova telomere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (ΔH), entropy (ΔS), and dissociation constant (KD-DNA) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T2G4), d(T4G4), d(G3T4G4), and d(G4T4G4) each formed monovalent protein complexes. In the case of d(T4G4T4G4), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity “A site” has a dissociation constant, KD-DNA(A)=13(±4) nM, while the low-affinity “B site” is characterized by KD-DNA(B)=5600(±600) nM at 25 °C. Nucleotide substitution variants verified that the A site corresponds principally with the 3′-terminal portion of d(T4G4T4G4). The relative contributions of entropy (ΔS) and enthalpy (ΔH) for binding reactions were DNA length-dependent as was heat capacity (ΔCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA–protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology. PMID:16678852

  12. Selective CGRP and adrenomedullin peptide binding by tethered RAMP-calcitonin receptor-like receptor extracellular domain fusion proteins

    PubMed Central

    Moad, Heather E; Pioszak, Augen A

    2013-01-01

    Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries. PMID:24115156

  13. P-glycoprotein substrate binding domains are located at the transmembrane domain/transmembrane domain interfaces: a combined photoaffinity labeling-protein homology modeling approach.

    PubMed

    Pleban, Karin; Kopp, Stephan; Csaszar, Edina; Peer, Michael; Hrebicek, Thomas; Rizzi, Andreas; Ecker, Gerhard F; Chiba, Peter

    2005-02-01

    P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps. PMID:15509712

  14. The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions.

    PubMed

    Dramićanin, Marija; López-Méndez, Blanca; Boskovic, Jasminka; Campos-Olivas, Ramón; Ramón-Maiques, Santiago

    2015-08-01

    MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition. PMID:26169224

  15. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    PubMed Central

    Dahms, Sven O.; Mayer, Magnus C.; Roeser, Dirk; Multhaup, Gerd; Than, Manuel E.

    2015-01-01

    Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2)2–(heparin)2 complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins. PMID:25760599

  16. Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

    PubMed Central

    Yang, C H; Tomkiel, J; Saitoh, H; Johnson, D H; Earnshaw, W C

    1996-01-01

    The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain. PMID:8668174

  17. GTPase Activating Protein (Sh3 Domain) Binding Protein 1 Regulates the Processing of MicroRNA-1 during Cardiac Hypertrophy

    PubMed Central

    He, Minzhen; Yang, Zhi; Abdellatif, Maha; Sayed, Danish

    2015-01-01

    Background MicroRNAs (miR) are small, posttranscriptional regulators, expressed as part of a longer primary transcript, following which they undergo nuclear and cytoplasmic processing by Drosha and Dicer, respectively, to form the functional mature ~20mer that gets incorporated into the silencing complex. Others and we have shown that mature miR-1 levels decrease with pressure-induced cardiac hypertrophy, however, there is little or no change in the primary transcript encompassing miR-1 stem-loop, suggesting critical regulatory step in microRNA processing. The objective of this study was to investigate the underlying mechanisms regulating miR-1 expression in cardiomyocytes. Results Here we report that GTPase–activating protein (SH3 domain) binding protein 1 (G3bp1), an endoribonuclease regulates miR-1 processing in cardiomyocytes. G3bp1 is upregulated during cardiac hypertrophy and restricts miR-1 processing by binding to its consensus sequence in the pre-miR-1-2 stem-loop. In accordance, exogenous G3bp1 is sufficient to reduce miR-1 levels, along with derepression of miR-1 targets; General transcription factor IIB (Gtf2b), cyclin dependent factor 9 (Cdk9) and eukaryotic initiation factor 4E (Eif4e). While Cdk9 and Gtf2b are essential for transcription, Eif4e is required for translation. Thus, downregulation of miR-1 is necessary for increase in these molecules. Similar to miR-1 knockdown, G3bp1 overexpression is not sufficient for development of cardiac hypertrophy. Conversely, knockdown of G3bp1 in hypertrophying cardiomyocytes inhibited downregulation of miR-1 and upregulation of its targets along with restricted hypertrophy, suggesting that G3bp1 is necessary for development of cardiac hypertrophy. These results indicate that G3bp1-mediated inhibition of miR-1 processing with growth stimulation results in decrease in mature miR-1 and, thereby, an increase of its targets, which play fundamental roles in the development of hypertrophy. Conclusion G3bp1

  18. The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein.

    PubMed Central

    Borg, J P; Ooi, J; Levy, E; Margolis, B

    1996-01-01

    The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP. PMID:8887653

  19. A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties

    PubMed Central

    Unverdorben, Felix; Hutt, Meike; Seifert, Oliver; Kontermann, Roland E.

    2015-01-01

    Background Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions. Methodology/Principal Findings Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics. Conclusions/Significance The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity. PMID:26430884

  20. Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

    NASA Astrophysics Data System (ADS)

    Liu, Feng; Roth, Richard A.

    1995-10-01

    To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

  1. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    SciTech Connect

    Dahms, Sven O.; Mayer, Magnus C.; Roeser, Dirk; Multhaup, Gerd; Than, Manuel E.

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  2. Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain

    PubMed Central

    Mao, Xiaoyan; Zhang, Tiancheng; Ji, Guanghui; Shi, Xin; Xia, Tian; Lu, Weijia; Zhang, Dapeng; Dai, Jianxin; Guo, Yajun

    2012-01-01

    Background Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed. Methods and Findings We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo. Conclusions The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes. PMID:22952786

  3. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev

    PubMed Central

    Cooper, Christopher D. O.; Newman, Joseph A.; Aitkenhead, Hazel; Allerston, Charles K.; Gileadi, Opher

    2015-01-01

    Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40–200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors. PMID:25866208

  4. Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

    PubMed Central

    Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

    2015-01-01

    Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

  5. Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain.

    PubMed

    Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A; Chen, Yuxing; Zhou, Cong-Zhao

    2015-01-01

    Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

  6. Electrophoretic behavior of DNA-methyl-CpG-binding domain protein complexes revealed by capillary electrophoreses laser-induced fluorescence.

    PubMed

    Zhong, Shangwei; Zou, Dandan; Zhao, Bailin; Zhang, Dapeng; Li, Xiangjun; Wang, Hailin

    2015-12-01

    The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors. PMID:26377303

  7. The first α-helical domain of the vesicle-inducing protein in plastids 1 promotes oligomerization and lipid binding.

    PubMed

    Otters, Stephanie; Braun, Paula; Hubner, Johanna; Wanner, Gerhardt; Vothknecht, Ute C; Chigri, Fatima

    2013-02-01

    The vesicle-inducing protein in plastids 1 (Vipp1) is an essential component for thylakoid biogenesis in cyanobacteria and chloroplasts. Vipp1 proteins share significant structural similarity with their evolutionary ancestor PspA (bacterial phage shock protein A), namely a predominantly α-helical structure, the formation of oligomeric high molecular weight complexes (HMW-Cs) and a tight association with membranes. Here, we elucidated domains of Vipp1 from Arabidopsis thaliana involved in homo-oligomerization as well as association with chloroplast inner envelope membranes. We could show that the 21 N-terminal amino acids of Vipp1, which form the first α-helix of the protein, are essential for assembly of the 2 MDa HMW-C but are not needed for formation of smaller subcomplexes. Interestingly, removal of this domain also interferes with association of the Vipp1 protein to the inner envelope. Fourier transform infrared spectroscopy of recombinant Vipp1 further indicates that Escherichia coli lipids bind tightly enough that they can be co-purified with the protein. This feature also depends on the presence of the first helix, which strongly supports an interaction of lipids with the Vipp1 HMW-C but not with smaller subcomplexes. Therefore, Vipp1 oligomerization appears to be a prerequisite for its membrane association. Our results further highlight structural differences between Vipp1 and PspA, which might be important in regard to their different function in thylakoid biogenesis and bacterial stress response, respectively. PMID:23053543

  8. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    SciTech Connect

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  9. Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

    PubMed

    Aarabi, Mahmoud; Balakier, Hanna; Bashar, Siamak; Moskovtsev, Sergey I; Sutovsky, Peter; Librach, Clifford L; Oko, Richard

    2014-10-01

    Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.- PMID:24970390

  10. Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

    SciTech Connect

    Yamada, Mototsugu Watanabe, Takashi; Baba, Nobuyoshi; Miyara, Takako; Saito, Jun; Takeuchi, Yasuo

    2008-04-01

    The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 86.39, c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent.

  11. In Caenorhabditis elegans, the RNA-binding domains of the cytoplasmic polyadenylation element binding protein FOG-1 are needed to regulate germ cell fates.

    PubMed Central

    Jin, S W; Arno, N; Cohen, A; Shah, A; Xu, Q; Chen, N; Ellis, R E

    2001-01-01

    FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3'-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself. PMID:11779801

  12. The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein.

    PubMed

    Suzuki, Hidetaka; Yamada, Seiko; Toyama, Yoshiharu; Takeda, Shigeki

    2010-09-01

    The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified. PMID:20478417

  13. Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion

    PubMed Central

    Watkins, Harriet A.; Baker, Edward N.

    2008-01-01

    The predicted ribonuclease (RNase) HI domain of the open reading frame Rv2228c from Mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (MBP) fusion. Expression was only observed for the MBP-fusion protein, which was purified using amylose affinity chromatography and gel filtration. The RNase HI domain could be cleaved from the MBP-fusion protein by factor Xa digestion, but was very unstable. In contrast, the fusion protein was stable, could be obtained in high yield and gave crystals which diffracted to 2.25 Å resolution. The crystals belong to space group P21 and have unit-cell parameters a = 73.63, b = 101.38, c = 76.09 Å, β = 109.0°. Two fusion-protein molecules of 57 417 Da were present in each asymmetric unit. PMID:18678948

  14. Protein Dynamics of the HIF-2α PAS-B Domain upon Heterodimerization and Ligand Binding

    PubMed Central

    Masetti, Matteo; Falchi, Federico; Recanatini, Maurizio

    2014-01-01

    Hypoxia-Inducible Factor (HIF) transcription factors are heterodimeric proteins involved in the regulation of oxygen homeostatis. Their upregulation has been related to several tumors with a remarkably poor clinical outcome. The recent discovery of a druggable cavity in the HIF-2α PAS-B domain has opened an unprecedented opportunity for targeting the HIF-2α transcription factor in view of pharmaceutical strategies. Coincidentally, a novel compound able to selectively disrupt the HIF heterodimerization with a submicromolar activity has been reported. In this work, we investigated the molecular mechanisms responsible for the inhibition by comparing the dynamical features of the HIF-2α PAS-B monomer and the HIF-2α PAS-B/HIF-1β PAS-B complex, in the ligand-bound and -unbound states. Plain and biased Molecular Dynamics were used to characterize the differential conformational changes both structurally and energetically. PMID:24736273

  15. The immunosuppressive domain of the transmembrane envelope protein gp41 of HIV-1 binds to human monocytes and B cells.

    PubMed

    Mühle, Michael; Kroniger, Tobias; Hoffmann, Kerstin; Denner, Joachim

    2016-06-01

    The induction of the acquired immunodeficiency syndrome by the human immunodeficiency virus-1 (HIV-1) is a complex process which is not yet understood in full detail. Still open is the question whether the highly conserved so-called immunosuppressive (Isu) domain in the transmembrane envelope (TM) protein gp41 of HIV-1 is actively participating in immunopathogenesis. Inactivated virus particles, recombinant gp41 and peptides corresponding to the Isu domain have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. Here we demonstrate, using fluorescence-activated cell sorting and competition experiments, that homopolymers of the Isu peptide of HIV-1 are binding specifically to human peripheral blood mononuclear cells, mainly to monocytes and B cells. These data suggest that a putative receptor might be involved in the immunomodulatory effects observed previously. PMID:26754765

  16. Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

    PubMed Central

    Kim, Doyoun; Hur, Jeonghwan; Park, Kwangsoo; Bae, Sangsu; Shin, Donghyuk; Ha, Sung Chul; Hwang, Hye-Yeon; Hohng, Sungchul; Lee, Joon-Hwa; Lee, Sangho; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2014-01-01

    Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate. PMID:24682817

  17. A highly conserved region of the Sendai virus nucleocapsid protein contributes to the NP-NP binding domain.

    PubMed

    Myers, T M; Pieters, A; Moyer, S A

    1997-03-17

    The nucleocapsid protein (NP) of Sendai virus is an essential component of both the nucleocapsid template and the NP-NP and NP0-P protein complexes required for viral RNA replication. When expressed alone in mammalian cells NP self-assembles into nucleocapsid-like particles which appear to contain cellular RNA. To identify putative NP-NP binding domains, fusions between the monomeric maltose-binding protein (MBP) and portions of NP were constructed. The fusion proteins which contain the central conserved region (CCR) (amino acids 258-357, MBP-NP1) and the N-terminal 255 amino acids (MBP-NP2) of NP both oligomerized, suggesting that these regions contain sequences important for NP-NP self-assembly. In addition, the MBP-NP1 fusion protein can function as an inhibitor of viral RNA replication. Complementary studies involving site-directed mutagenesis of the full-length NP protein have identified specific residues in the CCR which are essential for viral RNA replication in vitro. Two such replication-negative mutants, F324V and F324I, were defective in self-assembly, suggesting that the Phe residue at amino acid 324 is essential for the NP-NP interaction. A third mutant, NP260-1 (Y260D), self-assembled to form aberrant oligomers which exhibit an unusual helical structure and appear to lack any associated RNA. The mutants NP299-5 (L299I and I300V) and NP313-2 (I313F), in contrast, appear to form all the required protein complexes, but were inactive in viral RNA replication, suggesting that interactions specifically with Sendai RNA were disrupted. These data have thus identified specific residues in the CCR of the native NP protein which appear to be important for NP-NP or NP-RNA interactions and for genome replication. PMID:9126246

  18. Identification of co-evolving sites in the ligand binding domain of G protein-coupled receptors using mutual information

    NASA Astrophysics Data System (ADS)

    Fatakia, Sarosh N.; Costanzi, Stefano; Chow, Carson C.

    2008-03-01

    G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in humans. They are involved in signal transduction in numerous cellular processes and are the most common target for pharmacological intervention via activation or inhibition. Identification of functionally important sites is relevant for better understanding the ligand-receptor interaction and therefore for drug delivery. In a superfamily of proteins, functionally important but co-evolving sites are not easily identified in a multiple sequence alignment (MSA). Using a MSA of trans-membrane (TM) domains of GPCR superfamily, we identify sites which co-evolve, and may therefore be functionally important. Assigning the TM site as a node and the MI of site pairs as an inverse inter-node distance, a MI graph is established. Co-evolving sites are then identified via this graph. Nodes characterized by high connectivity are located within the commonly accepted ligand binding site of GPCRs, suggesting that concerted co-evolution of a number of neighboring residues gave rise to a multitude of subfamilies each recognizing a specific set of ligands. MI and graph analysis may serve as a tool for the identification of topologically conserved binding pockets in the families of evolutionarily related proteins.

  19. Quantitative analysis of multisite protein ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP

    PubMed Central

    Arai, Munehito; Ferreon, Josephine C.; Wright, Peter E.

    2012-01-01

    Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition, and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and lineshapes show that a primary dissociation constant as small as 1~10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 1010 M−1 s−1, approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions. PMID:22280219

  20. Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation*

    PubMed Central

    DeSantis, Morgan E.; Sweeny, Elizabeth A.; Snead, David; Leung, Eunice H.; Go, Michelle S.; Gupta, Kushol; Wendler, Petra; Shorter, James

    2014-01-01

    The homologous hexameric AAA+ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis. PMID:24280225

  1. Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

    PubMed

    Vetter, S W; Leclerc, E

    2001-08-01

    We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides. PMID:11488924

  2. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure.

    PubMed

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M Cristina

    2016-03-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  3. DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin

    PubMed Central

    Nowicka, Urszula; Zhang, Daoning; Walker, Olivier; Krutauz, Daria; Castañeda, Carlos A.; Chaturvedi, Apurva; Chen, Tony Y.; Reis, Noa; Glickman, Michael H.; Fushman, David

    2015-01-01

    SUMMARY Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle. PMID:25703377

  4. Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess six-cysteine-containing chitin-binding domains (CBDs) related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of...

  5. Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains*

    PubMed Central

    Barnes, Mark; van Rensburg, Gerrit; Li, Wai-Ming; Mehmood, Kashif; Mackedenski, Sebastian; Chan, Ching-Man; King, Dustin T.; Miller, Andrew L.; Lee, Chow H.

    2015-01-01

    The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function. PMID:25389298

  6. Kinetics of the Antibody Recognition Site in the Third IgG-Binding Domain of Protein G.

    PubMed

    Pratihar, Supriya; Sabo, T Michael; Ban, David; Fenwick, R Bryn; Becker, Stefan; Salvatella, Xavier; Griesinger, Christian; Lee, Donghan

    2016-08-01

    Protein dynamics occurring on a wide range of timescales play a crucial role in governing protein function. Particularly, motions between the globular rotational correlation time (τc ) and 40 μs (supra-τc window), strongly influence molecular recognition. This supra-τc window was previously hidden, owing to a lack of experimental methods. Recently, we have developed a high-power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 μs. For the first time, this method, performed under super-cooled conditions, enabled us to detect a global motion in the first β-turn of the third IgG-binding domain of protein G (GB3), which was extrapolated to 371±115 ns at 310 K. Furthermore, the same residues show the plasticity in the model-free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra-τc dynamics. This β-turn is involved in antibody binding, exhibiting the potential link of the observed supra-τc motion with molecular recognition. PMID:27345359

  7. Common antigenic domains in transferrin-binding protein 2 of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae type b.

    PubMed

    Stevenson, P; Williams, P; Griffiths, E

    1992-06-01

    There is now considerable evidence to show that in the Neisseria and Haemophilus species, membrane receptors specific for either transferrin or lactoferrin are involved in the acquisition of iron from these glycoproteins. In Neisseria meningitidis, the transferrin receptor appears to consist of two proteins, one of which (TBP 1) has an M(r) of 95,000 and the other of which (TBP 2) has an M(r) ranging from 68,000 to 85,000, depending on the strain; TBP 2 binds transferrin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, but TBP 1 does not do so. The relative contributions of these two proteins to the binding reaction observed with intact cells and to iron uptake are presently unknown. However, they are being considered as potential components of a group B meningococcal vaccine. Analogous higher- and lower-molecular-weight proteins associated with transferrin binding have been found in N. gonorrhoeae and Haemophilus influenzae. Previous work with polyclonal antibodies raised in mice with whole cells of iron-restricted N. meningitidis showed that the meningococcal TBP 2 exhibits considerable antigenic heterogeneity. Here, we report that antiserum against purified TBP 2 from one strain of N. meningitidis cross-reacts on immunoblotting with the TBP 2 of all meningococcal isolates examined, as well as with the TBP 2 of N. gonorrhoeae. This antiserum also cross-reacted with the TBP 2 of several strains of H. influenzae type b, thus showing the presence of common antigenic domains among these functionally equivalent proteins in different pathogens; no cross-reaction was detected with a purified sample of the human transferrin receptor. PMID:1587606

  8. DNA binding by the ETS-domain transcription factor PEA3 is regulated by intramolecular and intermolecular protein.protein interactions.

    PubMed

    Greenall, A; Willingham, N; Cheung, E; Boam, D S; Sharrocks, A D

    2001-05-11

    The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly. PMID:11278941

  9. Chromodomain-helicase-DNA binding protein 5, 7 and pronecrotic mixed lineage kinase domain-like protein serve as potential prognostic biomarkers in patients with resected pancreatic adenocarcinomas

    PubMed Central

    Seldon, Crystal S; Colbert, Lauren E; Hall, William A; Fisher, Sarah B; Yu, David S; Landry, Jerome C

    2016-01-01

    Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomain-helicase-DNA binding protein 5, chromodomain-helicase-DNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation, and should be validated in future studies. PMID:27096031

  10. Analysis of ligand binding and protein dynamics of human retinoid X receptor alpha ligand-binding domain by nuclear magnetic resonance.

    PubMed

    Lu, Jianyun; Cistola, David P; Li, Ellen

    2006-02-14

    Retinoid X receptors (RXRs) are nuclear receptors that can activate transcription as homodimers or as obligate heterodimeric partners of other nuclear receptors. While the crystal structures of the RXR ligand-binding domains (LBD) have been previously determined, the dynamics of activation is less well characterized at an atomic level. To probe the effect of ligand binding on RXR LBD dynamics, we initiated nuclear magnetic resonance studies of recombinant human RXRalpha LBD (T223-T462) with and without bound 9-cis-retinoic acid (9cRA). The 1HN, 15N, 13C(alpha), 13CO, and 13C(beta) resonance assignments were established for 164 of 240 residues in apo-RXRalpha LBD. Resonances corresponding to an additional 47 residues emerged upon 9cRA binding. These additional residues included those located in the vicinity of the ligand-binding pocket (helices H3, H5, and strands S1, S2), as well as residues located at the dimerization interface (helices H9 and H10). Thus 9cRA binding stabilized the ligand-binding pocket and had allosteric effects on the dimerization interface. Ligand-induced chemical shift perturbations outside the binding cavity were mapped to helix H3 and the AF-2 helix H12, indicating conformational changes in these regions. However, helix H11, a component of the tetramerization interface, and a large part of helix H10, a component of the dimerization interface, remained undetectable even after 9cRA binding. Although apo- and holo-hRXRalpha LBD existed predominantly as homodimers in solution, exchange between monomeric, dimeric, and tetrameric forms of the protein could have contributed to line broadening of cross-peaks corresponding to helices H10 and H11. 15N T1, T2, and steady-state {1H}-15N NOE data collected at 500 and 700 MHz static magnetic fields showed that the internal motions for the residues in the H1-H3 loop (K245-D263) were much less restricted than those in the protein core for both apo- and holo-forms. Significant exchange R(ex) contributions to

  11. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

    SciTech Connect

    He Yuxian . E-mail: yhe@nybloodcenter.org; Li Jingjing; Jiang Shibo

    2006-05-26

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

  12. Preclinical assessment of the receptor-binding domain of Plasmodium vivax Duffy-binding protein as a vaccine candidate in rhesus macaques.

    PubMed

    Moreno, A; Caro-Aguilar, I; Yazdani, S S; Shakri, A R; Lapp, S; Strobert, E; McClure, H; Chitnis, C E; Galinski, M R

    2008-08-12

    The receptor-binding domain of Plasmodium vivax Duffy-binding protein, region II (PvRII), is an attractive candidate for a vaccine against P. vivax malaria. Here, we have studied the safety and immunogenicity of recombinant PvRII in Macaca mulatta (rhesus monkeys). Recombinant PvRII with a C-terminal 6-histidine tag was expressed in E. coli, recovered from inclusion bodies, refolded into its functional conformation, purified to homogeneity and formulated with three adjuvants, namely, Alhydrogel, Montanide ISA 720 and the GSK proprietary Adjuvant System AS02A for use in immunogenicity studies. All the PvRII vaccine formulations tested were safe and highly immunogenic. The overall magnitude of the antibody response was significantly higher for both Montanide ISA 720 and AS02A formulations in comparison with Alhydrogel. Furthermore, there was a significant correlation between antibody recognition titers by ELISA and binding inhibition titers in in vitro binding assays. The PvRII vaccine formulations also induced IFN-gamma recall responses that were identified using ex vivo ELISPOT assays. These results provide support for further clinical development of a vaccine for P. vivax malaria based on recombinant PvRII. PMID:18573299

  13. Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity*

    PubMed Central

    Dick, Alexej; Graf, Laura; Olal, Daniel; von der Malsburg, Alexander; Gao, Song; Kochs, Georg; Daumke, Oliver

    2015-01-01

    Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action. PMID:25829498

  14. Role of nucleotide binding and GTPase domain dimerization in dynamin-like myxovirus resistance protein A for GTPase activation and antiviral activity.

    PubMed

    Dick, Alexej; Graf, Laura; Olal, Daniel; von der Malsburg, Alexander; Gao, Song; Kochs, Georg; Daumke, Oliver

    2015-05-15

    Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action. PMID:25829498

  15. High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold.

    PubMed

    Dryla, Agnieszka; Hoffmann, Bernd; Gelbmann, Dieter; Giefing, Carmen; Hanner, Markus; Meinke, Andreas; Anderson, Annaliesa S; Koppensteiner, Walter; Konrat, Robert; von Gabain, Alexander; Nagy, Eszter

    2007-01-01

    Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins. PMID:17041047

  16. A Conserved alpha-helical motif mediates the binding of diverse nuclear proteins to the SRC1 interaction domain of CBP.

    PubMed

    Matsuda, Sachiko; Harries, Janet C; Viskaduraki, Maria; Troke, Philip J F; Kindle, Karin B; Ryan, Colm; Heery, David M

    2004-04-01

    CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the alpha-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERalpha/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the alpha-helix Aalpha1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic alpha-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP. PMID:14722092

  17. Diversity in protein recognition by PTB domains.

    PubMed

    Forman-Kay, J D; Pawson, T

    1999-12-01

    Phosphotyrosine-binding (PTB) domains were originally identified as modular domains that recognize phosphorylated Asn-Pro-Xxx-p Tyr-containing proteins. Recent binding and structural studies of PTB domain complexes with target peptides have revealed a number of deviations from the previously described mode of interaction, with respect to both the sequences of possible targets and their structures within the complexes. This diversity of recognition by PTB domains extends and strengthens our general understanding of modular binding domain recognition. PMID:10607674

  18. FhCaBP2: a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains.

    PubMed

    Thomas, Charlotte M; Timson, David J

    2015-09-01

    FhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles. PMID:26152524

  19. The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

    SciTech Connect

    Loughlin, Fionna E.; Mansfield, Robyn E.; Vaz, Paula M.; McGrath, Aaron P.; Setiyaputra, Surya; Gamsjaeger, Roland; Chen, Eva S.; Morris, Brian J.; Guss, J. Mitchell; Mackay, Joel P.

    2009-09-02

    The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N{sub x})AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5{prime} splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine 'ladder.' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

  20. Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway. PMID:22303012

  1. Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)*

    PubMed Central

    Wang, Wuyang; Linsdell, Paul

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between “outwardly facing” and “inwardly facing” conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)2− ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)2−, access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional (“vestigial”) gates within the CFTR permeation pathway. PMID:22303012

  2. Crystal and solution studies of the "Plus-C" odorant-binding protein 48 from Anopheles gambiae: control of binding specificity through three-dimensional domain swapping.

    PubMed

    Tsitsanou, Katerina E; Drakou, Christina E; Thireou, Trias; Vitlin Gruber, Anna; Kythreoti, Georgia; Azem, Abdussalam; Fessas, Dimitrios; Eliopoulos, Elias; Iatrou, Kostas; Zographos, Spyros E

    2013-11-15

    Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases. PMID:24097978

  3. African Swine Fever Virus Protein A238L Interacts with the Cellular Phosphatase Calcineurin via a Binding Domain Similar to That of NFAT

    PubMed Central

    Miskin, James E.; Abrams, Charles C.; Dixon, Linda K.

    2000-01-01

    The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-κB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L. PMID:11000210

  4. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    PubMed Central

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  5. Crystal Structure of a Histidine Kinase Sensor Domain with Similarity to Periplasmic Binding Proteins

    SciTech Connect

    Cheung, J.; Le-Khac, M; Hendrickson, W

    2009-01-01

    Histidine kinase receptors are elements of the two-component signal transduction systems commonly found in bacteria and lower eukaryotes, where they are crucial for environmental adaption through the coupling of extracellular changes to intracellular responses. The typical two-component system consists of a membrane-spanning histidine kinase sensor and a cytoplasmic response regulator. In the calssic system, extracellular signals such as small molecule ligands and ions are detected by the periplasmic sensor domain of the histidine kinase receptor, which modulates the catalytic activity of the cytoplasmic histidine kinase domain and promotes ATP-dependent autophosphorylation of a conserved histidine residue. G. sulfurreducens genomic DNA was used.

  6. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    SciTech Connect

    Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Fujiwara, Toshinobu; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  7. Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.

    PubMed

    Wang, Hong; Hu, Liping; Dalen, Knut; Dorward, Heidi; Marcinkiewicz, Amy; Russell, Deanna; Gong, Dawei; Londos, Constantine; Yamaguchi, Tomohiro; Holm, Cecilia; Rizzo, Mark A; Brasaemle, Dawn; Sztalryd, Carole

    2009-11-13

    Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein. PMID:19717842

  8. Methyl-CpG-binding domain protein MBD7 is required for active DNA demethylation in Arabidopsis.

    PubMed

    Wang, Chunlei; Dong, Xiaomei; Jin, Dan; Zhao, Yusheng; Xie, Shaojun; Li, Xiaojie; He, Xinjian; Lang, Zhaobo; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2015-03-01

    Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (Arabidopsis thaliana) line expressing the Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) and Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenes, we isolated an mbd7 (for methyl-CpG-binding domain protein7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis. PMID:25593350

  9. The C-terminal Domain Supports a Novel Function for CETPI as a New Plasma Lipopolysaccharide-Binding Protein

    PubMed Central

    García-González, Victor; Gutiérrez-Quintanar, Nadia; Mas-Oliva, Jaime

    2015-01-01

    Described by our group a few years ago, the cholesteryl-ester transfer protein isoform (CETPI), exclusively expressed in the small intestine and present in human plasma, lacked a functional identification for a role of physiological relevance. Now, this study introduces CETPI as a new protein with the potential capability to recognise, bind and neutralise lipopolysaccharides (LPS). Peptides derived from the C-terminal domain of CETPI showed that CETPI not only might interact with several LPS serotypes but also might displace LPS bound to the surface of cells. Peptide VSAK, derived from the last 18 residues of CETPI, protected against the cytotoxic effect of LPS on macrophages. At high concentrations, when different cell types were tested in culture, it did not exhibit cytotoxicity by itself and it did prevent the expression of pro-inflammatory cytokines as well as the generation of oxidative stress conditions. In a rabbit model of septic shock, the infusion of peptide VSAK exerted a protective effect against the effects of LPS and reduced the presence of tumor necrosis factor-alpha (TNFα) in plasma. Therefore, CETPI is proposed as a new protein with the capability to advance the possibilities for better understanding and treatment of the dangerous effects of LPS in vivo. PMID:26537318

  10. Structure and dynamics of the N-terminal domain of the Cu(I) binding protein CusB.

    PubMed

    Ucisik, Melek N; Chakravorty, Dhruva K; Merz, Kenneth M

    2013-10-01

    CusCFBA is one of the metal efflux systems in Escherichia coli that is highly specific for its substrates, Cu(I) and Ag(I). It serves to protect the bacteria in environments that have lethal concentrations of these metals. The membrane fusion protein CusB is the periplasmic piece of CusCFBA, which has not been fully characterized by crystallography because of its extremely disordered N-terminal region. This region has both structural and functional importance because it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. Understanding metal uptake from the periplasm is critical to gain insight into the mechanism of the whole CusCFBA pump, which makes resolving a structure for the N-terminal region necessary because it contains the metal binding site. We ran extensive molecular dynamics simulations to reveal the structural and dynamic properties of both the apo and Cu(I)-bound versions of the CusB N-terminal region. In contrast to its functional companion CusF, Cu(I) binding to the N-terminus of CusB causes only a slight, local stabilization around the metal site. The trajectories were analyzed in detail, revealing extensive structural disorder in both the apo and holo forms of the protein. CusB was further analyzed by breaking the protein up into three subdomains according to the extent of the observed disorder: the N- and C-terminal tails, the central beta strand motif, and the M21-M36 loop connecting the two metal-coordinating methionine residues. Most of the observed disorder was traced back to the tail regions, leading us to hypothesize that the latter two subdomains (residues 13-45) may form a functionally competent metal-binding domain because the tail regions appear to play no role in metal binding. PMID:23988152

  11. Structure and Dynamics of the N-terminal Domain of the Cu(I) Binding Protein CusB

    PubMed Central

    Ucisik, Melek N.; Chakravorty, Dhruva K.; Merz, Kenneth M.

    2013-01-01

    CusCFBA is one of the metal efflux systems in Escherichia coli, which is highly specific for its substrates Cu(I) and Ag(I). It serves to protect the bacteria in environments that have lethal concentrations of these metals. The membrane fusion protein CusB is the periplasmic piece of CusCFBA, which has not been fully characterized by crystallography due to its extremely disordered N-terminal region. This region has both structural and functional importance as it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. Understanding metal uptake from the periplasm is critical to gain an insight to the mechanism of the whole CusCFBA pump, which makes resolving a structure for the N-terminal region necessary as it contains the metal binding site. We ran extensive molecular dynamics simulations to reveal the structural and dynamic properties of both apo and Cu(I)-bound versions of the CusB N-terminal region. In contrast to its functional companion CusF, Cu(I)-binding to the N-terminal of CusB causes only a slight, local stabilization around the metal site. The trajectories were analyzed in detail revealing extensive structural disorder in both the apo and holo forms of the protein. CusB was further analyzed by breaking the protein up into three subdomains according to the extent of the observed disorder: the N- and C-terminal tails, the central beta strand motif, and the M21–M36 loop connecting the two metal-coordinating methionine residues. Most of the observed disorder was traced back to the tail regions leading us to hypothesize that the latter two subdomains (residues 13–45) may form a functionally competent metal binding domain as the tail regions appear to play no role in metal binding. PMID:23988152

  12. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    PubMed

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M

    2016-05-20

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  13. Impaired memory CD8 T cell development in the absence of methyl-CpG-binding domain protein 2.

    PubMed

    Kersh, Ellen N

    2006-09-15

    Intracellular differentiation events that determine which cells develop into memory CD8 T cells are currently incompletely understood. Methyl-CpG-binding domain protein 2 (MBD2) is a transcriptional repressor that binds to methylated DNA and mediates the biological consequences of epigenetic gene methylation. The role of MBD2 during the differentiation of naive CD8 T cells into effector and memory cells was determined following acute infection of MBD2-deficient mice with lymphocytic choriomeningitis virus. Despite rapid viral clearance and an efficient primary effector CD8 T cell response, reduced numbers of Ag-specific memory CD8 T cells were observed. Importantly, the appearance of precursor memory cells (IL-7Ralphahigh) was delayed. The remaining MBD2(-/-) memory cells were not fully protective during rechallenge, and memory cell characteristics were altered with regard to surface markers (IL-7Ralpha, KLRG-1, CD27, and others) and cytokine production. The defect was CD8 T cell intrinsic, because memory cell development was also delayed when MBD2(-/-) CD8 T cells were adoptively transferred into SCID mice. These data demonstrate that MBD2 is a previously unrecognized intracellular factor required for the efficient generation of protective memory CD8 T cells. PMID:16951344

  14. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. PMID:24862193

  15. Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein.

    PubMed Central

    Emili, A; Greenblatt, J; Ingles, C J

    1994-01-01

    We have used protein-blotting and protein affinity chromatography to demonstrate that each of the two glutamine-rich activation domains of the human transcription factor Sp1 can bind specifically and directly to the C-terminal evolutionarily conserved domain of the human TATA box-binding protein (TBP). These activation domains of Sp1 also bind directly to Drosophila TBP but bind much less strongly to TBP from the yeast Saccharomyces cerevisiae. The abilities of the Sp1 activation domains to interact directly with the TBPs of various species correlate well with their abilities to activate transcription in extracts derived from the same species. We also show that a glutamine-rich transcriptional activating region of the Drosophila protein Antennapedia binds directly to TBP in a species-specific manner that reflects its ability to activate transcription in vivo. These results support the notion that TBP is a direct and important target of glutamine-rich transcriptional activators. Images PMID:8114696

  16. High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70.

    PubMed

    Morshauser, R C; Hu, W; Wang, H; Pang, Y; Flynn, G C; Zuiderweg, E R

    1999-06-25

    The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383-540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 A for the backbone atoms and 0.76 A for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions. PMID:10373374

  17. The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2

    PubMed Central

    Park, Young-Jun; Budiarto, Tanya; Wu, Meiting; Pardon, Els; Steyaert, Jan; Hol, Wim G. J.

    2012-01-01

    Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ∼20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1. PMID:22561373

  18. Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

    PubMed Central

    de Nobel, H; Lipke, P N; Kurjan, J

    1996-01-01

    The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin. Images PMID:8741846

  19. Fibronectin-based scaffold domain proteins that bind myostatin: a patent evaluation of WO2014043344.

    PubMed

    Walker, Ryan G; Thompson, Thomas B

    2015-05-01

    Muscular dystrophies (MD) are commonly characterized by progressive loss of muscle mass and function. It is hypothesized that therapeutic blockade of the TGF-β ligand myostatin, a negative regulator of muscle mass, will stimulate muscle growth and restore muscle function. Although many anti-myostatin targets are currently being pursued in the clinical setting, the efficacies of the tested molecules have shown mixed results. The patent WO2014043344 describes a novel approach for myostatin inhibition using a modified fibronectin type III domain that could potentially be used to treat MD and other muscle-related pathologies. PMID:25632990

  20. Identification of Novel Protein Kinase A Phosphorylation Sites in the M-domain of Human and Murine Cardiac Myosin Binding Protein-C Using Mass Spectrometry Analysis

    PubMed Central

    Jia, Weitao; Shaffer, Justin F.; Harris, Samantha P.; Leary, Julie A.

    2010-01-01

    Cardiac myosin binding protein-C (cMyBP-C) is a large multi-domain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people1. The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation dependent manner. The cMyBP-C motif or “M-domain” is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser275, Ser284, and Ser304), while three homologous sites exist in the murine isoform (Ser273, Ser282, and Ser302). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser307 that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 (~50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (~35 KDa, contains C1, M and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser307 and a novel human Ser311 can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo

  1. Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)*

    PubMed Central

    Makkinje, Anthony; Vanden Borre, Pierre; Near, Richard I.; Patel, Prayag S.; Lerner, Adam

    2012-01-01

    The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. PMID:22711540

  2. Autoantibodies define a family of proteins with conserved double-stranded RNA-binding domains as well as DNA binding activity.

    PubMed

    Satoh, M; Shaheen, V M; Kao, P N; Okano, T; Shaw, M; Yoshida, H; Richards, H B; Reeves, W H

    1999-12-01

    Cellular responses to viral infection are signaled by double-stranded (ds) RNA, which is not found in substantial amounts in uninfected cells. Although cellular dsRNA-binding proteins have been described, their characterization is incomplete. We show that dsRNA-binding proteins are prominent autoantigens. Sera from B6 and B10.S mice with pristane-induced lupus and human autoimmune sera immunoprecipitated a novel set of 130-, 110-, 90-, 80-, and 45-kDa proteins. The proteins were all major cellular poly(IC)-binding factors. N-terminal amino acid sequences of p110 and p90 were identical and matched nuclear factor (NF) 90 and M phase phosphoprotein 4. p45 and p90 were identified as the NF45.NF90 complex, which binds the interleukin-2 promoter as well as certain highly structured viral RNAs. NF90.NF45 and M phase phosphoprotein 4 belong to a large group of proteins with conserved dsRNA-binding motifs. Besides binding dsRNA, NF90.NF45, p110, and p130 had single-stranded and dsDNA binding activity. Some sera contained autoantibodies whose binding was inhibited by poly(IC) but not single-stranded DNA or vice versa, suggesting that the DNA- and RNA-binding sites are different. These autoantibodies will be useful probes of the function of dsRNA-binding proteins. Their interaction with dsRNA, an immunological adjuvant, also could promote autoimmunity. PMID:10574923

  3. Resveratrol induces apoptosis by directly targeting Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1)

    PubMed Central

    Oi, Naomi; Yuan, Jian; Malakhova, Margarita; Luo, Kuntian; Li, Yunhui; Ryu, Joohyun; Zhang, Lei; Bode, Ann M.; Xu, Zengguang; Li, Yan; Lou, Zhenkun; Dong, Zigang

    2014-01-01

    Resveratrol possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase activating protein SH3 domain binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol (RSVL)-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53 and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1. PMID:24998844

  4. The Relevance of Women's Diseases, Jun Activation-domain Binding Protein 1 (JAB1) and p27kip1

    PubMed Central

    Kim, Mijin; Lee, Hae-Hyeog

    2016-01-01

    The Jun activation-domain binding protein 1 (Jab1) recognize a potential coactivator of activator protein 1 (AP-1) such as c-fos, c-jun transcription factor and the fifth subunit of the COP9 signalosome complex. Also, Jab1 activate the c-jun gene resulted cell proliferation. Not only a powerful tumor suppressor but also regulator of apoptosis negative cdk inhibitor p27kip1 are involved in the cell cycle. This is Jab1 and p27kip1 interact with each other, Jab1 accelerate p27kip1 from nuclear to cytoplasm through ubiquitin/proteasome pathway. However, information about the relationship between Jab1 and p27kip1 is not known much. Taken together, the results of this study identify function and structure of Jab1 and p27kip1 were described in a recent article on the basis of relevant. Besides Jab1 and p27kip1 will organize the relationship between the disease and women. PMID:27152307

  5. Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein*

    PubMed Central

    Moktan, Hem; Guiraldelli, Michel F.; Eyster, Craig A.; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R. Daniel; Sung, Patrick; Zhou, Donghua H.; Pezza, Roberto J.

    2014-01-01

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. PMID:24711446

  6. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  7. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1996-03-05

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 15 figs.

  8. The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Córsico, Betina; Liou, Heng Ling; Storch, Judith

    2004-03-30

    Intestinal fatty acid binding protein (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms for the transfer of fatty acids (FAs) to acceptor membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP, transfer occurs by diffusion through the aqueous phase. Earlier, we had shown that the helical domain of IFABP is critical in determining its collisional FA transfer mechanism. In the study presented here, we have engineered a pair of chimeric proteins, one with the "body" (ligand binding domain) of IFABP and the alpha-helical region of LFABP (alphaLbetaIFABP) and the other with the ligand binding pocket of LFABP and the helical domain of IFABP (alphaIbetaLFABP). The objective of this work was to determine whether the change in the alpha-helical domain of each FABP would alter the rate and mechanism of transfer of FA from the chimeric proteins in comparison with those of the wild-type proteins. The fatty acid transfer properties of the FABP chimeras were examined using a fluorescence resonance transfer assay. The results showed a significant modification of the absolute rate of FA transfer from the chimeric proteins compared to that of the wild type, indicating that the slower rate of FA transfer observed for wild-type LFABP relative to that of wild-type IFABP is, in part, determined by the helical domain of the proteins. In addition to these quantitative changes, it was of great interest to observe that the apparent mechanism of FA transfer also changed when the alpha-helical domain was exchanged, with transfer from alphaLbetaIFABP occurring by aqueous diffusion and transfer from alphaIbetaLFABP occurring via protein-membrane collisional interactions. These results demonstrate that the alpha-helical region of LFABP is responsible for its diffusional mechanism of fatty acid transfer to membranes. PMID:15035630

  9. Heterologous expression and purification of biologically active domains 3 and 4 of human polymeric immunoglobulin receptor and its interaction with choline binding protein A of Streptococcus pneumoniae.

    PubMed

    Venables, Luanne; Govender, Sharlene; Oosthuizen, Vaughan

    2013-10-01

    Streptococcus pneumoniae, one of the common causes of pneumonia, colonises the epithelium via the interaction between a choline binding protein of S. pneumoniae and the human polymeric immunoglobulin receptor (pIgR). One of the functions of pIgR is to mediate the transcytosis of polymeric immunoglobulins from the basolateral to the apical surface of epithelial cells. S. pneumoniae invades human epithelial cells by exploiting the transcytosis machinery. Due to an increase in the prevalence of antibiotic resistant strains of S. pneumoniae, and the limitations and expense of the vaccines available, extensive research may provide insights into the potential of new therapeutic regimes. This study investigated the potential of pIgR domains as an alternative non-antibiotic immune therapy for treating pneumonia. The aim was to determine the binding affinity of recombinant D3D4 protein, the domains of pIgR responsible for binding S. pneumoniae, to recombinant R1R2 repeat domains of choline binding protein A of S. pneumoniae. Biologically active recombinant D3D4 was produced in Escherichia coli using a gel filtration chromatography refolding method, a novel approach for the refolding of pIgR domains, after the purification of inclusion bodies using nickel affinity chromatography. Surface Plasmon resonance (SPR) spectroscopy showed that purified recombinant D3D4 binds recombinant R1R2 with an equilibrium dissociation constant (KD) of 3.36×10(-7)M. PMID:23973337

  10. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  11. Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C.

    PubMed Central

    Ho, C; Slater, S J; Stagliano, B A; Stubbs, C D

    1999-01-01

    The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine

  12. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    SciTech Connect

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong

    2013-09-25

    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

  13. Asc1p, a WD40-domain containing adaptor protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes.

    PubMed Central

    Baum, Sonja; Bittins, Margarethe; Frey, Steffen; Seedorf, Matthias

    2004-01-01

    Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein-protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys(1067) at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs. PMID:15012629

  14. Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

    PubMed

    Urbina, Patricia; Bersch, Beate; De Angelis, Fabien; Derfoufi, Kheiro-Mouna; Prévost, Martine; Goormaghtigh, Erik; Vandenbussche, Guy

    2016-05-24

    Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site. PMID:27145046

  15. Superactivation of Pax6-mediated transactivation from paired domain-binding sites by dna-independent recruitment of different homeodomain proteins.

    PubMed

    Mikkola, I; Bruun, J A; Holm, T; Johansen, T

    2001-02-01

    The Pax6 genes encode evolutionary conserved transcription factors that act high up in the regulatory hierarchy controlling development of central organs such as the eyes and the central nervous system. These proteins contain two DNA-binding domains. The N-terminal paired domain is separated from a paired-type homeodomain by a linker region, and a transactivation domain is located C-terminal to the homeodomain. Vertebrate Pax6 genes express a paired-less isoform of Pax6 (Pax6DeltaPD) from an internal start codon in the coding region between the paired domain and homeodomain. We now provide evidence for an interaction between the full-length isoform and Pax6DeltaPD, which enhances the transactivation activity of Pax6 from paired domain-binding sites. The paired-like homeodomain protein Rax behaved similarly to Pax6DeltaPD. Both Pax6DeltaPD and Rax bound to the homeodomain of Pax6 in vitro in the absence of specific DNA binding. Coimmunoprecipitation experiments following cotransfection confirmed the existence of complexes between Pax6 and Pax6DeltaPD, Pax6 and Rax, and Pax6DeltaPD and Rax in vivo. Interestingly, the C-terminal subdomain of the paired domain and the homeodomain can interact with each other. The paired domain can also interact with itself. Surprisingly, GST pull-down assays revealed that the homeodomains of such diverse proteins as Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to Pax6, and several of these enhanced Pax6-mediated transactivation upon coexpression. Since many homeodomain proteins are coexpressed with Pax6 in several tissues during development, our results indicate the existence of novel regulatory interactions that may be important for fine tuning of gene regulation. PMID:11069920

  16. OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

    PubMed Central

    Lemaire, M; Ohayon, H; Gounon, P; Fujino, T; Béguin, P

    1995-01-01

    Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively. PMID:7730277

  17. The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding.

    PubMed

    Steger, G; Ham, J; Lefebvre, O; Yaniv, M

    1995-01-16

    The E2 transactivator of bovine papillomavirus type-1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co-operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co-operativity with co-expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild-type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre-initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N-terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID. PMID:7835344

  18. Cold Spots in Protein Binding.

    PubMed

    Shirian, Jason; Sharabi, Oz; Shifman, Julia M

    2016-09-01

    Understanding the energetics and architecture of protein-binding interfaces is important for basic research and could potentially facilitate the design of novel binding domains for biotechnological applications. It is well accepted that a few key residues at binding interfaces (binding hot spots) are responsible for contributing most to the free energy of binding. In this opinion article, we introduce a new concept of 'binding cold spots', or interface positions occupied by suboptimal amino acids. Such positions exhibit a potential for affinity enhancement through various mutations. We give several examples of cold spots from different protein-engineering studies and argue that identification of such positions is crucial for studies of protein evolution and protein design. PMID:27477052

  19. SH3-domain binding protein 1 in the tumor microenvironment promotes hepatocellular carcinoma metastasis through WAVE2 pathway

    PubMed Central

    Tao, Yiming; Hu, Kuan; Tan, Fengbo; Zhang, Sai; Zhou, Ming; Luo, Jia; Wang, Zhiming

    2016-01-01

    SH3-domain binding protein-1 (SH3BP1) specifically inactivating Rac1 and its target WAVE2 is required for cell motility. The present study shows SH3BP1 expression patterns in human HCC tissues and cell lines were examined. The regulation of SH3BP1 on HCC cell migration and invasion related to Rac1-WAVE2 signaling was characterized using in vitro and in vivo models. SH3BP1 overexpressed in HCC tissues and highly metastatic HCC cells was significantly associated vascular invasion (VI). SH3BP1 promoted VEGF secretion via Rac1-WAVE2 signaling, so as to exert an augmentation on cell invasion and microvessel formation. In three study cohorts with a total of 516 HCC patients, high SH3BP1 expression combined with high microvessel density (MVD) was confirmed as a powerful independent predictor of HCC prognosis in both training cohorts and validation cohort. Being an important angiogenic factor of HCC through Rac1-WAVE2 signaling, SH3BP1 promotes tumor invasion and microvessel formation contributing to HCC metastasis and recurrence. SH3BP1 is a novel WAVE2 regulator, a prognostic marker and a potential therapeutic target of HCC. PMID:26933917

  20. Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

    SciTech Connect

    Pak, J.; Sharon, C; Satkunarajah, M; Thierry, C; Cameron, C; Kelvin, D; Seetharaman, J; Cochrane, A; Plummer, F; et. al.

    2009-01-01

    The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.

  1. Recombinant Receptor Binding Domain Protein Induces Partial Protective Immunity in Rhesus Macaques Against Middle East Respiratory Syndrome Coronavirus Challenge☆

    PubMed Central

    Lan, Jiaming; Yao, Yanfeng; Deng, Yao; Chen, Hong; Lu, Guangwen; Wang, Wen; Bao, Linlin; Deng, Wei; Wei, Qiang; Gao, George F.; Qin, Chuan; Tan, Wenjie

    2015-01-01

    Background Development an effective vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) is urgent and limited information is available on vaccination in nonhuman primate (NHP) model. We herein report of evaluating a recombinant receptor-binding domain (rRBD) protein vaccine in a rhesus macaque model. Methods Nine monkeys were randomly assigned to high-dose, low-dose and mock groups,which were immunized with different doses of rRBD plus alum adjuvant or adjuvant alone at different time points (0, 8, 25 weeks). Immunological analysis was conducted after each immunisation. Monkeys were challenged with MERS-CoV at 14 days after the final immunisation followed by observation for clinical signs and chest X-rays. Nasal, oropharyngeal and rectal swabs were also collected for analyses. Monkeys were euthanized 3 days after challenge and multiple specimens from tissues were collected for pathological, virological and immunological tests. Conclusion Robust and sustained immunological responses (including neutralisation antibody) were elicited by the rRBD vaccination. Besides, rRBD vaccination alleviated pneumonia with evidence of reduced tissue impairment and clinical manifestation in monkeys. Furthermore, the rRBD vaccine decreased viral load of lung, trachea and oropharyngeal swabs of monkeys. These data in NHP paves a way for further development of an effective human vaccine against MERS-CoV infection. PMID:26629538

  2. Mutations in the TGFβ Binding-Protein-Like Domain 5 of FBN1 Are Responsible for Acromicric and Geleophysic Dysplasias

    PubMed Central

    Le Goff, Carine; Mahaut, Clémentine; Wang, Lauren W.; Allali, Slimane; Abhyankar, Avinash; Jensen, Sacha; Zylberberg, Louise; Collod-Beroud, Gwenaelle; Bonnet, Damien; Alanay, Yasemin; Brady, Angela F.; Cordier, Marie-Pierre; Devriendt, Koen; Genevieve, David; Kiper, Pelin Özlem Simsek; Kitoh, Hiroshi; Krakow, Deborah; Lynch, Sally Ann; Le Merrer, Martine; Mégarbane, André; Mortier, Geert; Odent, Sylvie; Polak, Michel; Rohrbach, Marianne; Sillence, David; Stolte-Dijkstra, Irene; Superti-Furga, Andrea; Rimoin, David L.; Topouchian, Vicken; Unger, Sheila; Zabel, Bernhard; Bole-Feysot, Christine; Nitschke, Patrick; Handford, Penny; Casanova, Jean-Laurent; Boileau, Catherine; Apte, Suneel S.; Munnich, Arnold; Cormier-Daire, Valérie

    2011-01-01

    Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes. PMID:21683322

  3. Identification of a Novel Calcium Binding Motif Based on the Detection of Sequence Insertions in the Animal Peroxidase Domain of Bacterial Proteins

    PubMed Central

    Santamaría-Hernando, Saray

    2012-01-01

    Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca2+ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33–79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca2+ binding with a KD of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life. PMID

  4. Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

    PubMed

    Santamaría-Hernando, Saray; Krell, Tino; Ramos-González, María-Isabel

    2012-01-01

    Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life. PMID

  5. Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals.

    PubMed

    Janeček, Štefan; Svensson, Birte; MacGregor, E Ann

    2011-10-10

    Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from several glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein kinase SNF1 complex, and an adaptor-regulator related to the SNF1/AMPK family, AKINβγ. CBM20s and CBM48s of amylolytic enzymes occur predominantly in the microbial world, whereas the non-amylolytic proteins containing these modules are mostly of plant and animal origin. Comparison of amino acid sequences and tertiary structures of CBM20 and CBM48 reveals the close relatedness of these SBDs and, in some cases, glycogen-binding domains (GBDs). The families CBM20 and CBM48 share both an ancestral form and the mode of starch/glycogen binding at one or two binding sites. Phylogenetic analyses demonstrate that they exhibit independent behaviour, i.e. each family forms its own part in an evolutionary tree, with enzyme specificity (protein function) being well represented within each family. The distinction between CBM20 and CBM48 families is not sharp since there are representatives in both CBM families that possess an intermediate character. These are, for example, CBM20s from hypothetical GH57 amylopullulanase (probably lacking the starch-binding site 2) and CBM48s from the GH13 pullulanase subfamily (probably lacking the starch/glycogen-binding site 1). The knowledge gained concerning the occurrence of these SBDs and GBDs through the range of taxonomy will support future experimental research. PMID:22112614

  6. Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae.

    PubMed

    Soares de Lima, Cristiana; Zulianello, Laurence; Marques, Maria Angela de Melo; Kim, Heejin; Portugal, Michelle Iespa; Antunes, Sérgio Luiz; Menozzi, Franco Dante; Ottenhoff, Tom Henricus Maria; Brennan, Patrick Joseph; Pessolani, Maria Cristina Vidal

    2005-07-01

    Binding of Mycobacterium leprae to and invasion of Schwann cells (SC) represent a crucial step that initiates nerve damage in leprosy. We and others have described that M. leprae colonization of the peripheral nerve system may be mediated in part by a surface-exposed histone-like protein (Hlp), characterized as a laminin-binding protein (LBP). Hlp/LBP has also been shown to play a role in the binding of mycobacteria to alveolar epithelial cells and macrophages. In the present study we report that M. leprae expresses Hlp/LBP protein during the course of human infection. Additionally, we analyzed the interaction of Hlp/LBP with the extracellular matrix and host cell surface. We show that Hlp/LBP, besides laminin, also binds heparin and heparan sulfate. Testing truncated recombinant Hlp molecules corresponding to the N-terminal (rHlp-N) and the C-terminal (rHlp-C) domains of the protein, we established that interaction of Hlp/LBP with laminin-2 and heparin is mainly mediated by the C-terminal domain of the protein. Moreover, the same domain was found to be involved in Hlp/LBP-mediating bacterial binding to human SC. Finally, evidence is shown suggesting that M. leprae produces a post-translationally modified Hlp/LBP containing methyllysine residues. Methylation of the lysine residues, however, seems not to affect the adhesive properties of Hlp/LBP. Taken together, our observations reinforce the involvement of Hlp/LBP as an adhesin in mycobacterial infections and define its highly positive C-terminal region as the major adhesive domain of this protein. PMID:15919224

  7. Crystallization and preliminary crystallographic studies of the copper-binding domain of the amyloid precursor protein of Alzheimer’s disease

    SciTech Connect

    Kong, Geoffrey K.-W.; Galatis, Denise; Barnham, Kevin J.; Polekhina, Galina; Adams, Julian J.; Masters, Colin L.; Cappai, Roberto; Parker, Michael W.; McKinstry, William J.

    2005-01-01

    The binding of Cu{sup 2+} ions to the copper-binding domain of the amyloid precursor protein of Alzheimer’s disease reduces the production of the amyloid β peptide, which is centrally involved in Alzheimer’s disease. Structural studies of the copper-binding domain will provide a basis for structure-based drug design that might prove useful in treating this devastating disease. Alzheimer’s disease is thought to be triggered by production of the amyloid β (Aβ) peptide through proteolytic cleavage of the amyloid precursor protein (APP). The binding of Cu{sup 2+} to the copper-binding domain (CuBD) of APP reduces the production of Aβ in cell-culture and animal studies. It is expected that structural studies of the CuBD will lead to a better understanding of how copper binding causes Aβ depletion and will define a potential drug target. The crystallization of CuBD in two different forms suitable for structure determination is reported here.

  8. Identification of Phosphotyrosine Binding Domain-Containing Proteins as Novel Downstream Targets of the EphA8 Signaling Function▿ †

    PubMed Central

    Shin, Jongdae; Gu, Changkyu; Park, Eunjeong; Park, Soochul

    2007-01-01

    Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway. PMID:17875921

  9. RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids.

    PubMed Central

    Wang, W; Riedel, K; Lynch, P; Chien, C Y; Montelione, G T; Krug, R M

    1999-01-01

    The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2', which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target. PMID:10024172

  10. A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP.

    PubMed Central

    Duncan, R; Collins, I; Tomonaga, T; Zhang, T; Levens, D

    1996-01-01

    The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c-myc gene. Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation is heretofore unknown. Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? Using chimeric GAL4-FBP fusion proteins we have shown that the carboxyl-terminal region (residues 448 to 644) is a potent transcriptional activation domain. This region contains three copies of a unique amino acid sequence motif containing tyrosine diads. Analysis of deletion mutants demonstrated that a single tyrosine motif alone (residues 609 to 644) was capable of activating transcription. The activation property of the C-terminal domain is repressed by the N-terminal 107 amino acids of FBP. These results show that FBP contains a transactivation domain which can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site. Furthermore, activation is mediated by a new motif which can be negatively regulated by a repression domain of FBP. PMID:8628294