Comparison of FDTD numerical computations and analytical multipole expansion method for plasmonics-active nanosphere dimers.

PubMed

Dhawan, Anuj; Norton, Stephen J; Gerhold, Michael D; Vo-Dinh, Tuan

2009-06-08

This paper describes a comparative study of finite-difference time-domain (FDTD) and analytical evaluations of electromagnetic fields in the vicinity of dimers of metallic nanospheres of plasmonics-active metals. The results of these two computational methods, to determine electromagnetic field enhancement in the region often referred to as "hot spots" between the two nanospheres forming the dimer, were compared and a strong correlation observed for gold dimers. The analytical evaluation involved the use of the spherical-harmonic addition theorem to relate the multipole expansion coefficients between the two nanospheres. In these evaluations, the spacing between two nanospheres forming the dimer was varied to obtain the effect of nanoparticle spacing on the electromagnetic fields in the regions between the nanostructures. Gold and silver were the metals investigated in our work as they exhibit substantial plasmon resonance properties in the ultraviolet, visible, and near-infrared spectral regimes. The results indicate excellent correlation between the two computational methods, especially for gold nanosphere dimers with only a 5-10% difference between the two methods. The effect of varying the diameters of the nanospheres forming the dimer, on the electromagnetic field enhancement, was also studied.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds

PubMed Central

Rueda, Daniel; Sheen, Patricia; Gilman, Robert H.; Bueno, Carlos; Santos, Marco; Pando-Robles, Victoria; Batista, Cesar V.; Zimic, Mirko

2014-01-01

Recombinant wild-pyrazinamidase from H37Rv M. tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer. PMID:25199451

Structure-activity relationships in defensin dimers: a novel link between beta-defensin tertiary structure and antimicrobial activity.

PubMed

Campopiano, Dominic J; Clarke, David J; Polfer, Nick C; Barran, Perdita E; Langley, Ross J; Govan, John R W; Maxwell, Alison; Dorin, Julia R

2004-11-19

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a beta-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mm NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have beta-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.

Effect of thermal sterilization on ferulic, coumaric and cinnamic acids: dimerization and antioxidant activity.

PubMed

Arrieta-Baez, Daniel; Dorantes-Álvarez, Lidia; Martinez-Torres, Rocio; Zepeda-Vallejo, Gerardo; Jaramillo-Flores, Maria Eugenia; Ortiz-Moreno, Alicia; Aparicio-Ozores, Gerardo

2012-10-01

Some phenolic compounds, such as ferulic acid and p-coumaric acid, exist in the form of free acids, in fruits, rice, corn and other grains. Thermal treatment (121 °C at 15-17 psi) for different times on ferulic, p-coumaric and cinnamic acids as well as equimolar mixtures of these acids was investigated. Ferulic and p-coumaric acids underwent decarboxylation, yielding dimeric products formed through their corresponding radical intermediates, while cinnamic acid was recovered unreacted. High-performance liquid chromatography analysis showed no cross-dimerization when equimolar mixtures of pairs of hydroxycinnamic acids were treated under the same conditions. Dimers were characterized as (E)-4',4″-(but-1-ene-1,3-diyl)bis(2'-methoxyphenol)) (dimer of 4-vinylguaiacol) and (E)-4,4'-(but-1-ene-1,3-diyl)diphenol) (dimer of 4-vinylphenol) by nuclear magnetic resonance and mass spectrometry. Sterilization by thermal processing produced dimers of ferulic and coumaric acid. The antioxidant activity of these dimers was greater than that of the respective hydroxycinnamic acids. These results may be relevant for fruits and grains that contain hydroxycinnamic acids and undergo sterilization processes such as canning. Copyright © 2012 Society of Chemical Industry.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress

PubMed Central

Topolska-Woś, Agnieszka M.; Shell, Steven M.; Kilańczyk, Ewa; Szczepanowski, Roman H.; Chazin, Walter J.; Filipek, Anna

2015-01-01

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.—Topolska-Woś, A. M., Shell, S. M., Kilańczyk, E., Szczepanowski, R. H., Chazin, W. J., Filipek, A. Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress. PMID:25609429

Elucidation of the active conformation of vancomycin dimers with antibacterial activity against vancomycin-resistant bacteria.

PubMed

Nakamura, Jun; Yamashiro, Hidenori; Hayashi, Sayaka; Yamamoto, Mami; Miura, Kenji; Xu, Shu; Doi, Takayuki; Maki, Hideki; Yoshida, Osamu; Arimoto, Hirokazu

2012-10-01

Covalently linked vancomycin dimers have attracted a great deal of attention among researchers because of their enhanced antibacterial activity against vancomycin-resistant strains. However, the lack of a clear insight into the mechanisms of action of these dimers hampers rational optimization of their antibacterial potency. Here, we describe the synthesis and antibacterial activity of novel vancomycin dimers with a constrained molecular conformation achieved by two tethers between vancomycin units. Conformational restriction is a useful strategy for studying the relationship between the molecular topology and biological activity of compounds. In this study, two vancomycin units were linked at three distinct positions of the glycopeptide (vancosamine residue (V), C terminus (C), and N terminus (N)) to form two types of novel vancomycin cyclic dimers. Active NC-VV-linked dimers with a stable conformation as indicated by molecular mechanics calculations selectively suppressed the peptidoglycan polymerization reaction of vancomycin-resistant Staphylococcus aureus in vitro. In addition, double-disk diffusion tests indicated that the antibacterial activity of these dimers against vancomycin-resistant enterococci might arise from the inhibition of enzymes responsible for peptidoglycan polymerization. These findings provide a new insight into the biological targets of vancomycin dimers and the conformational requirements for efficient antibacterial activity against vancomycin-resistant strains. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preferential recognition of undisruptable dimers of inducible nitric oxide synthase by a monoclonal antibody directed against an N-terminal epitope.

PubMed

Mazumdar, Tuhina; Eissa, N Tony

2005-02-15

Overproduction of NO by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer in which the subunits align in a head-to-head manner, with the N-terminal oxygenase domains forming the dimer interface and a zinc metal center stabilizing the dimer. Thus, dimerization represents a critical locus for therapeutic interventions for regulation of NO synthesis. We have recently shown that intracellular iNOS forms dimers that are "undisruptable (UD)" by heat, SDS, strong denaturants, and/or reducing agents. Our data further suggest that the zinc metal center plays a role in forming and/or stabilizing iNOS undisruptable dimers (UD-dimers). In this study, we show that a mAb directed against a unique epitope at the oxygenase domain of human iNOS preferentially recognizes UD-dimers. This observation has implications for the mechanism of formation and regulation of dimer formation of iNOS. Our data suggest that UD-dimers of iNOS, in spite of SDS-PAGE denaturation, still maintain features of the quaternary structure of iNOS particularly at its N-terminal end and including head-to-head contact of the oxygenase domains.

Kinetics and thermodynamics of the interchange of the morpheein forms of human porphobilinogen synthase.

PubMed

Selwood, Trevor; Tang, Lei; Lawrence, Sarah H; Anokhina, Yana; Jaffe, Eileen K

2008-03-11

A morpheein is a homo-oligomeric protein that can adopt different nonadditive quaternary assemblies (morpheein forms) with different functionalities. The human porphobilinogen synthase (PBGS) morpheein forms are a high activity octamer, a low activity hexamer, and two structurally distinct dimer conformations. Conversion between hexamer and octamer involves dissociation to dimers, conformational change at the dimer level, followed by association to the alternate assembly. The current work promotes an alternative and novel view of the physiologically relevant dimeric structures, which are derived from the crystal structures, but are distinct from the asymmetric units of their crystal forms. Using a well characterized heteromeric system (WT+F12L; Tang, L. et al. (2005) J. Biol. Chem. 280, 15786-15793), extensive study of the human PBGS morpheein reequilibration process now reveals that the intervening dimers do not dissociate to monomers. The morpheein equilibria of wild type (WT) human PBGS are found to respond to changes in pH, PBGS concentration, and substrate turnover. Notably, the WT enzyme is predominantly an octamer at neutral pH, but increasing pH results in substantial conversion to lower order oligomers. Most significantly, the free energy of activation for the conversion of WT+F12L human PBGS heterohexamers to hetero-octamers is determined to be the same as that for the catalytic conversion of substrate to product by the octamer, remarkably suggesting a common rate-limiting step for both processes, which is postulated to be the opening/closing of the active site lid.

Adenylyl cyclase G is activated by an intramolecular osmosensor.

PubMed

Saran, Shweta; Schaap, Pauline

2004-03-01

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

PubMed Central

Tzeng, E; Billiar, T R; Robbins, P D; Loftus, M; Stuehr, D J

1995-01-01

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8524846

A dimeric form of a small-sized protein binder exhibits enhanced anti-tumor activity through prolonged blood circulation.

PubMed

Kim, Tae Yoon; Seo, Hyo-Deok; Lee, Joong-Jae; Kang, Jung Ae; Kim, Woo Sik; Kim, Hye-Min; Song, Ha-Yeon; Park, Ji Min; Lee, Dong-Eun; Kim, Hak-Sung

2018-06-10

Small-sized non-antibody scaffolds have attracted considerable interest as alternatives to immunoglobulin antibodies. However, their short half-life is considered a drawback in the development of therapeutic agents. Here we demonstrate that a homo-dimeric form of a repebody enhances the anti-tumor activity than a monomeric form through prolonged blood circulation. Spytag and spycatcher were genetically fused to the C-terminus of a respective human IL-6-specific repebody, and the resulting two repebody constructs were mixed at an equimolar ratio to produce a homo-dimeric form through interaction between spytag and spycatcher. The homo-dimeric repebody was detected as a single band in the SDS-PAGE analysis with an expected molecular size (78 kDa), showing high stability and homogeneity. The dimeric repebody was shown to simultaneously accommodate two hIL-6 molecules, and its binding affinity for hIL-6 was estimated to be comparable to a monomeric repebody. The serum concentration of the dimeric repebody was observed to be about 5.5 times higher than a monomeric repebody, consequently leading to considerably higher tumor suppression effect in human tumor xenograft mice. The present approach can be effectively used for prolonging the blood half-life of small-sized protein binders, resulting in enhanced therapeutic efficacy. Copyright © 2018 Elsevier B.V. All rights reserved.

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase: DAWIDZIAK et al.

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

Dawidziak, Daria M.; Sanchez, Jacint G.; Wagner, Jonathan M.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

Dimer formation and transcription activation in the sporulation response regulator Spo0A.

PubMed

Lewis, Richard J; Scott, David J; Brannigan, James A; Ladds, Joanne C; Cervin, Marguerite A; Spiegelman, George B; Hoggett, James G; Barák, Imrich; Wilkinson, Anthony J

2002-02-15

The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A. Copyright 2002 Elsevier Science Ltd.

Dynamic Cholesterol-Conditioned Dimerization of the G Protein Coupled Chemokine Receptor Type 4

PubMed Central

Kranz, Franziska

2016-01-01

G protein coupled receptors (GPCRs) allow for the transmission of signals across biological membranes. For a number of GPCRs, this signaling was shown to be coupled to prior dimerization of the receptor. The chemokine receptor type 4 (CXCR4) was reported before to form dimers and their functionality was shown to depend on membrane cholesterol. Here, we address the dimerization pattern of CXCR4 in pure phospholipid bilayers and in cholesterol-rich membranes. Using ensembles of molecular dynamics simulations, we show that CXCR4 dimerizes promiscuously in phospholipid membranes. Addition of cholesterol dramatically affects the dimerization pattern: cholesterol binding largely abolishes the preferred dimer motif observed for pure phospholipid bilayers formed mainly by transmembrane helices 1 and 7 (TM1/TM5-7) at the dimer interface. In turn, the symmetric TM3,4/TM3,4 interface is enabled first by intercalating cholesterol molecules. These data provide a molecular basis for the modulation of GPCR activity by its lipid environment. PMID:27812115

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

PubMed Central

Yeung, A T; Mattes, W B; Grossman, L

1986-01-01

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction. PMID:3960727

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

DOE PAGES

Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T.; ...

2016-04-07

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Here, although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-raymore » scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG.« less

Physical basis behind achondroplasia, the most common form of human dwarfism.

PubMed

He, Lijuan; Horton, William; Hristova, Kalina

2010-09-24

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism*

PubMed Central

He, Lijuan; Horton, William; Hristova, Kalina

2010-01-01

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia. PMID:20624921

High Molecular Weight Complex Analysis of Epstein-Barr Virus Latent Membrane Protein 1 (LMP-1): Structural Insights into LMP-1’s Homo-Oligomerization and Lipid Raft Association

PubMed Central

Wrobel, Christopher M.; Geiger, Timothy R.; Nix, Rebecca N.; Robitaille, Aaron M.; Balser, Sandra; Cervantes, Alfredo; Gonzalez, Miguel; Martin, Jennifer M.

2013-01-01

LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein-Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1’s TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers. PMID:24075898

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics.

PubMed

Chek, Min Fey; Kim, Sun-Yong; Mori, Tomoyuki; Arsad, Hasni; Samian, Mohammed Razip; Sudesh, Kumar; Hakoshima, Toshio

2017-07-13

Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

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

Zoghbi, M. E.; Altenberg, G. A.

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we usedmore » luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.« less

Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization.

PubMed

Chamachi, Neharika G; Chakrabarty, Suman

2016-08-04

The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion-prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation.

Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

PubMed

Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

2016-07-01

Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation.

PubMed

Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T; Reger, Albert S; Sankaran, Banumathi; Casteel, Darren E; Herberg, Friedrich W; Kim, Choel

2016-05-03

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Copyright © 2016 Elsevier Ltd. All rights reserved.

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

PubMed

Osipov, Alexey V; Kasheverov, Igor E; Makarova, Yana V; Starkov, Vladislav G; Vorontsova, Olga V; Ziganshin, Rustam Kh; Andreeva, Tatyana V; Serebryakova, Marina V; Benoit, Audrey; Hogg, Ronald C; Bertrand, Daniel; Tsetlin, Victor I; Utkin, Yuri N

2008-05-23

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Control activity of yeast geranylgeranyl diphosphate synthase from dimer interface through H-bonds and hydrophobic interaction.

PubMed

Chang, Chih-Kang; Teng, Kuo-Hsun; Lin, Sheng-Wei; Chang, Tao-Hsin; Liang, Po-Huang

2013-04-23

Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased k(cat) values (200-250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS.

Human monoclonal antibody homodimers. Effect of valency on in vitro and in vivo antibacterial activity.

PubMed

Wolff, E A; Esselstyn, J; Maloney, G; Raff, H V

1992-04-15

Human IgG1 mAb dimers specific for either group B streptococci or Escherichia coli K1 bacteria were formed using chemical cross-linkers. The effect of antibody valency on biologic efficacy was investigated by comparing the IgG dimers against the corresponding IgG monomers. Binding activity and relative avidity were assessed using Ag binding and competition ELISA, and functional activity was analyzed using opsonophagocytic assays. These in vitro assays revealed that the dimers were greater than or equal to 50-fold more active than the monomers. A neonatal rat infection model showed the in vivo protective efficacy of the dimers was greater than or equal to 20-fold greater than that of the monomers. Enhancing the activity of mAb by chemical cross-linking may be a useful strategy for salvaging low affinity IgG mAb that possess poor functional properties.

An ion mobility-mass spectrometry investigation of monocyte chemoattractant protein-1

NASA Astrophysics Data System (ADS)

Schenauer, Matthew R.; Leary, Julie A.

2009-10-01

In the present article we describe the gas-phase dissociation behavior of the dimeric form of monocyte chemoattractant protein-1 (MCP-1) using quadrupole-traveling wave ion mobility spectrometry-time of flight mass spectrometry (q-TWIMS-TOF MS) (Waters Synapt(TM)). Through investigation of the 9+ charge state of the dimer, we were able to monitor dissociation product ion (monomer) formation as a function of activation energy. Using ion mobility, we were able to observe precursor ion structural changes occurring throughout the activation process. Arrival time distributions (ATDs) for the 5+ monomeric MCP-1 product ions, derived from the gas-phase dissociation of the 9+ dimer, were then compared with ATDs obtained for the 5+ MCP-1 monomer isolated directly from solution. The results show that the dissociated monomer is as compact as the monomer arising from solution, regardless of the trap collision energy (CE) used in the dissociation. The solution-derived monomer, when collisionally activated, also resists significant unfolding within measure. Finally, we compared the collisional activation data for the MCP-1 dimer with an MCP-1 dimer non-covalently bound to a single molecule of the semi-synthetic glycosaminoglycan (GAG) analog Arixtra(TM); the latter a therapeutic anti-thrombin III-activating pentasaccharide. We observed that while dimeric MCP-1 dissociated at relatively low trap CEs, the Arixtra-bound dimer required much higher energies, which also induced covalent bond cleavage in the bound Arixtra molecule. Both the free and Arixtra-bound dimers became less compact and exhibited longer arrival times with increasing trap CEs, albeit the Arixtra-bound complex at slightly higher energies. That both dimers shifted to longer arrival times with increasing activation energy, while the dissociated MCP-1 monomers remained compact, suggests that the longer arrival times of the Arixtra-free and Arixtra-bound dimers may represent a partial breach of non-covalent interactions between the associated MCP-1 monomers, rather than extensive unfolding of individual subunits. The fact that Arixtra preferentially binds MCP-1 dimers and prevents dimer dissociation at comparable activation energies to the Arixtra-free dimer, may suggest that the drug interacts across the two monomers, thereby inhibiting their dissociation.

Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity.

PubMed

Wagner, R; Gonzalez, D H; Podesta, F E; Andreo, C S

1987-05-04

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e.g. 200-400 mM NaCl) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time-dependent manner. Turnover conditions and glucose 6-phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.

Oligomerization of a molecular chaperone modulates its activity

PubMed Central

Kawagoe, Soichiro; Ishimori, Koichiro

2018-01-01

Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell. PMID:29714686

Oral Fusobacterium nucleatum subsp. polymorphum binds to human salivary α-amylase.

PubMed

Zulfiqar, M; Yamaguchi, T; Sato, S; Oho, T

2013-12-01

Fusobacterium nucleatum acts as an intermediate between early and late colonizers in the oral cavity. In this study, we showed that F. nucleatum subsp. polymorphum can bind to a salivary component with a molecular weight of approximately 110 kDa and identified the protein and another major factor of 55 kDa, as salivary α-amylase by time-of-flight mass spectrometry and immuno-reactions. Salivary α-amylase is present in both monomeric and dimeric forms and we found that formation of the dimer depends on copper ions. The F. nucleatum adhered to both monomeric and dimeric salivary α-amylases, but the numbers of bacteria bound to the dimeric form were more than those bound to the monomeric form. The degree of adherence of F. nucleatum to four α-amylases from different sources was almost the same, however its binding to β-amylase was considerably decreased. Among four α-amylase inhibitors tested, acarbose and type 1 and 3 inhibitors derived from wheat flour showed significant activity against the adhesion of F.nucleatum to monomeric and dimeric amylases, however voglibose had little effect. Moreover F. nucleatum cells inhibited the enzymatic activity of salivary α-amylase in a dose-dependent manner. These results suggest that F. nucleatum plays more important and positive role as an early colonizer for maturation of oral microbial colonization. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

PubMed Central

Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

2017-01-01

Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed

Bieth, E; Gabus, C; Darlix, J L

1990-01-11

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed Central

Bieth, E; Gabus, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12. Images PMID:2155394

Characterization and antioxidant activity of gallic acid derivative

NASA Astrophysics Data System (ADS)

Malinda, Krissan; Sutanto, Hery; Darmawan, Akhmad

2017-11-01

Peroxidase enzyme was used to catalyze the dimerization process of gallic acid. The structure of the dimerization product was characterized by 1H NMR and LC-MS-MS. The mechanism of gallic acid dimerization was also discussed. It was proposed that ellagic acid was formed through an oxidative coupling mechanism that lead to the formation of a C-C bond and followed by an intramolecular Fischer esterification mechanism that lead to the formation of two C-O bonds. Moreover, the antioxidant activity of gallic acid and ellagic acid were also studied. Gallic acid and ellagic acid exhibited the DPPH radical scavenging activity with IC50 values of 13.2 μM and 15.9 μM, respectively.

Structure and mechanism of human DNA polymerase [eta

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

Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji

2010-11-03

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assistmore » translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.« less

Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization*

PubMed Central

Guan, Rongbin; Feng, Xiuyan; Wu, Xueqing; Zhang, Meilin; Zhang, Xuesen; Hébert, Terence E.; Segaloff, Deborah L.

2009-01-01

Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904–5914). In this study, bioluminescence resonance energy transfer (BRET2) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET2 to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET2 analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET2 signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR. PMID:19147490

Dimerization Interface of 3-Hydroxyacyl-CoA Dehydrogenase Tunes the Formation of Its Catalytic Intermediate

PubMed Central

Jin, Ying-Hua; Fan, Jun; Sun, Fei

2014-01-01

3-hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.35) is a homodimeric enzyme localized in the mitochondrial matrix, which catalyzes the third step in fatty acid β-oxidation. The crystal structures of human HAD and subsequent complexes with cofactor/substrate enabled better understanding of HAD catalytic mechanism. However, numerous human diseases were found related to mutations at HAD dimerization interface that is away from the catalytic pocket. The role of HAD dimerization in its catalytic activity needs to be elucidated. Here, we solved the crystal structure of Caenorhabditis elegans HAD (cHAD) that is highly conserved to human HAD. Even though the cHAD mutants (R204A, Y209A and R204A/Y209A) with attenuated interactions on the dimerization interface still maintain a dimerization form, their enzymatic activities significantly decrease compared to that of the wild type. Such reduced activities are in consistency with the reduced ratios of the catalytic intermediate formation. Further molecular dynamics simulations results reveal that the alteration of the dimerization interface will increase the fluctuation of a distal region (a.a. 60–80) that plays an important role in the substrate binding. The increased fluctuation decreases the stability of the catalytic intermediate formation, and therefore the enzymatic activity is attenuated. Our study reveals the molecular mechanism about the essential role of the HAD dimerization interface in its catalytic activity via allosteric effects. PMID:24763278

Structural basis of death domain signaling in the p75 neurotrophin receptor

PubMed Central

Lin, Zhi; Tann, Jason Y; Goh, Eddy TH; Kelly, Claire; Lim, Kim Buay; Gao, Jian Fang; Ibanez, Carlos F

2015-01-01

Death domains (DDs) mediate assembly of oligomeric complexes for activation of downstream signaling pathways through incompletely understood mechanisms. Here we report structures of complexes formed by the DD of p75 neurotrophin receptor (p75NTR) with RhoGDI, for activation of the RhoA pathway, with caspase recruitment domain (CARD) of RIP2 kinase, for activation of the NF-kB pathway, and with itself, revealing how DD dimerization controls access of intracellular effectors to the receptor. RIP2 CARD and RhoGDI bind to p75NTR DD at partially overlapping epitopes with over 100-fold difference in affinity, revealing the mechanism by which RIP2 recruitment displaces RhoGDI upon ligand binding. The p75NTR DD forms non-covalent, low-affinity symmetric dimers in solution. The dimer interface overlaps with RIP2 CARD but not RhoGDI binding sites, supporting a model of receptor activation triggered by separation of DDs. These structures reveal how competitive protein-protein interactions orchestrate the hierarchical activation of downstream pathways in non-catalytic receptors. DOI: http://dx.doi.org/10.7554/eLife.11692.001 PMID:26646181

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution.

PubMed Central

Chang, B S; Beauvais, R M; Arakawa, T; Narhi, L O; Dong, A; Aparisio, D I; Carpenter, J F

1996-01-01

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface. PMID:8968609

His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.

PubMed

Haglin, Elizabeth R; Yang, Wen; Briegel, Ariane; Thompson, Lynmarie K

2017-11-07

Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes.

Proteolysis of truncated hemolysin A yields a stable dimerization interface

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

Novak, Walter R. P.; Bhattacharyya, Basudeb; Grilley, Daniel P.

2017-02-21

Wild-type and variant forms of HpmA265 (truncated hemolysin A) fromProteus mirabilisreveal a right-handed, parallel β-helix capped and flanked by segments of antiparallel β-strands. The low-salt crystal structures form a dimeric structureviathe implementation of on-edge main-chain hydrogen bonds donated by residues 243–263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formedviamain-chain hydrogen bonds donated by residues 203–215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel β-sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interfacemore » is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge β-strand positioning used in template-assisted hemolytic activity.« less

Rapid dimerization of quercetin through an oxidative mechanism in the presence of serum albumin decreases its ability to induce cytotoxicity in MDA-MB-231 cells

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

Pham, Anh; Bortolazzo, Anthony; White, J. Brandon, E-mail: Brandon.White@sjsu.edu

Highlights: Black-Right-Pointing-Pointer Quercetin cannot be detected intracellularly despite killing MDA-MB-231 cells. Black-Right-Pointing-Pointer Quercetin forms a heterodimer through oxidation in media with serum. Black-Right-Pointing-Pointer The quercetin heterodimer does not kill MDA-MB-231 cells. Black-Right-Pointing-Pointer Ascorbic acid stabilizes quercetin increasing cell death in quercetin treated cells. Black-Right-Pointing-Pointer Quercetin, and not a modified form, is responsible for apoptosis and cell death. -- Abstract: Quercetin is a member of the flavonoid family and has been previously shown to have a variety of anti-cancer activities. We and others have reported anti-proliferation, cell cycle arrest, and induction of apoptosis of cancer cells after treatment with quercetin. Quercetinmore » has also been shown to undergo oxidation. However, it is unclear if quercetin or one of its oxidized forms is responsible for cell death. Here we report that quercetin rapidly oxidized in cell culture media to form a dimer. The quercetin dimer is identical to a dimer that is naturally produced by onions. The quercetin dimer and quercetin-3-O-glucopyranoside are unable to cross the cell membrane and do not kill MDA-MB-231 cells. Finally, supplementing the media with ascorbic acid increases quercetin's ability to induce cell death probably by reduction oxidative dimerization. Our results suggest that an unmodified quercetin is the compound that elicits cell death.« less

Muscle-type 6-phosphofructo-1-kinase and aldolase associate conferring catalytic advantages for both enzymes.

PubMed

Marcondes, Mariah Celestino; Sola-Penna, Mauro; Torres, Renan da Silva Gianoti; Zancan, Patricia

2011-06-01

6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism. Copyright © 2011 Wiley Periodicals, Inc.

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

PubMed Central

Fong, Yu Hang; Wong, Ho Chun; Yuen, Man Hon; Lau, Pak Ho; Chen, Yu Wai; Wong, Kam-Bo

2013-01-01

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. PMID:24115911

Cross-linking of serine racemase dimer by reactive oxygen species and reactive nitrogen species.

PubMed

Wang, Wei; Barger, Steven W

2012-06-01

Serine racemase (SR) is the only identified enzyme in mammals responsible for isomerization of L-serine to D-serine, a coagonist at N-methyl-D-aspartate (NMDA) receptors in the forebrain. Our previous data showed that an apparent SR dimer resistant to sodium dodecyl sulfate and β-mercaptoethanol was elevated in microglial cells after proinflammatory activation. Because the activation of microglia is typically associated with an oxidative burst, oxidative cross-linking between SR subunits was speculated. In this study, an siRNA technique was employed to confirm the identity of this SR dimer band. The oxidative species potentially responsible for the cross-linking was investigated with recombinant SR protein. The data indicate that nitric oxide, peroxynitrite, and hydroxyl radical were the likely candidates, whereas superoxide and hydrogen peroxide per se failed to contribute. Furthermore, the mechanism of formation of SR dimer by peroxynitrite oxidation was studied by mass spectrometry. A disulfide bond between Cys₆ and Cys₁₁₃ was identified in 3-morpholinosydnonimine hydrochloride (SIN-1)-treated SR monomer and dimer. Activity assays indicated that SIN-1 treatment decreased SR activity, confirming our previous conclusion that noncovalent dimer is the most active form of SR. These findings suggest a compensatory feedback in which the consequences of neuroinflammation might dampen D-serine production to limit excitotoxic stimulation of NMDA receptors. Copyright © 2012 Wiley Periodicals, Inc.

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

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

Hickey, John M.; Lovell, Scott; Battaile, Kevin P.

2013-05-29

Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to thismore » interaction have not been elucidated. We hypothesize that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two {alpha}-helices present in all other OmpR/PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.« less

Covalent Dimer Species of β-Defensin Defr1 Display Potent Antimicrobial Activity against Multidrug-Resistant Bacterial Pathogens▿

PubMed Central

Taylor, Karen; McCullough, Bryan; Clarke, David J.; Langley, Ross J.; Pechenick, Tali; Hill, Adrian; Campopiano, Dominic J.; Barran, Perdita E.; Dorin, Julia R.; Govan, John R. W.

2007-01-01

Beta defensins comprise a family of cationic, cysteine-rich antimicrobial peptides, predominantly expressed at epithelial surfaces. Previously we identified a unique five-cysteine defensin-related peptide (Defr1) that, when synthesized, is a mixture of dimeric isoforms and exhibits potent antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Here we report that Defr1 displays antimicrobial activity against an extended panel of multidrug-resistant nosocomial pathogens for which antimicrobial treatment is limited or nonexistent. Defr1 fractions were collected by high-pressure liquid chromatography and analyzed by gel electrophoresis and mass spectrometry. Antimicrobial activity was initially investigated with the type strain Pseudomonas aeruginosa PAO1. All fractions tested displayed equivalent, potent antimicrobial activity levels comparable with that of the unfractionated Defr1. However, use of an oxidized, monomeric six-cysteine analogue (Defr1 Y5C), or of reduced Defr1, gave diminished antimicrobial activity. These results suggest that the covalent dimer structure of Defr1 is crucial to antimicrobial activity; this hypothesis was confirmed by investigation of a synthetic one-cysteine variant (Defr1-1cys). This gave an activity profile similar to that of synthetic Defr1 but only in an oxidized, dimeric form. Thus, we have shown that covalent, dimeric molecules based on the Defr1 β-defensin sequence demonstrate antimicrobial activity even in the absence of the canonical cysteine motif. PMID:17353239

Direct Assessment of the Effect of the Gly380Arg Achondroplasia Mutation on FGFR3 Dimerization Using Quantitative Imaging FRET

PubMed Central

Placone, Jesse; Hristova, Kalina

2012-01-01

The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small, but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the absence of ligand. PMID:23056398

I222 crystal form of despentapeptide (B26-B30) insulin provides new insights into the properties of monomeric insulin.

PubMed

Whittingham, Jean L; Youshang, Zhang; Záková, Lenka; Dodson, Eleanor J; Turkenburg, Johan P; Brange, Jens; Dodson, G Guy

2006-05-01

Despentapeptide (des-B26-B30) insulin (DPI), an active modified insulin, has been crystallized in the presence of 20% acetic acid pH 2. A crystal structure analysis to 1.8 A spacing (space group I222) revealed that the DPI molecule, which is unable to make beta-strand interactions for physiological dimer formation and is apparently monomeric in solution, formed an alternative lattice-generated dimer. The formation of this dimer involved interactions between surfaces which included the B9-B19 alpha-helices (usually buried by the dimer-dimer contacts within the native hexamer). The two crystallographically independent molecules within the dimer were essentially identical and were similar in conformation to T-state insulin as seen in the T(6) insulin hexamer. An unusual feature of each molecule in the dimer was the presence of two independent conformations at the B-chain C-terminus (residues B20-B25). Both conformations were different from that of native insulin, involving a 3.5 A displacement of the B20-B23 beta-turn and a repositioning of residue PheB25 such that it made close van der Waals contact with the main body of the molecule, appearing to stabilize the B-chain C-terminus.

Structure of the dimerization domain of DiGeorge Critical Region 8

PubMed Central

Senturia, Rachel; Faller, Michael; Yin, Sheng; Loo, Joseph A; Cascio, Duilio; Sawaya, Michael R; Hwang, Daniel; Clubb, Robert T; Guo, Feng

2010-01-01

Maturation of microRNAs (miRNAs, ∼22nt) from long primary transcripts [primary miRNAs (pri-miRNAs)] is regulated during development and is altered in diseases such as cancer. The first processing step is a cleavage mediated by the Microprocessor complex containing the Drosha nuclease and the RNA-binding protein DiGeorge critical region 8 (DGCR8). We previously reported that dimeric DGCR8 binds heme and that the heme-bound DGCR8 is more active than the heme-free form. Here, we identified a conserved dimerization domain in DGCR8. Our crystal structure of this domain (residues 298–352) at 1.7 Å resolution demonstrates a previously unknown use of a WW motif as a platform for extensive dimerization interactions. The dimerization domain of DGCR8 is embedded in an independently folded heme-binding domain and directly contributes to association with heme. Heme-binding-deficient DGCR8 mutants have reduced pri-miRNA processing activity in vitro. Our study provides structural and biochemical bases for understanding how dimerization and heme binding of DGCR8 may contribute to regulation of miRNA biogenesis. PMID:20506313

Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

DOE PAGES

Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.; ...

2016-03-07

Activation triggers the exchange of subunits in Ca 2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This convertsmore » the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.« less

Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

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

Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.

Activation triggers the exchange of subunits in Ca 2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This convertsmore » the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.« less

Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II

PubMed Central

Bhattacharyya, Moitrayee; Stratton, Margaret M; Going, Catherine C; McSpadden, Ethan D; Huang, Yongjian; Susa, Anna C; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L; Barros, Tiago; Schulman, Howard; Williams, Evan R; Kuriyan, John

2016-01-01

Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. DOI: http://dx.doi.org/10.7554/eLife.13405.001 PMID:26949248

Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.

PubMed

Monsey, John; Shen, Wei; Schlesinger, Paul; Bose, Ron

2010-03-05

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

Differential Stability of Dimeric and Monomeric Cytochrome c Oxidase Exposed to Elevated Hydrostatic Pressure†

PubMed Central

Staničová, Jana; Sedlák, Erik; Musatov, Andrej; Robinson, Neal C.

2007-01-01

Detergent-solubilized dimeric and monomeric cytochrome c oxidase (CcO) have significantly different quaternary stability when exposed to 2−3 kbar of hydrostatic pressure. Dimeric, dodecyl maltoside-solubilized cytochrome c oxidase is very resistant to elevated hydrostatic pressure with almost no perturbation of its quaternary structure or functional activity after release of pressure. In contrast to the stability of dimeric CcO, 3 kbar of hydrostatic pressure triggers multiple structural and functional alterations within monomeric cytochrome c oxidase. The perturbations are either irreversible or slowly reversible since they persist after the release of high pressure. Therefore, standard biochemical analytical procedures could be used to quantify the pressure-induced changes after the release of hydrostatic pressure. The electron transport activity of monomeric cytochrome c oxidase decreases by as much as 60% after exposure to 3 kbar of hydrostatic pressure. The irreversible loss of activity occurs in a time- and pressure-dependent manner. Coincident with the activity loss is a sequential dissociation of four subunits as detected by sedimentation velocity, high-performance ion-exchange chromatography, and reversed-phase and SDS–PAGE subunit analysis. Subunits VIa and VIb are the first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the resulting 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO containing all 13 subunits. However, dimeric CcO, in which the association of VIa and VIb is stabilized, is not susceptible to pressure-induced inactivation. We conclude that dissociation of subunit III and/or VIIa must be responsible for pressure-induced inactivation of CcO since VIa and VIb can be removed from monomeric CcO without significant activity loss. These results are the first to clearly demonstrate an important structural role for the dimeric form of cytochrome c oxidase, i.e., stabilization of its quaternary structure. PMID:17530783

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

PubMed Central

Joseph, Prem Raj B.; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M.; Garofalo, Roberto P.; Rajarathnam, Krishna

2013-01-01

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. PMID:24048001

A High-Resolution Crystal Structure of a Psychrohalophilic α–Carbonic Anhydrase from Photobacterium profundum Reveals a Unique Dimer Interface

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

Somalinga, Vijayakumar; Buhrman, Greg; Arun, Ashikha

Bacterial α–carbonic anhydrases (α-CA) are zinc containing metalloenzymes that catalyze the rapid interconversion of CO2 to bicarbonate and a proton. We report the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, Photobacterium profundum. Size exclusion chromatography of the purified P. profundum α–CA (PprCA) reveals that the protein is a heterogeneous mix of monomers and dimers. Furthermore, an “in-gel” carbonic anhydrase activity assay, also known as protonography, revealed two distinct bands corresponding to monomeric and dimeric forms of PprCA that are catalytically active. The crystal structure of PprCA was determined in its native form and reveals a highlymore » conserved “knot-topology” that is characteristic of α–CA’s. Similar to other bacterial α–CA’s, PprCA also crystallized as a dimer. Furthermore, dimer interface analysis revealed the presence of a chloride ion (Cl-) in the interface which is unique to PprCA and has not been observed in any other α–CA’s characterized so far. Molecular dynamics simulation and chloride ion occupancy analysis shows 100% occupancy for the Cl- ion in the dimer interface. Zinc coordinating triple histidine residues, substrate binding hydrophobic patch residues, and the hydrophilic proton wire residues are highly conserved in PprCA and are identical to other well-studied α–CA’s.« less

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

PubMed

Fulton, Melody D; Hanold, Laura E; Ruan, Zheng; Patel, Sneha; Beedle, Aaron M; Kannan, Natarajan; Kennedy, Eileen J

2018-03-15

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Carbodiimide EDC induces cross-links that stabilize RNase A C-dimer against dissociation: EDC adducts can affect protein net charge, conformation, and activity.

PubMed

López-Alonso, Jorge P; Diez-García, Fernando; Font, Josep; Ribó, Marc; Vilanova, Maria; Scholtz, J Martin; González, Carlos; Vottariello, Francesca; Gotte, Giovanni; Libonati, Massimo; Laurents, Douglas V

2009-08-19

RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDC-treated RNase A monomer were found to carry an increased number of positive charges (about 6 ± 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH(2) monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling*

PubMed Central

Singh, Deo R.; Ahmed, Fozia; King, Christopher; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B.; Hristova, Kalina

2015-01-01

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. PMID:26363067

[Tykerb for breast cancer].

PubMed

Suzuki, Yasuhiro; Saito, Yuki; Okamura, Takuho; Tokuda, Yutaka

2011-06-01

There are four members of the ErbB family: the epidermal growth factor(EGF)receptor(also called HER1 or EGFR), HER2, HER3 and HER4. Dimerization is the process whereby two HER receptor molecules associate to form a noncovalent complex. HER dimers are the active receptor forms required for transmission of external stimuli to the interior of the cell. HER dimerization occurs upon ligand binding and both HER homodimers and heterodimers can be formed in the process. However, HER2 appears to be the preferred dimerization partner of the other HER family members. Fifteen∼20% of all breast cancers are HER2 positive and have a poor prognosis. Trastuzumab is an excellent, rationally-designed targeted cancer treatment. It is a recombinant, humanized, anti-HER2 monoclonal antibody that specifically binds to the extracellular area of HER2. However, the overall trastuzumab response rate is low, and the causes of trastuzumab resistance are poorly understood. Thus, there is a need for alternative anti-HER2 strategies for trastuzumab-resistant disease. Lapatinib is an orally administered small-molecule, reversible inhibitor of both EGFR and HER2 tyrosine kinase, and its activities include subsequent inhibition of its down- stream MAPK-ERK1/2, and the AKT signaling pathway. Lapatinib is more active when used in combination with capecitabine. For women with trastuzumab pre-treated HER2-positive breast cancer, Here, I will review the basics of EGFR and HER, and the treatment strategy for HER2-positive breast cancer with lapatinib.

RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E).

PubMed

Poulikakos, Poulikos I; Persaud, Yogindra; Janakiraman, Manickam; Kong, Xiangju; Ng, Charles; Moriceau, Gatien; Shi, Hubing; Atefi, Mohammad; Titz, Bjoern; Gabay, May Tal; Salton, Maayan; Dahlman, Kimberly B; Tadi, Madhavi; Wargo, Jennifer A; Flaherty, Keith T; Kelley, Mark C; Misteli, Tom; Chapman, Paul B; Sosman, Jeffrey A; Graeber, Thomas G; Ribas, Antoni; Lo, Roger S; Rosen, Neal; Solit, David B

2011-11-23

Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)

PubMed Central

Poulikakos, Poulikos I.; Persaud, Yogindra; Janakiraman, Manickam; Kong, Xiangju; Ng, Charles; Moriceau, Gatien; Shi, Hubing; Atefi, Mohammad; Titz, Bjoern; Gabay, May Tal; Salton, Maayan; Dahlman, Kimberly B.; Tadi, Madhavi; Wargo, Jennifer A.; Flaherty, Keith T.; Kelley, Mark C.; Misteli, Tom; Chapman, Paul B.; Sosman, Jeffrey A.; Graeber, Thomas G.; Ribas, Antoni; Lo, Roger S.; Rosen, Neal; Solit, David B.

2011-01-01

Summary Activated RAS promotes dimerization of members of the RAF kinase family1-3. ATP-competitive RAF inhibitors activate ERK signaling4-7 by transactivating RAF dimers4. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumor-specific inhibition of ERK signaling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E)8. However, resistance invariably develops. Here, we identify a novel resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumors of six of 19 patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner. PMID:22113612

Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.

PubMed

Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B; Bach, Ronald R; Nelsestuen, Gary L

2005-04-26

Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF-fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

NASA Astrophysics Data System (ADS)

Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

2015-03-01

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

Cleavage of Disulfide Bonds in Mouse Spermatogenic Cell-Specific Type 1 Hexokinase Isozyme Is Associated with Increased Hexokinase Activity and Initiation of Sperm Motility1

PubMed Central

Nakamura, Noriko; Miranda-Vizuete, Antonio; Miki, Kiyoshi; Mori, Chisato; Eddy, Edward M.

2008-01-01

During epididymal transit, sperm acquire the ability to initiate rapid forward progressive motility on release into the female reproductive tract or physiological media. Glycolysis is the primary source of the ATP necessary for this motility in the mouse, and several novel glycolytic enzymes have been identified that are localized to the principal piece region of the flagellum. One of these is the spermatogenic cell-specific type 1 hexokinase isozyme (HK1S), the only member of the hexokinase enzyme family detected in sperm. Hexokinase activity was found to be lower in immotile sperm immediately after removal from the cauda epididymis (quiescent) than in sperm incubated in physiological medium for 5 min and showing rapid forward progressive motility (activated). However, incubating sperm in medium containing diamide, an inhibitor of disulfide bond reduction, resulted in lower motility and HK activity than in controls. HK1S was present in dimer and monomer forms in extracts of quiescent sperm but mainly as a monomer in motile sperm. A dimer-size band detected in quiescent sperm with phosphotyrosine antibody was not detected in activated sperm, and the monomer-size band was enhanced. In addition, the general protein oxido-reductase thioredoxin-1 was able to catalyze the in vitro conversion of HK1S dimers to the monomeric form. These results strongly suggest that cleavage of disulfide bonds in HK1S dimers contributes to the increases in HK activity and motility that occur when mouse sperm become activated. PMID:18509164

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

PubMed

Friberg, Anders; Thumann, Sybille; Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D; Sattler, Michael; Kempkes, Bettina

2015-05-01

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

PubMed

Hjörleifsson, Jens G; Ásgeirsson, Bjarni

2017-09-26

The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7-10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the K i for inorganic phosphate (product inhibition) and the K M for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.

A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner.

PubMed

Li, Qi; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Chen, Yanyu; Zhou, Jian-Min; Dou, Daolong

2016-05-31

Oomycete pathogens produce a large number of effectors to promote infection. Their mode of action are largely unknown. Here we show that a Phytophthora sojae effector, PsCRN63, suppresses flg22-induced expression of FRK1 gene, a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation, indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arabidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici. The callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants. Genes involved in PTI were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms an dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed, the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63.

Dimeric Structure of the Blue Light Sensor Protein Photozipper in the Active State.

PubMed

Ozeki, Kohei; Tsukuno, Hiroyuki; Nagashima, Hiroki; Hisatomi, Osamu; Mino, Hiroyuki

2018-02-06

The light oxygen voltage-sensing (LOV) domain plays a crucial role in blue light (BL) sensing in plants and microorganisms. LOV domains are usually associated with the effector domains and regulate the activities of effector domains in a BL-dependent manner. Photozipper (PZ) is monomeric in the dark state. BL induces reversible dimerization of PZ and subsequently increases its affinity for the target DNA sequence. In this study, we report the analyses of PZ by pulsed electron-electron double resonance (PELDOR). The neutral flavin radical was formed by BL illumination in the presence of dithiothreitol in the LOV-C254S (without the bZIP domain) and PZ-C254S mutants, where the cysteine residue responsible for adduct formation was replaced with serine. The magnetic dipole interactions of 3 MHz between the neutral radicals were detected in both LOV-C254S and PZ-C254S, indicating that these mutants are dimeric in the radical state. The PELDOR simulation showed that the distance between the radical pair is close to that estimated from the dimeric crystal structure in the "light state" [Heintz, U., and Schlichting, I. (2016) eLife 5, e11860], suggesting that in the radical state, LOV domains in PZ-C254S form a dimer similar to that of LOV-C254S, which lacks the bZIP domain.

A structural basis for the regulatory inactivation of DnaA.

PubMed

Xu, Qingping; McMullan, Daniel; Abdubek, Polat; Astakhova, Tamara; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Elsliger, Marc-Andre; Feuerhelm, Julie; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Johnson, Hope A; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L; Sefcovic, Natasha; Trame, Christine; van den Bedem, Henry; Weekes, Dana; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2009-01-16

Regulatory inactivation of DnaA is dependent on Hda (homologous to DnaA), a protein homologous to the AAA+ (ATPases associated with diverse cellular activities) ATPase region of the replication initiator DnaA. When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA-ATP into inactive DnaA-ADP. The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 A resolution reveals that Hda resembles typical AAA+ ATPases. The arrangement of the two subdomains in Hda (residues 1-174 and 175-241) differs dramatically from that of DnaA. A CDP molecule anchors the Hda domains in a conformation that promotes dimer formation. The Hda dimer adopts a novel oligomeric assembly for AAA+ proteins in which the arginine finger, crucial for ATP hydrolysis, is fully exposed and available to hydrolyze DnaA-ATP through a typical AAA+ type of mechanism. The sliding clamp binding motifs at the N-terminus of each Hda monomer are partially buried and combine to form an antiparallel beta-sheet at the dimer interface. The inaccessibility of the clamp binding motifs in the CDP-bound structure of Hda suggests that conformational changes are required for Hda to form a functional complex with the clamp. Thus, the CDP-bound Hda dimer likely represents an inactive form of Hda.

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

Competition between Anion Binding and Dimerization Modulates Staphylococcus aureus Phosphatidylinositol-specific Phospholipase C Enzymatic Activity*

PubMed Central

Cheng, Jiongjia; Goldstein, Rebecca; Stec, Boguslaw; Gershenson, Anne; Roberts, Mary F.

2012-01-01

Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane. PMID:23038258

Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization.

PubMed

Gong, Xin; Qian, Hongwu; Shao, Wei; Li, Jingxian; Wu, Jianping; Liu, Jun-Jie; Li, Wenqi; Wang, Hong-Wei; Espenshade, Peter; Yan, Nieng

2016-11-01

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins.

PubMed

Joseph, Prem Raj B; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M; Garofalo, Roberto P; Rajarathnam, Krishna

2013-09-17

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization.

PubMed

Hameed, Uzma; Price, Ian; Ikram-Ul-Haq; Ke, Ailong; Wilson, David B; Mirza, Osman

2017-10-01

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb 1+ , K 1+ and Ca 2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases. Copyright © 2017 Elsevier B.V. All rights reserved.

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.

PubMed

Qiu, Yue; Ogawa, Haruo; Miyagi, Masaru; Misono, Kunio S

2004-02-13

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

PubMed

Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

2016-09-06

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

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

Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed thatmore » hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.« less

Pestivirus Npro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

PubMed Central

Holthauzen, Luis Marcelo F.; Ruggli, Nicolas

2016-01-01

ABSTRACT Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/β) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the Npro-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site. IMPORTANCE The pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of the Npro interaction with IRF3, including the IRF3 species (inactive monomer versus activated dimer) that Npro targets for degradation, is largely unknown. We show that classical swine fever virus Npro and porcine IRF3 directly interact in solution and that full-length IRF3 is required for interaction with Npro. Additionally, Npro interacts with a constitutively active form of IRF3 bound to its transcriptional cofactor, the CREB-binding protein. This is the first study to demonstrate that Npro is able to bind both inactive IRF3 monomer and activated IRF3 dimer and thus likely targets both IRF3 species for ubiquitination and proteasomal degradation. PMID:27334592

Heterodimerization with Jun family members regulates c-Fos nucleocytoplasmic traffic.

PubMed

Malnou, Cécile E; Salem, Tamara; Brockly, Frédérique; Wodrich, Harald; Piechaczyk, Marc; Jariel-Encontre, Isabelle

2007-10-19

c-Fos proto-oncoprotein forms AP-1 transcription complexes with heterodimerization partners such as c-Jun, JunB, and JunD. Thereby, it controls essential cell functions and exerts tumorigenic actions. The dynamics of c-Fos intracellular distribution is poorly understood. Hence, we have combined genetic, cell biology, and microscopic approaches to investigate this issue. In addition to a previously characterized basic nuclear localization signal (NLS) located within the central DNA-binding domain, we identified a second NLS within the c-Fos N-terminal region. This NLS is non-classic and its activity depends on transportin 1 in vivo. Under conditions of prominent nuclear localization, c-Fos can undergo nucleocytoplasmic shuttling through an active Crm-1 exportin-independent mechanism. Dimerization with the Jun proteins inhibits c-Fos nuclear exit. The strongest effect is observed with c-Jun probably in accordance with the relative stabilities of the different c-Fos:Jun dimers. Retrotransport inhibition is not caused by binding of dimers to DNA and, therefore, is not induced by indirect effects linked to activation of c-Fos target genes. Monomeric, but not dimeric, Jun proteins also shuttle actively. Thus, our work unveils a novel regulation operating on AP-1 by demonstrating that dimerization is crucial, not only for active transcription complex formation, but also for keeping them in the compartment where they exert their transcriptional function.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

PubMed Central

Xie, Q W; Leung, M; Fuortes, M; Sassa, S; Nathan, C

1996-01-01

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:8643499

Design and synthesis of small molecule agonists of EphA2 receptor.

PubMed

Petty, Aaron; Idippily, Nethrie; Bobba, Viharika; Geldenhuys, Werner J; Zhong, Bo; Su, Bin; Wang, Bingcheng

2018-01-01

Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure-activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor. Published by Elsevier Masson SAS.

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling.

PubMed

Singh, Deo R; Ahmed, Fozia; King, Christopher; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B; Hristova, Kalina

2015-11-06

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

PubMed

Stranava, Martin; Man, Petr; Skálová, Tereza; Kolenko, Petr; Blaha, Jan; Fojtikova, Veronika; Martínek, Václav; Dohnálek, Jan; Lengalova, Alzbeta; Rosůlek, Michal; Shimizu, Toru; Martínková, Markéta

2017-12-22

The heme-based oxygen sensor histidine kinase Af GcHK is part of a two-component signal transduction system in bacteria. O 2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His 183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH - and -CN - complexes of Af GcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN - and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length Af GcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of Af GcHK. We conclude that Af GcHK functions as an ensemble of molecules sampling at least two conformational states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of autophosphorylation of mycobacterial PknB explored by molecular dynamics simulations.

PubMed

Damle, Nikhil P; Mohanty, Debasisa

2014-07-22

Mycobacterial Ser/Thr kinase, PknB, is essential for the growth of the pathogen. Unphosphorylated PknB is catalytically inactive, and its activation requires autophosphorylation of Thr residues on the activation loop. Autophosphorylation can in principle take place via two distinct mechanisms. Intermolecular trans autophosphorylation involves dimerization and phosphorylation of the activation loop of one chain in the catalytic pocket of the other chain. On the other hand, intramolecular cis autophosphorylation involves phosphorylation of the activation loop of the kinases in its own catalytic pocket within a monomer. On the basis of the crystal structure of PknB in the front-to-front dimeric form, it is currently believed that activation of PknB involves trans autophosphorylation. However, because of the lack of coordinates of the activation loop in the crystal structures, atomic details of the conformational changes associated with activation are yet to be deciphered. Therefore, to understand the conformational transitions associated with activation via autophosphorylation, a series of explicit solvent molecular dynamics simulations with a duration of 1 μs have been performed on each of the phosphorylated and nonphosphorylated forms of the PknB catalytic domain in monomeric and dimeric states. Simulations on phosphorylated PknB revealed a differential network of crucial electrostatic and hydrophobic residues that stabilize the phosphorylated form in the active conformation. Interestingly, in our simulations on nonphosphorylated monomers, the activation loop was observed to fold into its own active site, thereby opening the novel possibility of activation through intramolecular cis autophosphorylation. Thus, our simulations suggest that autophosphorylation of PknB might also involve cis initiation followed by trans amplification as reported for other eukaryotic kinases based on recent reaction kinetics studies.

Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-06-01

Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

Porphyrin-induced photogeneration of hydrogen peroxide determined using the luminol chemiluminescence method in aqueous solution: A structure-activity relationship study related to the aggregation of porphyrin.

PubMed

Komagoe, Keiko; Katsu, Takashi

2006-02-01

A luminol chemiluminescence method was used to evaluate the porphyrin-induced photogeneration of hydrogen peroxide (H2O2). This method enabled us to detect H202 in the presence of a high concentration of porphyrin, which was not possible using conventional colorimetry. The limit of detection was about 1 microM. We compared the ability to generate H2O2, using uroporphyrin (UP), hexacarboxylporphyrin (HCP), coproporphyrin (CP), hematoporphyrin (HP), mesoporphyrin (MP), and protoporphyrin (PP). The amount of H2O2 photoproduced was strongly related to the state of the porphyrin in the aqueous solution. UP and HCP, which existed predominantly in a monomeric form, had a good ability to produce H2O2. HP and MP, existing as dimers, showed weak activity. CP, forming a mixture of monomer and dimer, had a moderate ability to produce H2O2. PP, which was highly aggregated, had a good ability. These results demonstrated that the efficiency of porphyrins to produce H2O2 was strongly dependent on their aggregated form, and the dimer suppressed the production of H2O2.

Quantitative fluorescence nanoscopy for cancer biomedicine

NASA Astrophysics Data System (ADS)

Huang, Tao; Nickerson, Andrew; Peters, Alec; Nan, Xiaolin

2015-08-01

Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.

Integrated analysis reveals that STAT3 is central to the crosstalk between HER/ErbB receptor signaling pathways in human mammary epithelial cells

DOE PAGES

Gong, Chunhong; Zhang, Yi; Shankaran, Harish; ...

2014-10-02

Human epidermal growth factor receptors (HER, also known as ErbB) drive cellular proliferation, pro-survival and stress responses by activating several downstream kinases, in particular ERK, p38, JNK (SAPK), the PI3K/AKT, as well as various transcriptional regulators such as STAT3. When co-expressed, first three members of HER family (HER1-3) can form homo- and hetero-dimers. Based on the considerable evidence which suggest that every receptor dimer activates intracellular signaling pathways differentially, we hypothesized that the HER dimerization pattern is a better predictor of downstream signaling than the total receptor activation levels. We validated our hypothesis using a combination of model-based analysis tomore » quantify the HER dimerization patterns and multi-factorial experiments where HER dimerization patterns and signaling crosstalk were rationally perturbed. We have measured the activation of HER1-3 receptors and of the sentinel signaling proteins ERK, AKT, p38, JNK, STAT3 as a function of time in a panel of human mammary epithelial (HME) cells expressing different levels of HER1-3 stimulated with various ligand combinations. Our analysis using multiple ways of clustering the activation data has confirmed that the HER receptor dimer is a better predictor of the signaling through p38, ERK and AKT pathways than the total HER receptor expression and activation levels. Targeted inhibition studies to identify the causal effects allowed us to obtain a consensus regulatory interaction model, which revealed that STAT3 occupies a central role in the crosstalk between the studied pathways.« less

The Globular Tail Domain of Myosin-5a Functions as a Dimer in Regulating the Motor Activity.

PubMed

Zhang, Wen-Bo; Yao, Lin-Lin; Li, Xiang-Dong

2016-06-24

Myosin-5a contains two heavy chains, which are dimerized via the coiled-coil regions. Thus, myosin-5a comprises two heads and two globular tail domains (GTDs). The GTD is the inhibitory domain that binds to the head and inhibits its motor function. Although the two-headed structure is essential for the processive movement of myosin-5a along actin filaments, little is known about the role of GTD dimerization. Here, we investigated the effect of GTD dimerization on its inhibitory activity. We found that the potent inhibitory activity of the GTD is dependent on its dimerization by the preceding coiled-coil regions, indicating synergistic interactions between the two GTDs and the two heads of myosin-5a. Moreover, we found that alanine mutations of the two conserved basic residues at N-terminal extension of the GTD not only weaken the inhibitory activity of the GTD but also enhance the activation of myosin-5a by its cargo-binding protein melanophilin (Mlph). These results are consistent with the GTD forming a head to head dimer, in which the N-terminal extension of the GTD interacts with the Mlph-binding site in the counterpart GTD. The Mlph-binding site at the GTD-GTD interface must be exposed prior to the binding of Mlph. We therefore propose that the inhibited Myo5a is equilibrated between the folded state, in which the Mlph-binding site is buried, and the preactivated state, in which the Mlph-binding site is exposed, and that Mlph is able to bind to the Myo5a in preactivated state and activates its motor function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Biological Insights of the Dopaminergic Stabilizer ACR16 at the Binding Pocket of Dopamine D2 Receptor.

PubMed

Ekhteiari Salmas, Ramin; Seeman, Philip; Aksoydan, Busecan; Stein, Matthias; Yurtsever, Mine; Durdagi, Serdar

2017-04-19

The dopamine D2 receptor (D2R) plays an important part in the human central nervous system and it is considered to be a focal target of antipsychotic agents. It is structurally modeled in active and inactive states, in which homodimerization reaction of the D2R monomers is also applied. The ASP2314 (also known as ACR16) ligand, a D2R stabilizer, is used in tests to evaluate how dimerization and conformational changes may alter the ligand binding space and to provide information on alterations in inhibitory mechanisms upon activation. The administration of the D2R agonist ligand ACR16 [ 3 H](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ((+)PHNO) revealed K i values of 32 nM for the D2 high R and 52 μM for the D2 low R. The calculated binding affinities of ACR16 with post processing molecular dynamics (MD) simulations analyses using MM/PBSA for the monomeric and homodimeric forms of the D2 high R were -9.46 and -8.39 kcal/mol, respectively. The data suggests that the dimerization of the D2R leads negative cooperativity for ACR16 binding. The dimerization reaction of the D2 high R is energetically favorable by -22.95 kcal/mol. The dimerization reaction structurally and thermodynamically stabilizes the D2 high R conformation, which may be due to the intermolecular forces formed between the TM4 of each monomer, and the result strongly demonstrates dimerization essential for activation of the D2R.

The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion.

PubMed

Schulte, Kathrin; Pawlowski, Nikolaus; Faelber, Katja; Fröhlich, Chris; Howard, Jonathan; Daumke, Oliver

2016-03-02

The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action.

Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

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

S Menon; S Wang

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, themore » switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.« less

Crystal Structure of Ripk4 Reveals Dimerization-Dependent Kinase Activity.

PubMed

Huang, Christine S; Oberbeck, Nina; Hsiao, Yi-Chun; Liu, Peter; Johnson, Adam R; Dixit, Vishva M; Hymowitz, Sarah G

2018-05-01

Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities. We describe the structure of the murine Ripk4 (MmRipk4) kinase domain, in ATP- and inhibitor-bound forms. The crystallographic dimer of MmRipk4 is similar to those of RIPK2 and BRAF, and we show that the intact dimeric entity is required for MmRipk4 catalytic activity through a series of engineered mutations and cell-based assays. We also assess the impact of BPS mutations on protein structure and activity to elucidate the molecular origins of the disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

PubMed

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

Insight into the evolution of nidovirus endoribonuclease based on the finding that Nsp15 from porcine deltacoronavirus functions as a dimer.

PubMed

Zheng, Anjun; Shi, Yuejun; Shen, Zhou; Wang, Gang; Shi, Jiale; Xiong, Qiqi; Fang, Liurong; Xiao, Shaobo; Fu, Zhen F; Peng, Guiqing

2018-06-10

Nidovirus endoribonucleases (NendoUs) include Nsp15 from coronaviruses and Nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E and MHV Nsp15 indicate that it mainly forms a functional hexamer, whereas Nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine deltacoronavirus (PDCoV) Nsp15 primarily exists as dimers and monomers in vitro. Biological experiments reveal that a PDCoV Nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (NTD, Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV Nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV Nsp15. This result may explain why PDCoV Nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells and that NendoU from arterivirus gained ability to form dimers and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV Nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Disentangling mechanisms involved in collagen pyridinoline cross-linking: The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

PubMed Central

Gjaltema, Rutger A. F.; van der Stoel, Miesje M.; Boersema, Miriam; Bank, Ruud A.

2016-01-01

Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen Iα1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer. PMID:27298363

Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

PubMed

Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

2015-08-07

The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis

PubMed Central

Pesch, Martina; Schultheiß, Ilka; Klopffleisch, Karsten; Clemen, Christoph S.; Hülskamp, Martin

2015-01-01

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and TRANSPARENT TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, TRIPTYCHON (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes. PMID:25926482

Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

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

Pioszak, Augen A.; Harikumar, Kaleeckal G.; Parker, Naomi R.

2010-06-25

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an {alpha}-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure,more » PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.« less

Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

PubMed Central

Debyser, Z.; De Clercq, E.

1996-01-01

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

Determination of Abraham model solute descriptors for the monomeric and dimeric forms of trans-cinnamic acid using measured solubilities from the Open Notebook Science Challenge.

PubMed

Bradley, Jean-Claude; Abraham, Michael H; Acree, William E; Lang, Andrew Sid; Beck, Samantha N; Bulger, David A; Clark, Elizabeth A; Condron, Lacey N; Costa, Stephanie T; Curtin, Evan M; Kurtu, Sozit B; Mangir, Mark I; McBride, Matthew J

2015-01-01

Calculating Abraham descriptors from solubility values requires that the solute have the same form when dissolved in all solvents. However, carboxylic acids can form dimers when dissolved in non-polar solvents. For such compounds Abraham descriptors can be calculated for both the monomeric and dimeric forms by treating the polar and non-polar systems separately. We illustrate the method of how this can be done by calculating the Abraham descriptors for both the monomeric and dimeric forms of trans-cinnamic acid, the first time that descriptors for a carboxylic acid dimer have been obtained. Abraham descriptors were calculated for the monomeric form of trans-cinnamic acid using experimental solubility measurements in polar solvents from the Open Notebook Science Challenge together with a number of water-solvent partition coefficients from the literature. Similarly, experimental solubility measurements in non-polar solvents were used to determine Abraham descriptors for the trans-cinnamic acid dimer. Abraham descriptors were calculated for both the monomeric and dimeric forms of trans-cinnamic acid. This allows for the prediction of further solubilities of trans-cinnamic acid in both polar and non-polar solvents with an error of about 0.10 log units. Graphical abstractMolar concentration of trans-cinnamic acid in various polar and non-polar solvents.

Graded-index optical dimer formed by optical force

DOE PAGES

Akbarzadeh, Alireza; Koschny, Thomas; Kafesaki, Maria; ...

2016-05-30

We propose an optical dimer formed from two spherical lenses bound by the pressure that light exerts on matter. With the help of the method of force tracing, we find the required graded-index profiles of the lenses for the existence of the dimer. We study the dynamics of the opto-mechanical interaction of lenses under the illumination of collimated light beams and quantitatively validate the performance of the proposed dimer. We also examine the stability of the dimer due to the lateral misalignments and we show how restoring forces bring the dimer into lateral equilibrium. The dimer can be employed inmore » various practical applications such as optical manipulation, sensing and imaging.« less

Graded-index optical dimer formed by optical force

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

Akbarzadeh, Alireza; Koschny, Thomas; Kafesaki, Maria

We propose an optical dimer formed from two spherical lenses bound by the pressure that light exerts on matter. With the help of the method of force tracing, we find the required graded-index profiles of the lenses for the existence of the dimer. We study the dynamics of the opto-mechanical interaction of lenses under the illumination of collimated light beams and quantitatively validate the performance of the proposed dimer. We also examine the stability of the dimer due to the lateral misalignments and we show how restoring forces bring the dimer into lateral equilibrium. The dimer can be employed inmore » various practical applications such as optical manipulation, sensing and imaging.« less

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

PubMed

Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A facile route to the synthesis of anilinic electroactive colloidal hydrogels for neural tissue engineering applications.

PubMed

Zarrintaj, Payam; Urbanska, Aleksandra M; Gholizadeh, Saman Seyed; Goodarzi, Vahabodin; Saeb, Mohammad Reza; Mozafari, Masoud

2018-04-15

An innovative drug-loaded colloidal hydrogel was synthesized for applications in neural interfaces in tissue engineering by reacting carboxyl capped aniline dimer and gelatin molecules. Dexamethasone was loaded into the gelatin-aniline dimer solution as a model drug to form an in situ drug-loaded colloidal hydrogel. The conductivity of the hydrogel samples fluctuated around 10 -5  S/cm which appeared suitable for cellular activities. Cyclic voltammetry was used for electroactivity determination, in which 2 redox states were observed, suggesting that the short chain length and steric hindrance prevented the gel from achieving a fully oxidized state. Rheological data depicted the modulus decreasing with aniline dimer increment due to limited hydrogen bonds accessibility. Though the swelling ratio of pristine gelatin (600%) decreased by the introduction and increasing the concentration of aniline dimer because of its hydrophobic nature, it took the value of 300% at worst, which still seems promising for drug delivery uses. Degradation rate of hydrogel was similarly decreased by adding aniline dimer. Drug release was evaluated in passive and stimulated patterns demonstrating tendency of aniline dimer to form a vesicle that controls the drug release behavior. The optimal cell viability, proper cell attachment and neurite extension was achieved in the case of hydrogel containing 10 wt% aniline dimer. Based on tissue/organ behavior, it was promisingly possible to adjust the characteristics of the hydrogels for an optimal drug release. The outcome of this simple and effective approach can potentially offer additional tunable characteristics for recording and stimulating purposes in neural interfaces. Copyright © 2018 Elsevier Inc. All rights reserved.

Mechanisms for dominance: Adh heterodimer formation in heterozygotes between ENU or x-ray induced null alleles and normal alleles in drosophila melanogaster

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

Jiang, J.C.; Lee, W.R.; Chang, S.H.

1992-01-01

To study mechanisms for dominance of phenotype, eight ENU- and four x-ray-induced mutations at the alcohol dehydrogenase (Adh) locus were analyzed for partial dominance in their interaction with normal alleles. All ENU and one of the x-ray mutations were single base substitutions; the other three x-ray mutations were 9-21 base deletions. All but one of the 12 mutant alleles were selected for this study because they produced detectable mutant polypeptides, but seven of the 11 producing a peptide could not form dimers with the normal peptide and the enzyme activity of heterozygotes was about half that of normal homozygotes. Fourmore » mutations formed dimers with a decreased catalytic efficiency and two of these were near the limit of detectability; these two also inhibited the formation of normal homodimers. The mutant alleles therefore show multiple mechanisms leading to partial enzyme expression in heterozygotes and a wide range of dominance ranging from almost complete recessive to nearly dominant. All amino acid changes in mutant peptides that form dimers are located between amino acids 182 and 194, so this region is not critical for dimerization. It may, however, be an important surface domain for catalyzation. 34 refs., 8 figs., 2 tabs.« less

A Structural Basis for the Regulatory Inactivation of DnaA

PubMed Central

Xu, Qingping; McMullan, Daniel; Abdubek, Polat; Astakhova, Tamara; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Elsliger, Marc-Andre; Feuerhelm, Julie; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Trame, Christine; van den Bedem, Henry; Weekes, Dana; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

2009-01-01

Summary Regulatory inactivation of DnaA is dependent on Hda, a protein homologous to the AAA+ ATPase region of the replication initiator DnaA. When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA-ATP into inactive DnaA-ADP. The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 Å resolution reveals that Hda resembles typical AAA+ ATPases. The arrangement of the two subdomains in Hda (residues 1-174, 175-241) differs dramatically from that of DnaA. A CDP molecule anchors the Hda domains in a conformation which promotes dimer formation. The Hda dimer adopts a novel oligomeric assembly for AAA+ proteins in which the arginine finger, crucial for ATP hydrolysis, is fully exposed and available to hydrolyze DnaA-ATP through a typical AAA+ type mechanism. The sliding clamp binding motifs at the N-terminus of each Hda monomer are partially buried and combine to form an antiparallel β-sheet at the dimer interface. The inaccessibility of the clamp binding motifs in the CDP bound structure of Hda suggests that conformational changes are required for Hda to form a functional complex with the clamp. Thus, the CDP-bound Hda dimer likely represents an inactive form of Hda. PMID:19000695

Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

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

Svintradze, David V.; Virginia Commonwealth University, Richmond, VA 23219-1540; Peterson, Darrell L.

Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces,more » which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.« less

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

PubMed Central

Feng, Y X; Fu, W; Winter, A J; Levin, J G; Rein, A

1995-01-01

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA. PMID:7884897

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

PubMed

Feng, Y X; Fu, W; Winter, A J; Levin, J G; Rein, A

1995-04-01

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA.

Conditional Toxin Splicing Using a Split Intein System.

PubMed

Alford, Spencer C; O'Sullivan, Connor; Howard, Perry L

2017-01-01

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

Disruption of the HIV-1 protease dimer with interface peptides: structural studies using NMR spectroscopy combined with [2-(13)C]-Trp selective labeling.

PubMed

Frutos, Silvia; Rodriguez-Mias, Ricard A; Madurga, Sergio; Collinet, Bruno; Reboud-Ravaux, Michèle; Ludevid, Dolors; Giralt, Ernest

2007-01-01

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a four-stranded antiparallel beta-sheet involving the N- and C- termini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer. This strategy has been explored by several groups who targeted the four-stranded antiparallel beta-sheet that contributes close to 75% of the dimerization energy. Interface peptides corresponding to native monomer N- or C-termini of several of their mimetics demonstrated, mainly on the basis of kinetic analyses, to act as dimerization inhibitors. However, to the best of our knowledge, neither X-ray crystallography nor NMR structural studies of the enzyme-inhibitor complex have been performed to date. In this article we report a structural study of the dimerization inhibition of HIV-1 PR by NMR using selective Trp side chain labeling.

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

PubMed Central

Burchett, Gina G.; Folsom, Charles G.; Lane, Kimberly T.

2015-01-01

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins. PMID:26121040

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase.

PubMed

Burchett, Gina G; Folsom, Charles G; Lane, Kimberly T

2015-01-01

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.

The complex of hypericin with β-lactoglobulin has antimicrobial activity with potential applications in dairy industry.

PubMed

Rodríguez-Amigo, Beatriz; Delcanale, Pietro; Rotger, Gabriel; Juárez-Jiménez, Jordi; Abbruzzetti, Stefania; Summer, Andrea; Agut, Montserrat; Luque, F Javier; Nonell, Santi; Viappiani, Cristiano

2015-01-01

Using a combination of molecular modeling and spectroscopic experiments, the naturally occurring, pharmacologically active hypericin compound is shown to form a stable complex with the dimeric form of β-lactoglobulin (β-LG). Binding is predicted to occur at the narrowest cleft found at the interface between monomers in the dimeric β-LG. The complex is able to preserve the fluorescence and singlet oxygen photosensitizing properties of the dye. The equilibrium constant for hypericin binding has been determined as Ka=1.40±0.07µM(-1), equivalent to a dissociation constant, Kd=0.71±0.03µM. The complex is active against Staphylococcus aureus bacteria. Overall, the results are encouraging for pursuing the potential application of the complex between hypericin and β-LG as a nanodevice with bactericidal properties for disinfection. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex.

PubMed

Schiering, N; Casale, E; Caccia, P; Giordano, P; Battistini, C

2000-11-07

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.

Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

PubMed Central

Corrada, Dario; Soshilov, Anatoly A.; Denison, Michael S.

2016-01-01

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR. PMID:27295348

Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation.

PubMed

Corrada, Dario; Soshilov, Anatoly A; Denison, Michael S; Bonati, Laura

2016-06-01

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.

Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications.

PubMed

Sánchez-Martín, M; Pandiella, A

2012-07-01

Deregulation of ErbB/HER receptor tyrosine kinases has been linked to several types of cancer. The mechanism of activation of these receptors includes establishment of receptor dimers. Here, we have analyzed the action of different small molecule HER tyrosine kinase inhibitors (TKIs) on HER receptor dimerization. Breast cancer cell lines were treated with distinct TKIs and the formation of HER2-HER3 dimers was analyzed by coimmunoprecipitation and western blot or by Förster resonance energy transfer assays. Antibody-dependent cellular cytotoxicity was analyzed by measuring the release of lactate dehydrogenase and cell viability. Lapatinib and neratinib interfered with ligand-induced dimerization of HER receptors; while pelitinib, gefitinib, canertinib or erlotinib did not. Moreover, lapatinib and neratinib were able to disrupt previously formed receptor dimers. Structural analyses allowed the elucidation of the mechanism by which some TKIs prevent the formation of HER receptor dimers, while others do not. Experiments aimed at defining the functional importance of dimerization indicated that TKIs that impeded dimerization prevented down-regulation of HER2 receptors, and favored the action of trastuzumab. We postulate that TKIs that prevent dimerization and down-regulation of HER2 may augment the antitumoral action of trastuzumab, and this mechanism of action should be considered in the treatment of HER2 positive tumors which combine TKIs with antireceptor antibodies. Copyright © 2011 UICC.

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

PubMed Central

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

Dimers in α-pinene secondary organic aerosol: effect of hydroxyl radical, ozone, relative humidity and aerosol acidity

NASA Astrophysics Data System (ADS)

Kristensen, K.; Cui, T.; Zhang, H.; Gold, A.; Glasius, M.; Surratt, J. D.

2014-04-01

The formation of secondary organic aerosol (SOA) from both ozonolysis and hydroxyl radical (OH)-initiated oxidation of α-pinene under conditions of high nitric oxide (NO) concentrations with varying relative humidity (RH) and aerosol acidity was investigated in the University of North Carolina dual outdoor smog chamber facility. SOA formation from ozonolysis of α-pinene was enhanced relative to that from OH-initiated oxidation in the presence of initially high-NO conditions. However, no effect of RH on SOA mass was evident. Ozone (O3)-initiated oxidation of α-pinene in the presence of ammonium sulfate (AS) seed coated with organic aerosol from OH-initiated oxidation of α-pinene showed reduced nucleation compared to ozonolysis in the presence of pure AS seed aerosol. The chemical composition of α-pinene SOA was investigated by ultra-performance liquid chromatography/electrospray ionization high-resolution quadrupole time-of-flight mass spectrometry (UPLC/ESI-HR-Q-TOFMS), with a focus on the formation of carboxylic acids and high-molecular weight dimers. A total of eight carboxylic acids and four dimers were identified, constituting between 8 and 12% of the total α-pinene SOA mass. OH-initiated oxidation of α-pinene in the presence of nitrogen oxides (NOx) resulted in the formation of highly oxidized carboxylic acids, such as 3-methyl-1,2,3-butanetricarboxylic acid (MBTCA) and diaterpenylic acid acetate (DTAA). The formation of dimers was observed only in SOA produced from the ozonolysis of α-pinene in the absence of NOx, with increased concentrations by a factor of two at higher RH (50-90%) relative to lower RH (30-50%). The increased formation of dimers correlates with an observed increase in new particle formation at higher RH due to nucleation. Increased aerosol acidity was found to have a negligible effect on the formation of the dimers. SOA mass yield did not influence the chemical composition of SOA formed from α-pinene ozonolysis with respect to carboxylic acids and dimers. The results support the formation of the high-molecular weight dimers through gas-phase reactions of the stabilized Criegee Intermediate (sCI) formed from the ozonolysis of α-pinene. The high molecular weight and polar nature of dimers formed in the gas phase may explain increased particle number concentration as a result of homogenous nucleation. Since three of these dimers (i.e. pinyl-diaterpenyl dimer (MW 358), pinyl-diaterebyl dimer (MW 344) and pinonyl-pinyl dimer (MW 368)) have been observed in both laboratory-generated and ambient fine organic aerosol samples, we conclude that the dimers observed in this study can be used as tracers for the O3-initiated oxidation of α-pinene, and are therefore indicative of enhanced anthropogenic activities, and that the high molecular weight and low volatility dimers result in homogenous nucleation under laboratory conditions, increasing the particle number concentration.

Dimerization of BTas is required for the transactivational activity of bovine foamy virus

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

Tan Juan; Qiao Wentao; Xu Fengwen

2008-06-20

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46-62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activitymore » on the LTR and IP. Furthermore, BTas ({delta}46-62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV.« less

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase*

PubMed Central

Zhang, Shengnan; Fitzpatrick, Paul F.

2016-01-01

Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25–117) is the regulatory domain of PheH lacking residues 1–24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25–117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25–117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. PMID:26823465

Designer interface peptide grafts target estrogen receptor alpha dimerization

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

Chakraborty, S.; Asare, B.K.; Biswas, P.K., E-mail: pbiswas@tougaloo.edu

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide “I-box” derived from ER residues 503–518 specifically blocks ER dimerization.more » Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479–485), LQQQHQRLAQ (residues 497–506), and LSHIRHMSNK (residues 511–520) and reported the suitability of using LQQQHQRLAQ (ER 497–506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. - Highlights: • Designer peptide grafts retain core molecular recognition motif during MD simulations. • Designer peptide grafts with Poly-ALA helix form stable complexes with estrogen receptor in silico. • Inhibitor peptides significantly decrease estrogen induced cell proliferation of ER positive breast cancer cells in vitro.« less

Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

PubMed

Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

2016-08-26

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. Copyright © 2016. Published by Elsevier Inc.

Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate.

PubMed

Hiraga, Kazumi; Ueno, Yoshie; Oda, Kohei

2008-05-01

In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.

Bowman-Birk proteinase inhibitor from Cajanus cajan seeds: purification, characterization, and insecticidal properties.

PubMed

Prasad, Elaprolu R; Merzendorfer, H; Madhurarekha, C; Dutta-Gupta, A; Padmasree, K

2010-03-10

A red gram proteinase inhibitor (RgPI) was purified from red gram ( Cajanus cajan ) seeds by using ammonium sulfate precipitation and ion-exchange, affinity, and gel filtration chromatography. SDS-PAGE under nonreducing condition revealed two protein bands with molecular masses of approximately 8.5 and approximately 16.5 kDa corresponding to monomeric and dimeric forms of RgPI, respectively. Similarly, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry also confirmed the presence of dimer as well as other oligomeric forms: trimer, tetramer, and pentamer. Reduction of RgPI with dithiothreitol (DTT) led to the dissociation of the dimeric and oligomeric forms. Native-PAGE and two-dimensional gel electrophoresis indicated the existence of isoinhibitors with pI values of 5.95, 6.25, 6.50, 6.90, and 7.15, respectively. The MALDI-TOF-TOF mass spectrum and N-terminal sequence 'DQHHSSKACC' suggested that the isolated RgPI is a member of the Bowman-Birk inhibitor family. RgPI exhibited noncompetitive type inhibitory activity against bovine pancreatic trypsin and chymotrypsin, with inhibition constants of 292 and 2265 nM, respectively. It was stable up to a temperature of 80 degrees C and was active over a wide pH range between 2 and 12. However, reduction with DTT or 2-mercaptoethanol resulted in loss of inhibitory activity against trypsin and chymotrypsin. It also decreased the activity of larval midgut trypsin-like proteinases in Manduca sexta . Its insecticidal property was further confirmed by reduction in the growth and development of these larvae, when supplemented in the diet.

Coiled-coil forming peptides for the induction of silver nanoparticles

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

Božič Abram, Sabina; Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000; Aupič, Jana

Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at aroundmore » 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.« less

The Role of Dimerization in Raf Signaling | Center for Cancer Research

Cancer.gov

One frequently mutated pathway in a variety of cancers and developmental disorders is the Ras-Raf-MEK-ERK cascade. Normally, binding of a growth factor to its receptor switches on Ras, which, in turn, activates one or more of the Raf kinase family members, A-Raf, B-Raf, and C-Raf. Rafs perpetuate the signal by phosphorylating and activating MEK, another kinase that phosphorylates a third kinase, ERK. ERK then phosphorylates a number of key growth-, survival-, or differentiation-promoting targets. Of the proteins in the cascade, Rafs have the most complex regulatory mechanisms, including the ability to form dimers. Because the role that dimerization plays in Raf function has been unclear, researchers working with Deborah Morrison, Ph.D., Chief of CCR’s Laboratory of Cell and Developmental Signaling, decided to investigate its significance in normal and disease-associated Raf signaling.

FT-IR spectra of the anti-HIV nucleoside analogue d4T (Stavudine). Solid state simulation by DFT methods and scaling by different procedures

NASA Astrophysics Data System (ADS)

Alcolea Palafox, M.; Kattan, D.; Afseth, N. K.

2018-04-01

A theoretical and experimental vibrational study of the anti-HIV d4T (stavudine or Zerit) nucleoside analogue was carried out. The predicted spectra in the three most stable conformers in the biological active anti-form of the isolated state were compared. Comparison of the conformers with those of the natural nucleoside thymidine was carried out. The calculated spectra were scaled by using different scaling procedures and three DFT methods. The TLSE procedure leads to the lowest error and is thus recommended for scaling. With the population of these conformers the IR gas-phase spectra were predicted. The crystal unit cell of the different polymorphism forms of d4T were simulated through dimer forms by using DFT methods. The scaled spectra of these dimer forms were compared. The FT-IR spectrum was recorded in the solid state in the 400-4000 cm-1 range. The respective vibrational bands were analyzed and assigned to different normal modes of vibration by comparison with the scaled vibrational values of the different dimer forms. Through this comparison, the polymorphous form of the solid state sample was identified. The study indicates that d4T exist only in the ketonic form in the solid state. The results obtained were in agreement with those determined in related anti-HIV nucleoside analogues.

Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics

PubMed Central

Motta, Stefano

2018-01-01

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses. PMID:29489822

Crystal structure and function of an unusual dimeric Hsp20.1 provide insight into the thermal protection mechanism of small heat shock proteins.

PubMed

Liu, Liang; Chen, Jiyun; Yang, Bo; Wang, Yonghua

2015-03-06

Small heat shock proteins (sHSPs) are ubiquitous chaperones that play a vital role in protein homeostasis. sHSPs are characterized by oligomeric architectures and dynamic exchange of subunits. The flexible oligomeric assembling associating with function remains poorly understood. Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a β6 strand to differentiate nonmetazoan sHSPs from metazoan sHSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of β6, Hsp20.1 dimer does not depend on β7 strand for forming dimer interface as metazoan sHSPs, nor dissociates to monomers. This is in contrast to other published sHSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein. Together, the structure and function of Hsp20.1 give more insight into the thermal protection mechanism of sHSPs. Copyright © 2015 Elsevier Inc. All rights reserved.

Targeting cysteine-mediated dimerization of the MUC1-C oncoprotein in human cancer cells

PubMed Central

RAINA, DEEPAK; AHMAD, REHAN; RAJABI, HASAN; PANCHAMOORTHY, GOVIND; KHARBANDA, SURENDER; KUFE, DONALD

2012-01-01

The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas and contributes to the malignant phenotype. The MUC1-C transmembrane subunit contains a CQC motif in the cytoplasmic domain that has been implicated in the formation of dimers and in its oncogenic function. The present study demonstrates that MUC1-C forms dimers in human breast and lung cancer cells. MUC1-C dimerization was detectable in the cytoplasm and was independent of MUC1-N, the N-terminal mucin subunit that extends outside the cell. We show that the MUC1-C cytoplasmic domain forms dimers in vitro that are disrupted by reducing agents. Moreover, dimerization of the MUC1-C subunit in cancer cells was blocked by reducing agents and increased by oxidative stress, supporting involvement of the CQC motif in forming disulfide bonds. In support of these observations, mutation of the MUC1-C CQC motif to AQA completely blocked MUC1-C dimerization. Importantly, this study was performed with MUC1-C devoid of fluorescent proteins, such as GFP, CFP and YFP. In this regard, we show that GFP, CFP and YFP themselves form dimers that are readily detectable with cross-linking agents. The present results further demonstrate that a cell-penetrating peptide that targets the MUC1-C CQC cysteines blocks MUC1-C dimerization in cancer cells. These findings provide definitive evidence that: i) the MUC1-C cytoplasmic domain cysteines are necessary and sufficient for MUC1-C dimerization, and ii) these CQC motif cysteines represent an Achilles’ heel for targeting MUC1-C function. PMID:22200620

Hsa-miR-1587 G-quadruplex formation and dimerization induced by NH4+, molecular crowding environment and jatrorrhizine derivatives.

PubMed

Tan, Wei; Yi, Long; Zhu, Zhentao; Zhang, Lulu; Zhou, Jiang; Yuan, Gu

2018-03-01

A guanine-rich human mature microRNA, miR-1587, was discovered to form stable intramolecular G-quadruplexes in the presence of K + , Na + and low concentration of NH 4 + (25mM) by electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism (CD) spectroscopy. Furthermore, under high concentration of NH 4 + (100mM) or molecular crowding environments, miR-1587 formed a dimeric G-quadruplex through 3'-to-3' stacking of two monomeric G-quadruplex subunits with one ammonium ion sandwiched between the interfaces. Specifically, two synthesized jatrorrhizine derivatives with terminal amine groups could also induce the dimerization of miR-1587 G-quadruplex and formed 1:1 and 2:1 complexes with the dimeric G-quadruplex. In contrast, jatrorrhizine could bind with the dimeric miR-1587 G-quadruplex, but could not induce dimerization of miR-1587 G-quadruplex. These results provide a new strategy to regulate the functions of miR-1587 through induction of G-quadruplex formation and dimerization. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel inhibitor of STAT3 homodimerization selectively suppresses STAT3 activity and malignant transformation

PubMed Central

Zhang, Xiaolei; Sun, Ying; Pireddu, Roberta; Yang, Hua; Urlam, Murali K.; Lawrence, Harshani R.; Guida, Wayne C.; Lawrence, Nicholas J.; Sebti, Saïd M.

2014-01-01

STAT3-STAT3 dimerization, which involves reciprocal binding of the STAT3-SH2 domain to phosphorylated tyrosine-705 (Y-705), is required for STAT3 nuclear translocation, DNA binding and transcriptional regulation of downstream target genes. Here we describe a small molecule S3I-1757 capable of disrupting STAT3-STAT3 dimerization, activation and malignant transforming activity. Fluorescence polarization assays and molecular modeling suggest that S3I-1757 interacts with the Y-705 binding site in the SH2 domain and displaces fluorescein-labelled GpYLPQTV phosphotyrosine peptide from binding to STAT3. We generated HA-tagged STAT3 and FLAG-tagged STAT3 and showed using co-immunoprecipitation and co-localization studies that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analogue, S3I-1756, that does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over AKT1 and ERK1/2 (MAPK3/1), nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding and transcriptional activation and suppresses the expression levels of STAT3 target genes such as Bcl-xL (BCL2L1), survivin (BIRC5), cyclin D1 (CCND1) and MMP9. Furthermore, S3I-1757 but not S3I-1756 inhibits anchorage-dependent and -independent growth, migration and invasion of human cancer cells which depend on STAT3. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues cells from the effects of S3I-1757 inhibition. Thus, we have developed S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyper activated STAT3 and suppressing malignant transformation in human cancer cells that depend on STAT3. PMID:23322008

The Rate of Vitamin A Dimerization in Lipofuscinogenesis, Fundus Autofluorescence, Retinal Senescence and Degeneration.

PubMed

Washington, Ilyas; Saad, Leonide

2016-01-01

One of the earliest events preceding several forms of retinal degeneration is the formation and accumulation of vitamin A dimers in the retinal pigment epithelium (RPE) and underlying Bruch's membrane (BM). Such degenerations include Stargardt disease, Best disease, forms of retinitis pigmentosa, and age-related macular degeneration (AMD). Since their discovery in the 1990's, dimers of vitamin A, have been postulated as chemical triggers driving retinal senescence and degeneration. There is evidence to suggest that the rate at which vitamin A dimerizes and the eye's response to the dimerization products may dictate the retina's lifespan. Here, we present outstanding questions, finding the answers to which may help to elucidate the role of vitamin A dimerization in retinal degeneration.

Overall conformation of covalently stabilized domain-swapped dimer of human cystatin C in solution

NASA Astrophysics Data System (ADS)

Murawska, Magdalena; Szymańska, Aneta; Grubb, Anders; Kozak, Maciej

2017-11-01

Human cystatin C (HCC), a small protein, plays a crucial role in inhibition of cysteine proteases. The most common structural form of human cystatin C in crystals is a dimer, which has been evidenced both for the native protein and its mutants. In these structures, HCC dimers were formed through the mechanism of domain swapping. The structure of the monomeric form of human cystatin C was determined for V57N mutant and the mutant with the engineered disulfide bond (L47C)-(G69C) (known as stab1-HCC). On the basis of stab1-HCC, a number of covalently stabilized oligomers, including also dimers have been obtained. The aim of this study was to analyze the structure of the covalently stabilized dimer HCC in solution by the small angle X-ray scattering (SAXS) technique and synchrotron radiation. Experimental data confirmed that in solution this protein forms a dimer, which is characterized by the radius of gyration RG = 3.1 nm and maximum intramolecular distance Dmax = 10.3 nm. Using the ab initio method and program DAMMIN, we propose a low resolution structure of stabilized covalently cystatin C in solution. Stab-HCC dimer adopts in solution an elongated conformation, which is well reconstructed by the ab initio model.

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

DOE PAGES

Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; ...

2015-05-15

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

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

Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane

PubMed Central

Hellriegel, Christian; Caiolfa, Valeria R.; Corti, Valeria; Sidenius, Nicolai; Zamai, Moreno

2011-01-01

We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.—Hellriegel, C., Caiolfa, V. R., Corti, V., Sidenius, N., Zamai, M. Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. PMID:21602447

Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

PubMed Central

Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

2015-01-01

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

NASA Astrophysics Data System (ADS)

Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

2016-02-01

The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

PubMed

Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

1990-12-05

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

Modulation of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

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

Shi, X.; Shao, C; Zhang, X

2009-01-01

Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more ofmore » these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.« less

Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of β-arrestin.

PubMed

Smith, Thomas H; Li, Julia G; Dores, Michael R; Trejo, JoAnn

2017-08-18

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate β-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls β-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for β-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes β-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibitory effects of p-cresol and p-hydroxy anisole dimers on expression of the cyclooxygenase-2 gene and lipopolysaccharide-stimulated activation of nuclear factor-κB in RAW264.7 cells.

PubMed

Murakami, Yukio; Kawata, Akifumi; Ito, Shigeru; Katayama, Tadashi; Fujisawa, Seiichiro

2014-01-01

Phenolic compounds, particularly dihydroxybiphenyl-related compounds, possess efficient anti-oxidative and anti-inflammatory activity. We investigated the anti-inflammatory activity of 2,2'-dihydroxy-5,5'-dimethylbiphenol (p-cresol dimer), 2,2'-dihydroxy-5,5'-dimethoxybiphenol (pHA dimer), p-cresol, p-hydroxyanisole (pHA) and 2-t-butyl-4-hydroxyanisole (BHA). The cytotoxicity of the investigated compounds against RAW264.7 cells was determined using a cell counting kit (CCK-8). Their inhibitory effects on cyclooxygenase-2 (Cox2) mRNA expression stimulated by lipopolysaccharide (LPS) were determined using northern blot analysis, and their inhibition of LPS-stimulated nuclear factor-kappa B (Nf-κb) activation was evaluated using enzyme-linked immunosorbent assay-like microwell colorimetric transcription factor activity assay. The molecular orbital energy was calculated on the basis of density function theory BLYP/6-31G*. The cytotoxicity of the compounds declined in the order pHA dimer > p-cresol dimer > BHA > p-cresol > pHA. The inhibitory effect on Cox2 expression and Nf-κb activation was enhanced by p-cresol dimer and pHA dimer, particularly the former, suggesting potent anti-inflammatory activity, whereas p-cresol and pHA showed weak activity, and BHA no activity. Both p-cresol dimer and pHA dimer were highly electronegative, as determined by quantum chemical calculations. Dimerization of p-cresol and pHA enhances their anti-inflammatory activity. p-Cresol dimer and pHA dimer, particularly the former, are potent anti-inflammatory agents. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Tomato y-glutamilhydrolases: expression, characterization, and evidence for heterodimer formation

USDA-ARS?s Scientific Manuscript database

It is known that GGH is active as a dimmer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in-vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These i...

Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has Improved In Vitro Serum Stability

PubMed Central

Nair, Vinojini B.; Bathgate, Ross A. D.; Separovic, Frances; Samuel, Chrishan S.; Hossain, Mohammed Akhter; Wade, John D.

2015-01-01

Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin. PMID:25685807

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases.

PubMed

Naldi, Marina; Baldassarre, Maurizio; Nati, Marina; Laggetta, Maristella; Giannone, Ferdinando Antonino; Domenicali, Marco; Bernardi, Mauro; Caraceni, Paolo; Bertucci, Carlo

2015-08-10

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

PubMed

Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

2016-11-01

Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Evolution of magnetization due to asymmetric dimerization: theoretical considerations and application to aberrant oligomers formed by apoSOD1(2SH).

PubMed

Sekhar, Ashok; Bain, Alex D; Rumfeldt, Jessica A O; Meiering, Elizabeth M; Kay, Lewis E

2016-02-17

A set of coupled differential equations is presented describing the evolution of magnetization due to an exchange reaction whereby a pair of identical monomers form an asymmetric dimer. In their most general form the equations describe a three-site exchange process that reduces to two-site exchange under certain limiting conditions that are discussed. An application to the study of sparsely populated, transiently formed sets of aberrant dimers, symmetric and asymmetric, of superoxide dismutase is presented. Fits of concentration dependent CPMG relaxation dispersion profiles provide measures of the dimer dissociation constants and both on- and off-rates. Dissociation constants on the order of 70 mM are extracted from fits of the data, with dimeric populations of ∼2% and lifetimes of ∼6 and ∼2 ms for the symmetric and asymmetric complexes, respectively. This work emphasizes the important role that NMR relaxation experiments can play in characterizing very weak molecular complexes that remain invisible to most biophysical approaches.

Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

PubMed Central

2010-01-01

Background Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. Results In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between asymmetric and symmetric dimers in response to changes in either biochemical modifications (e.g. proteolytic cleavage) or other biological cues. Such transitions may contribute to the broad range of protein-protein interactions and functions attributed to class II chaperones. PMID:20633281

Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion.

PubMed

Madsen, Jens Kvist; Kaspersen, Jørn Døvling; Andersen, Camilla Bertel; Nedergaard Pedersen, Jannik; Andersen, Kell Kleiner; Pedersen, Jan Skov; Otzen, Daniel E

2017-08-15

We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlL's thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.

Inhibitory activity of synthesized acetylated Procyanidin B1 analogs against HeLa S3 cells proliferation.

PubMed

Okamoto, Syuhei; Ishihara, Sayaka; Okamoto, Taisuke; Doi, Syoma; Harui, Kota; Higashino, Yusuke; Kawasaki, Takashi; Nakajima, Noriyuki; Saito, Akiko

2014-02-04

Proanthocyanidins, also known as condensed tannins and/or oligomeric flavonoids, occur in many edible plants and have various interesting biological activities. Previously, we reported a synthetic method for the preparation of various procyanidins in pure form and described their biological activities. Here, we describe the synthesis of procyanidin B1 acetylated analogs and discuss their inhibition activities against HeLa S3 cell proliferation. Surprisingly, the lower-unit acetylated procyanidin B1 strongly inhibited the proliferation of HeLa S3 cells. This molecule showed much stronger inhibitory activity than did epigallocatechin-3-O-gallate (EGCG), green tea polyphenol, and dimeric compounds that included EGCG as a unit. This result suggests that the phenolic hydroxyl groups of the upper-units in flavan-3-ols are important for their inhibitory activity against cancer cell proliferation and that a hydrophobic lower unit dimer enhances this activity.

Dimerization of the EphA1 Receptor Tyrosine Kinase Transmembrane Domain: Insights into the Mechanism of Receptor Activation

PubMed Central

2014-01-01

EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape. PMID:25286141

Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

PubMed

Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

2003-08-29

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C

DOE PAGES

Gruschus, James M.; Jiang, Zhiping; Yap, Thai Leong; ...

2015-01-16

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (K d = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63–66 and 74–76, which is distinct from the region known tomore » enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. In conclusion, for the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.« less

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-02-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed Central

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation. Images PMID:2153242

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

NASA Astrophysics Data System (ADS)

Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

2015-09-01

The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

Engineered stabilization and structural analysis of the autoinhibited conformation of PDE4

DOE PAGES

Cedervall, Peder; Aulabaugh, Ann; Geoghegan, Kieran F.; ...

2015-03-09

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the existence of over 25 variants of PDE4, broadly classified as long, short, and supershort forms. We report the X-ray crystal structure of long form PDE4B containing UCR1, UCR2, and the catalytic domain, crystallized as a dimer in which a disulfide bond cross-linksmore » cysteines engineered into UCR2 and the catalytic domain. Biochemical and mass spectrometric analyses showed that the UCR2-catalytic domain interaction occurs in trans, and established that this interaction regulates the catalytic activity of PDE4. By elucidating the key structural determinants of dimerization, we show that only long forms of PDE4 can be regulated by this mechanism. The results also provide a structural basis for the long-standing observation of high- and low-affinity binding sites for the prototypic inhibitor rolipram.« less

Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state.

PubMed

Ou, Wen-bin; Wang, Ri-Sheng; Lu, Jie; Zhou, Hai-Meng

2003-11-03

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.

Exploring Molecular Mechanisms of Paradoxical Activation in the BRAF Kinase Dimers: Atomistic Simulations of Conformational Dynamics and Modeling of Allosteric Communication Networks and Signaling Pathways

PubMed Central

Tse, Amanda; Verkhivker, Gennady M.

2016-01-01

The recent studies have revealed that most BRAF inhibitors can paradoxically induce kinase activation by promoting dimerization and enzyme transactivation. Despite rapidly growing number of structural and functional studies about the BRAF dimer complexes, the molecular basis of paradoxical activation phenomenon is poorly understood and remains largely hypothetical. In this work, we have explored the relationships between inhibitor binding, protein dynamics and allosteric signaling in the BRAF dimers using a network-centric approach. Using this theoretical framework, we have combined molecular dynamics simulations with coevolutionary analysis and modeling of the residue interaction networks to determine molecular determinants of paradoxical activation. We have investigated functional effects produced by paradox inducer inhibitors PLX4720, Dabrafenib, Vemurafenib and a paradox breaker inhibitor PLX7904. Functional dynamics and binding free energy analyses of the BRAF dimer complexes have suggested that negative cooperativity effect and dimer-promoting potential of the inhibitors could be important drivers of paradoxical activation. We have introduced a protein structure network model in which coevolutionary residue dependencies and dynamic maps of residue correlations are integrated in the construction and analysis of the residue interaction networks. The results have shown that coevolutionary residues in the BRAF structures could assemble into independent structural modules and form a global interaction network that may promote dimerization. We have also found that BRAF inhibitors could modulate centrality and communication propensities of global mediating centers in the residue interaction networks. By simulating allosteric communication pathways in the BRAF structures, we have determined that paradox inducer and breaker inhibitors may activate specific signaling routes that correlate with the extent of paradoxical activation. While paradox inducer inhibitors may facilitate a rapid and efficient communication via an optimal single pathway, the paradox breaker may induce a broader ensemble of suboptimal and less efficient communication routes. The central finding of our study is that paradox breaker PLX7904 could mimic structural, dynamic and network features of the inactive BRAF-WT monomer that may be required for evading paradoxical activation. The results of this study rationalize the existing structure-functional experiments by offering a network-centric rationale of the paradoxical activation phenomenon. We argue that BRAF inhibitors that amplify dynamic features of the inactive BRAF-WT monomer and intervene with the allosteric interaction networks may serve as effective paradox breakers in cellular environment. PMID:27861609

A novel inhibitor of STAT3 homodimerization selectively suppresses STAT3 activity and malignant transformation.

PubMed

Zhang, Xiaolei; Sun, Ying; Pireddu, Roberta; Yang, Hua; Urlam, Murali K; Lawrence, Harshani R; Guida, Wayne C; Lawrence, Nicholas J; Sebti, Saïd M

2013-03-15

STAT3-STAT3 dimerization, which involves reciprocal binding of the STAT3-SH2 domain to phosphorylated tyrosine-705 (Y-705), is required for STAT3 nuclear translocation, DNA binding, and transcriptional regulation of downstream target genes. Here, we describe a small molecule S3I-1757 capable of disrupting STAT3-STAT3 dimerization, activation, and malignant transforming activity. Fluorescence polarization assay and molecular modeling suggest that S3I-1757 interacts with the phospho-Y-705-binding site in the SH2 domain and displaces fluorescein-labeled GpYLPQTV phosphotyrosine peptide from binding to STAT3. We generated hemagglutinin (HA)-tagged STAT3 and FLAG-tagged STAT3 and showed using coimmunoprecipitation and colocalization studies that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor (EGFR) binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analog, S3I-1756, which does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over AKT1 and ERK1/2 (MAPK3/1), nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding, and transcriptional activation and suppresses the expression levels of STAT3 target genes, such as Bcl-xL (BCL2L1), survivin (BIRC5), cyclin D1 (CCND1), and matrix metalloproteinase (MMP)-9. Furthermore, S3I-1757, but not S3I-1756, inhibits anchorage-dependent and -independent growth, migration, and invasion of human cancer cells, which depend on STAT3. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues cells from the effects of S3I-1757 inhibition. Thus, we have developed S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyperactivated STAT3 and suppressing malignant transformation in human cancer cells that depend on STAT3.

H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms

PubMed Central

1992-01-01

We analyzed the production of Torpedo marmorata acetylcholinesterase (AChE) in transfected COS cells. We report that the presence of an aspartic acid at position 397, homologous to that observed in other cholinesterases and related enzymes (Krejci, E., N. Duval, A. Chatonnet, P. Vincens, and J. Massoulie. 1991. Proc. Natl. Acad. Sci. USA. 88:6647-6651), is necessary for catalytic activity. The presence of an asparagine in the previously reported cDNA sequence (Sikorav, J.L., E. Krejci, and J. Massoulie. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1865-1873) was most likely due to a cloning error (codon AAC instead of GAC). We expressed the T and H subunits of Torpedo AChE, which differ in their COOH-terminal region and correspond respectively to the collagen-tailed asymmetric forms and to glycophosphatidylinositol-anchored dimers of Torpedo electric organs, as well as a truncated T subunit (T delta), lacking most of the COOH- terminal peptide. The transfected cells synthesized similar amounts of AChE immunoreactive protein at 37 degrees and 27 degrees C. However AChE activity was only produced at 27 degrees C and, even at this temperature, only a small proportion of the protein was active. We analyzed the molecular forms of active AChE produced at 27 degrees C. The H polypeptides generated glycophosphatidylinositol-anchored dimers, resembling the corresponding natural AChE form. The cells also released non-amphiphilic dimers G2na. The T polypeptides generated a series of active forms which are not produced in Torpedo electric organs: G1a, G2a, G4a, and G4na cellular forms and G2a and G4na secreted forms. The amphiphilic forms appeared to correspond to type II forms (Bon, S., J. P. Toutant, K. Meflah, and J. Massoulie. 1988. J. Neurochem. 51:776- 785; Bon, S., J. P. Toutant, K. Meflah, and J. Massoulie. 1988. J. Neurochem. 51:786-794), which are abundant in the nervous tissue and muscles of higher vertebrates (Bon, S., T. L. Rosenberry, and J. Massoulie. 1991. Cell. Mol. Neurobiol. 11:157-172). The H and T catalytic subunits are thus sufficient to account for all types of known AChE forms. The truncated T delta subunit yielded only non- amphiphilic monomers, demonstrating the importance of the T COOH- terminal peptide in the formation of oligomers, and in the hydrophobic character of type II forms. PMID:1639848

Higher-order architecture of rhodopsin in intact photoreceptors and its implication for phototransduction kinetics.

PubMed

Gunkel, Monika; Schöneberg, Johannes; Alkhaldi, Weaam; Irsen, Stephan; Noé, Frank; Kaupp, U Benjamin; Al-Amoudi, Ashraf

2015-04-07

The visual pigment rhodopsin belongs to the family of G protein-coupled receptors that can form higher oligomers. It is controversial whether rhodopsin forms oligomers and whether oligomers are functionally relevant. Here, we study rhodopsin organization in cryosections of dark-adapted mouse rod photoreceptors by cryoelectron tomography. We identify four hierarchical levels of organization. Rhodopsin forms dimers; at least ten dimers form a row. Rows form pairs (tracks) that are aligned parallel to the disk incisures. Particle-based simulation shows that the combination of tracks with fast precomplex formation, i.e. rapid association and dissociation between inactive rhodopsin and the G protein transducin, leads to kinetic trapping: rhodopsin first activates transducin from its own track, whereas recruitment of transducin from other tracks proceeds more slowly. The trap mechanism could produce uniform single-photon responses independent of rhodopsin lifetime. In general, tracks might provide a platform that coordinates the spatiotemporal interaction of signaling molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions

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

Kumar, Shiva; Krishnamoorthy, Kalyanaraman; Mudeppa, Devaraja G.

P. falciparum orotate phosphoribosyltransferase, a potential target for antimalarial drugs and a conduit for prodrugs, crystallized as a structure with eight molecules per asymmetric unit that included some unique parasite-specific auto-inhibitory interactions between catalytic dimers. The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained aftermore » some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in the PfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein–protein interactions that occlude the catalytic active sites. The latter could potentially modulate PfOPRTase activity in parasites and possibly provide new insights for blocking PfOPRTase functions.« less

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

PubMed

Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

2017-07-19

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity

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

Moulaei, Tinoush; Shenoy, Shilpa R.; Giomarelli, Barbara

2010-10-28

Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviralmore » activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.« less

The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase.

PubMed

Gilmour, R; Goodhew, C F; Pettigrew, G W; Prazeres, S; Moura, J J; Moura, I

1994-06-15

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.

Model-based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3.

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

Shankaran, Harish; Zhang, Yi; Tan, Yunbing

2013-08-22

The HER/ErbB family of receptor tyrosine kinases drive critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panelmore » of human mammary epithelial cells expressing varying levels of EGFR, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of epithelial cells lines with known HER expression levels. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1/1 and HER1/2 dimers, and not HER1/3 dimers, ii) HER1/2 and HER2/3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2/3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.« less

Purification of Cu/Zn superoxide dismutase from Piper betle leaf and its characterization in the oral cavity.

PubMed

Liu, Yu-Ching; Lee, Miau-Rong; Chen, Chao-Jung; Lin, Yung-Chang; Ho, Heng-Chien

2015-03-04

The aim of this study was to purify protein(s) from Piper betle leaf for identification and further characterization. A functionally unknown protein was purified to apparent homogeneity with a molecular mass of 15.7 kDa and identified as Cu/Zn superoxide dismutase (SOD). The purified SOD appeared to be monomeric and converted to its dimeric form with increased enzymatic activity in betel nut oral extract. This irreversible conversion was mainly induced by slaked lime, resulting from the increase in pH of the oral cavity. Oral extract from chewing areca nut alone also induced SOD dimerization due to the presence of arginine. The enhanced activity of the SOD dimer was responsible for the continuous production of hydrogen peroxide in the oral cavity. Thus, SOD may contribute to oral carcinogenesis through the continuous formation of hydrogen peroxide in the oral cavity, in spite of its protective role against cancer in vivo.

The Aggregation Paths and Products of Aβ42 Dimers Are Distinct from Those of the Aβ42 Monomer.

PubMed

O'Malley, Tiernan T; Witbold, William M; Linse, Sara; Walsh, Dominic M

2016-11-08

Extracts of Alzheimer's disease (AD) brain that contain what appear to be sodium dodecyl sulfate-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated the Aβ monomer, consist primarily of Aβ42, and resist denaturation by chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in two ways: by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then used a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by the Aβ monomer. The results suggest that the cross-links disfavor fibril formation from Aβ dimers, thereby enhancing the concentration of soluble aggregates akin to those in aqueous extracts of AD brain. Thus, it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates.

The aggregation paths and products of Aβ42 dimers are distinct from Aβ42 monomer

PubMed Central

O'Malley, Tiernan T.; Witbold, William M.; Linse, Sara; Walsh, Dominic M.

2017-01-01

Extracts of Alzheimer's disease (AD) brain that contain what appear to be SDS-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated from Aβ monomer, consist primarily of Aβ42 and resist denaturation by powerful chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in only one of two different ways - either by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then applied a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by Aβ monomer. These results suggest that Aβ dimers allow the formation of soluble aggregates akin to those in aqueous extracts of AD brain. Thus it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates. PMID:27750419

ACTIVE ERK1 IS DIMERISED IN VIVO: BISPHOSPHODIMERS GENERATE PEAK KINASE ACTIVITY AND MONOPHOSPHODIMERS MAINTAIN BASAL ERK1 ACTIVITY

PubMed Central

Philipova, Rada; Whitaker, Michael

2012-01-01

SUMMARY ERK1 and ERK2 are widely involved in cell signalling. Using a recombinant approach, it has been shown that exogenous ERK2 is capable of dimerisation and that preventing dimerisation reduces its nuclear accumulation on stimulation. Dimerisation occurs on phosphorylation; the dimer partner of phosphorylated ERK2 may be either phosphorylated or unphosphorylated. It has been assumed that monophosphodimers are hemiactive. Here we show that ERK1 is capable of dimerisation both in vivo and in vitro. Dimerisation of human recombinant ERK1 in vitro requires both ERK1 phosphorylation and cellular cofactor(s); it leads to the formation of a high molecular weight complex that can be dissociated by treatment with β-mercaptoethanol. We demonstrate for the first time in both sea urchin embryos and human cells that native ERK forms dimers and that high ERK kinase activity is largely associated with bisphosphodimers, not with monophosphodimers or phosphorylated monomers: the activity of the bisphosphodimer is about 20-fold higher than that of the phosphorylated monomer in vitro and the bisphosphodimer shows 5 to 7-fold higher in vivo activity than the basal activity attributable to the monophosphodimer. Thus phosphorylation of both partners in the dimer is a hallmark of ERK activation. Judgments made about ERK kinase activity associated with phosphorylated monomers are at best a proxy for ERK activity. PMID:16317051

Disrupting a key hydrophobic pair in the oligomerization interface of the actinoporins impairs their pore‐forming activity

PubMed Central

Mesa‐Galloso, Haydeé; Delgado‐Magnero, Karelia H.; Cabezas, Sheila; López‐Castilla, Aracelys; Hernández‐González, Jorge E.; Pedrera, Lohans; Alvarez, Carlos; Peter Tieleman, D.; García‐Sáez, Ana J.; Lanio, Maria E.; Valiente, Pedro A.

2017-01-01

Abstract Crystallographic data of the dimeric and octameric forms of fragaceatoxin C (FraC) suggested the key role of a small hydrophobic protein–protein interaction surface for actinoporins oligomerization and pore formation in membranes. However, site‐directed mutagenesis studies supporting this hypothesis for others actinoporins are still lacking. Here, we demonstrate that disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII), and sticholysin II (StII) impairs the pore formation activity of these proteins. Our results allow for the extension of the importance of FraC protein–protein interactions in the stabilization of the oligomeric intermediates of StII and EqtII pointing out that all of these proteins follow a similar pathway of membrane disruption. These findings support the hybrid pore proposal as the universal model of actinoporins pore formation. Moreover, we reinforce the relevance of dimer formation, which appears to be a functional intermediate in the assembly pathway of some different pore‐forming proteins. PMID:28000294

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

PubMed

Fossé, P; Motté, N; Roumier, A; Gabus, C; Muriaux, D; Darlix, J L; Paoletti, J

1996-12-24

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Primary and secondary dimer interfaces of the fibroblast growth factor receptor 3 transmembrane domain: characterization via multiscale molecular dynamics simulations.

PubMed

Reddy, Tyler; Manrique, Santiago; Buyan, Amanda; Hall, Benjamin A; Chetwynd, Alan; Sansom, Mark S P

2014-01-21

Receptor tyrosine kinases are single-pass membrane proteins that form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. Fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of the cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface that is more highly populated in heterodimer and mutant configurations that may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer to allow interactions of the arginine side chain with lipid headgroup phosphates.

Primary and Secondary Dimer Interfaces of the FGFR3 Transmembrane Domain: Characterization via Multiscale Molecular Dynamics Simulations

PubMed Central

Reddy, Tyler; Manrique, Santiago; Buyan, Amanda; Hall, Benjamin A.; Chetwynd, Alan; Sansom, Mark S.P.

2016-01-01

Receptor tyrosine kinases are single pass membrane proteins which form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. The fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position relative of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface which is more highly populated in heterodimer and mutant configurations which may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer so as to enable interactions of the arginine sidechain with lipid head group phosphates. PMID:24397339

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

PubMed

Lorenzón, Esteban N; Santos-Filho, Norival A; Ramos, Matheus A S; Bauab, Tais M; Camargo, Ilana L B C; Cilli, Eduardo M

2016-01-01

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

Dimer esters in α-pinene secondary organic aerosol: effect of hydroxyl radical, ozone, relative humidity and aerosol acidity

NASA Astrophysics Data System (ADS)

Kristensen, K.; Cui, T.; Zhang, H.; Gold, A.; Glasius, M.; Surratt, J. D.

2013-12-01

The formation of secondary organic aerosol (SOA) from both ozonolysis and hydroxyl radical (OH)-initiated oxidation of α-pinene under conditions of high nitric oxide (NO) concentrations with varying relative humidity (RH) and aerosol acidity was investigated in the University of North Carolina dual outdoor smog chamber facility. SOA formation from ozonolysis of α-pinene was enhanced relative to that from OH-initiated oxidation in the presence of initially high NO conditions. However, no effect of RH on SOA mass was evident. Ozone (O3)-initiated oxidation of α-pinene in the presence of ammonium sulfate (AS) seed coated with organic aerosol from OH-initiated oxidation of α-pinene showed reduced nucleation compared to ozonolysis in the presence of pure AS seed aerosol. The chemical composition of α-pinene SOA was investigated by ultra-performance liquid chromatography/electrospray ionization high-resolution quadrupole time-of-flight mass spectrometry (UPLC/ESI-HR-Q-TOFMS), with a focus on the formation of carboxylic acids and high-molecular weight dimer esters. A total of eight carboxylic acids and four dimer esters were identified, constituting between 8 and 12% of the total α-pinene SOA mass. OH-initiated oxidation of α-pinene in the presence of nitrogen oxides (NOx) resulted in the formation of highly oxidized carboxylic acids, such as 3-methyl-1,2,3-butanetricarboxylic acid (MBTCA) and diaterpenylic acid acetate (DTAA). The formation of dimer esters was observed only in SOA produced from the ozonolysis of α-pinene in the absence of NOx, with increased concentrations by a~factor of two at higher RH (50-90%) relative to lower RH (30-50%). The increased formation of dimer esters correlates with an observed increase in new particle formation at higher RH due to nucleation. Increased aerosol acidity was found to have a negligible effect on the formation of the dimer esters. SOA mass yield did not influence the chemical composition of SOA formed from α-pinene ozonolysis with respect to carboxylic acids and dimer esters. The results support the formation of the high-molecular weight dimer esters through gas-phase reactions of the stabilized Criegee Intermediate (sCI) formed from the ozonolysis of α-pinene. The high molecular weight and polar nature of dimer esters formed in the gas-phase may explain increased particle number concentration as a~result of homogenous nucleation. Since three of these dimer esters (i.e., pinyl-diaterpenyl ester (MW 358), pinyl-diaterebyl ester (MW 344) and pinonyl-pinyl ester (MW 368)) have been observed in both laboratory-generated and ambient fine organic aerosol samples, we conclude that the dimer esters observed in this study can be used as tracers for the O3-initiated oxidation of α-pinene, and are therefore indicative of enhanced anthropogenic activities, and that the high molecular weight and low volatility esters result in homogenous nucleation under laboratory conditions, increasing the particle number concentration.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

PubMed

Feilen, Lukas P; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

Molecular dynamics simulations and structure-based network analysis reveal structural and functional aspects of G-protein coupled receptor dimer interactions.

PubMed

Baltoumas, Fotis A; Theodoropoulou, Margarita C; Hamodrakas, Stavros J

2016-06-01

A significant amount of experimental evidence suggests that G-protein coupled receptors (GPCRs) do not act exclusively as monomers but also form biologically relevant dimers and oligomers. However, the structural determinants, stoichiometry and functional importance of GPCR oligomerization remain topics of intense speculation. In this study we attempted to evaluate the nature and dynamics of GPCR oligomeric interactions. A representative set of GPCR homodimers were studied through Coarse-Grained Molecular Dynamics simulations, combined with interface analysis and concepts from network theory for the construction and analysis of dynamic structural networks. Our results highlight important structural determinants that seem to govern receptor dimer interactions. A conserved dynamic behavior was observed among different GPCRs, including receptors belonging in different GPCR classes. Specific GPCR regions were highlighted as the core of the interfaces. Finally, correlations of motion were observed between parts of the dimer interface and GPCR segments participating in ligand binding and receptor activation, suggesting the existence of mechanisms through which dimer formation may affect GPCR function. The results of this study can be used to drive experiments aimed at exploring GPCR oligomerization, as well as in the study of transmembrane protein-protein interactions in general.

Molecular dynamics simulations and structure-based network analysis reveal structural and functional aspects of G-protein coupled receptor dimer interactions

NASA Astrophysics Data System (ADS)

Baltoumas, Fotis A.; Theodoropoulou, Margarita C.; Hamodrakas, Stavros J.

2016-06-01

A significant amount of experimental evidence suggests that G-protein coupled receptors (GPCRs) do not act exclusively as monomers but also form biologically relevant dimers and oligomers. However, the structural determinants, stoichiometry and functional importance of GPCR oligomerization remain topics of intense speculation. In this study we attempted to evaluate the nature and dynamics of GPCR oligomeric interactions. A representative set of GPCR homodimers were studied through Coarse-Grained Molecular Dynamics simulations, combined with interface analysis and concepts from network theory for the construction and analysis of dynamic structural networks. Our results highlight important structural determinants that seem to govern receptor dimer interactions. A conserved dynamic behavior was observed among different GPCRs, including receptors belonging in different GPCR classes. Specific GPCR regions were highlighted as the core of the interfaces. Finally, correlations of motion were observed between parts of the dimer interface and GPCR segments participating in ligand binding and receptor activation, suggesting the existence of mechanisms through which dimer formation may affect GPCR function. The results of this study can be used to drive experiments aimed at exploring GPCR oligomerization, as well as in the study of transmembrane protein-protein interactions in general.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction

PubMed Central

Feilen, Lukas P.; Haubrich, Kevin; Strecker, Paul; Probst, Sabine; Eggert, Simone; Stier, Gunter; Sinning, Irmgard; Konietzko, Uwe; Kins, Stefan; Simon, Bernd; Wild, Klemens

2017-01-01

Physiological function and pathology of the Alzheimer’s disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling. PMID:28553201

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

PubMed

Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C

2018-05-07

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P 3 or PI(3,4)P 2 Our results indicate that dimerization plays a significant role in Ci-VSP function. © 2018 Rayaprolu et al.

Changes in Quaternary Structure in the Signaling Mechanisms of PAS Domains

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

Ayers, Rebecca A.; Moffat, Keith

2008-12-15

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group (P1) and at resolutions (1.5--1.8 {angstrom}) higher than the resolutions of those previously obtained. Interestingly, bjFixLHmore » can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated {approx}175 deg. relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to 2 deg. relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.« less

Man o' War Mutation in UDP-α-D-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

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

Walsh, Jr., Richard M.; Polizzi, Samuel J.; Kadirvelraj, Renuka

The man o’ war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-D-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ~16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies showmore » that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-D-4-keto-xylose, is not reduced to the final product, UDP-α-D-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.« less

Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure

PubMed Central

Nakabayashi, Makoto; Kataoka, Misumi; Mishima, Yumiko; Maeno, Yuka; Ishikawa, Kazuhiko

2014-01-01

β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed. PMID:24598756

Engineering a lifetime-based activatable probe for photoacoustic imaging

NASA Astrophysics Data System (ADS)

Morgounova, Ekaterina; Shao, Qi; Hackel, Benjamin; Ashkenazi, Shai

2013-02-01

High-resolution, high-penetration depth activatable probes are needed for in-vivo imaging of enzyme activity. In this paper, we will describe the contrast mechanism of a new photoacoustic activatable probe that changes its excitation lifetime upon activation. The excitation decay of methylene blue (MB), a chromophore commonly used in therapeutic and diagnostic applications, is probed by photoacoustic lifetime contrast imaging (PLCI). The monomer of the dye presents a high-quantum yield of intersystem-crossing and long lifetime (70 μs) whereas the dimer is statically quenched with a short lifetime (a few ns). This forms the basis of a highly sensitive contrast mechanism between monomers and dimers. Two dimerization models - one using sodium sulfate, the other using sodium dodecyl sulfate - were applied to control the monomer-to-dimer ratio in MB solutions. Preliminary results show that the photoacoustic signal of a dimer solution is efficiently suppressed (< 20 dB) due to their short lifetime compared to the monomer sample. Flash-photolysis of the same solutions reveals a 99% decrease in transient absorption confirming PLCI results. This contrast mechanism can be applied to design a MB dual-labeled activatable probe bound by an enzyme-specific cleavable peptide linker. When the probe is cleaved by its target, MB molecules will separate by molecular diffusion and recover their long excitation lifetime enabling their detection by PLCI. Our long-term goal is to investigate enzyme-specific imaging in small animals and establish pre-clinical data for translational research and implementation of the technology in clinical applications.

Tamoxifen metabolism in rat liver microsomes: identification of a dimeric metabolite derived from free radical intermediates by liquid chromatography/mass spectrometry.

PubMed

Jones, R M; Yuan, Z X; Lim, C K

1999-01-01

Tamoxifen has been shown to be a potent liver carcinogen in rats, and generates covalent DNA adducts. On-line high performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been used to further study the metabolites of tamoxifen formed by rat liver microsomes in the presence of NADPH with a view to identifying potential reactive metabolites which may be responsible for the formation of DNA adducts, and liver carcinogenesis. A metabolite has been detected with a protonated molecule at m/z 773. The mass of this compound is consistent with a dimer of hydroxylated tamoxifen (m/z 388). Analysis of 4-hydroxytamoxifen incubated with a rat liver microsomal preparation showed the formation of a similar metabolite with an apparent MH+ ion at m/z 773, believed to be a dimer of 4-hydroxytamoxifen formed by a free radical reaction. The retention time for this metabolite from 4-hydroxytamoxifen is identical to that of the tamoxifen metabolite, suggesting that these two compounds are the same. The levels of the dimer were higher when 4-hydroxytamoxifen was used as substrate and, in addition, two isomers were detected. It is proposed that tamoxifen was first converted to arene oxides which react with DNA or to 4-hydroxytamoxifen, either directly or via 3,4-epoxytamoxifen, which then undergoes activation via a free radical reaction to give reactive intermediates which can then react with DNA and protein, or with themselves, to give the dimers (m/z 773).

Structural modeling of the AhR:ARNT complex in the bHLH-PASA-PASB region elucidates the key determinants of dimerization.

PubMed

Corrada, Dario; Denison, Michael S; Bonati, Laura

2017-05-02

Elucidation of the dimerization process of the aryl hydrocarbon receptor (AhR) with the AhR nuclear translocator (ARNT) is crucial for understanding the mechanisms underlying the functional activity of AhR, including mediation of the toxicity of environmental contaminants. In this work, for the first time a structural model of the AhR:ARNT dimer encompassing the entire bHLH-PASA-PASB domain region is proposed. It is developed by using a template-based modeling approach, relying on the recently available crystallographic structures of two dimers of homologous systems in the bHLH-PAS family of proteins: the CLOCK:BMAL1 and the HIF2α:ARNT heterodimers. The structural and energetic characteristics of the modeled AhR:ARNT protein-protein interface are determined by evaluating the variations in solvent accessible surface area, the total binding free energy and the per-residue free energy contributions obtained by the MM-GBSA method and the Energy Decomposition Analysis. The analyses of the intricate network of inter-domain interactions at the dimerization interfaces provide insights into the key determinants of dimerization. These are confirmed by comparison of the computational findings with the available experimental mutagenesis and functional analysis data. The results presented here on the AhR:ARNT dimer structure and interactions provide a framework to start analyzing the mechanism of AhR transformation into its functional DNA binding form.

Kinetics of the monomer-dimer reaction of yeast hexokinase PI.

PubMed

Hoggett, J G; Kellett, G L

1992-10-15

Kinetic studies of the glucose-dependent monomer-dimer reaction of yeast hexokinase PI at pH 8.0 in the presence of 0.1 M-KCl have been carried out using the fluorescence temperature-jump technique. A slow-relaxation effect was observed which was attributed from its dependence on enzyme concentration to the monomer-dimer reaction; the reciprocal relaxation times tau-1 varied from 3 s-1 at low concentrations of glucose to 42 s-1 at saturating concentrations. Rate constants for association (kass.) and dissociation (kdiss.) were determined as a function of glucose concentration using values of the equilibrium association constant of the monomer-dimer reaction derived from sedimentation ultracentrifugation studies under similar conditions, and also from the dependence of tau-2 on enzyme concentration. kass. was almost independent of glucose concentration and its value (2 x 10(5) M-1.s-1) was close to that expected for a diffusion-controlled process. The influence of glucose on the monomer-dimer reaction is entirely due to effects on kdiss., which increases from 0.21 s-1 in the absence of glucose to 25 s-1 at saturating concentrations. The monomer and dimer forms of hexokinase have different affinities and Km values for glucose, and the results reported here imply that there may be a significant lag in the response of the monomer-dimer reaction to changes in glucose concentrations in vivo with consequent hysteretic effects on the hexokinase activity.

Kinetics of the monomer-dimer reaction of yeast hexokinase PI.

PubMed Central

Hoggett, J G; Kellett, G L

1992-01-01

Kinetic studies of the glucose-dependent monomer-dimer reaction of yeast hexokinase PI at pH 8.0 in the presence of 0.1 M-KCl have been carried out using the fluorescence temperature-jump technique. A slow-relaxation effect was observed which was attributed from its dependence on enzyme concentration to the monomer-dimer reaction; the reciprocal relaxation times tau-1 varied from 3 s-1 at low concentrations of glucose to 42 s-1 at saturating concentrations. Rate constants for association (kass.) and dissociation (kdiss.) were determined as a function of glucose concentration using values of the equilibrium association constant of the monomer-dimer reaction derived from sedimentation ultracentrifugation studies under similar conditions, and also from the dependence of tau-2 on enzyme concentration. kass. was almost independent of glucose concentration and its value (2 x 10(5) M-1.s-1) was close to that expected for a diffusion-controlled process. The influence of glucose on the monomer-dimer reaction is entirely due to effects on kdiss., which increases from 0.21 s-1 in the absence of glucose to 25 s-1 at saturating concentrations. The monomer and dimer forms of hexokinase have different affinities and Km values for glucose, and the results reported here imply that there may be a significant lag in the response of the monomer-dimer reaction to changes in glucose concentrations in vivo with consequent hysteretic effects on the hexokinase activity. Images Fig. 1. PMID:1445216

Immunoassays distinguishing between HNL/NGAL released in urine from kidney epithelial cells and neutrophils.

PubMed

Mårtensson, Johan; Xu, Shengyuan; Bell, Max; Martling, Claes-Roland; Venge, Per

2012-10-09

The distinction between monomeric human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), secreted by injured kidney tubular cells, and dimeric HNL/NGAL, released by activated neutrophils, is important to accurately diagnose acute kidney injury (AKI). 132 urine samples from 44 intensive care unit (ICU) patients and five urine samples from non-ICU patients with urinary tract infections (UTIs) were analyzed by two monoclonal enzyme-linked immunosorbent assays (ELISA-1 and ELISA-2). The presence of monomeric and/or dimeric HNL/NGAL in each sample was visualized by Western blotting. The ELISA-1 detected both monomeric and dimeric HNL/NGAL whereas the ELISA-2 almost exclusively detected dimeric HNL/NGAL with an area under the receiver-operating characteristics curve (AuROC) of 0.90. The ELISA-1/ELISA-2 ratio detected the monomeric form with an AuROC of 0.92. In 32 AKI patients, dimer-specific ELISA-2 levels decreased pre-AKI whereas the monomer-specific ELISA-1/ELISA-2 ratio gradually increased beyond AKI diagnosis. High ELISA-2 levels and/or low ELISA-1/ELISA-2 ratios detected a predominance of dimeric HNL/NGAL in urine from the patients with UTIs. In combination, our two ELISAs distinguish monomeric HNL/NGAL, produced by the kidney epithelium, from dimeric HNL/NGAL, released by neutrophils during AKI development, as well as reduce the confounding effect of neutrophil involvement when bacteriuria is present. Copyright © 2012 Elsevier B.V. All rights reserved.

Analytical expressions for the correlation function of a hard sphere dimer fluid

NASA Astrophysics Data System (ADS)

Kim, Soonho; Chang, Jaeeon; Kim, Hwayong

A closed form expression is given for the correlation function of a hard sphere dimer fluid. A set of integral equations is obtained from Wertheim's multidensity Ornstein-Zernike integral equation theory with Percus-Yevick approximation. Applying the Laplace transformation method to the integral equations and then solving the resulting equations algebraically, the Laplace transforms of the individual correlation functions are obtained. By the inverse Laplace transformation, the radial distribution function (RDF) is obtained in closed form out to 3D (D is the segment diameter). The analytical expression for the RDF of the hard dimer should be useful in developing the perturbation theory of dimer fluids.

Glycine transporter dimers: evidence for occurrence in the plasma membrane.

PubMed

Bartholomäus, Ingo; Milan-Lobo, Laura; Nicke, Annette; Dutertre, Sébastien; Hastrup, Hanne; Jha, Alok; Gether, Ulrik; Sitte, Harald H; Betz, Heinrich; Eulenburg, Volker

2008-04-18

Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.

Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase

PubMed Central

Bourne, Yves; Redford, Susan M.; Steinman, Howard M.; Lepock, James R.; Tainer, John A.; Getzoff, Elizabeth D.

1996-01-01

Eukaryotic Cu,Zn superoxide dismutases (CuZnSODs) are antioxidant enzymes remarkable for their unusually stable β-barrel fold and dimer assembly, diffusion-limited catalysis, and electrostatic guidance of their free radical substrate. Point mutations of CuZnSOD cause the fatal human neurodegenerative disease amyotrophic lateral sclerosis. We determined and analyzed the first crystallographic structure (to our knowledge) for CuZnSOD from a prokaryote, Photobacterium leiognathi, a luminescent symbiont of Leiognathid fish. This structure, exemplifying prokaryotic CuZnSODs, shares the active-site ligand geometry and the topology of the Greek key β-barrel common to the eukaryotic CuZnSODs. However, the β-barrel elements recruited to form the dimer interface, the strategy used to forge the channel for electrostatic recognition of superoxide radical, and the connectivity of the intrasubunit disulfide bond in P. leiognathi CuZnSOD are discrete and strikingly dissimilar from those highly conserved in eukaryotic CuZnSODs. This new CuZnSOD structure broadens our understanding of structural features necessary and sufficient for CuZnSOD activity, highlights a hitherto unrecognized adaptability of the Greek key β-barrel building block in evolution, and reveals that prokaryotic and eukaryotic enzymes diverged from one primordial CuZnSOD and then converged to distinct dimeric enzymes with electrostatic substrate guidance. PMID:8917495

Estrogen Receptor α L543A,L544A Mutation Changes Antagonists to Agonists, Correlating with the Ligand Binding Domain Dimerization Associated with DNA Binding Activity*

PubMed Central

Arao, Yukitomo; Hamilton, Katherine J.; Coons, Laurel A.; Korach, Kenneth S.

2013-01-01

A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα. PMID:23733188

The role of STATs in lung carcinogenesis: an emerging target for novel therapeutics.

PubMed

Karamouzis, Michalis V; Konstantinopoulos, Panagiotis A; Papavassiliou, Athanasios G

2007-05-01

The signal transducer and activator of transcription (STAT) proteins are a family of latent cytoplasmic transcription factors, which form dimers when activated by cytokine receptors, tyrosine kinase growth factor receptors as well as non-receptor tyrosine kinases. Dimeric STATs translocate to the nucleus, where they bind to specific DNA-response elements in the promoters of target genes, thereby inducing unique gene expression programs often in association with other transcription regulatory proteins. The functional consequence of different STAT proteins activation varies, as their target genes play diverse roles in normal cellular/tissue functions, including growth, apoptosis, differentiation and angiogenesis. Certain activated STATs have been implicated in human carcinogenesis, albeit only few studies have focused into their role in lung tumours. Converging evidence unravels their molecular interplays and complex multipartite regulation, rendering some of them appealing targets for lung cancer treatment with new developing strategies.

Crystal Structures of the Receiver Domain of the Response Regulator PhoP from Escherichia coli in the Absence and Presence of the Phosphoryl Analog Beryllofluoride▿

PubMed Central

Bachhawat, Priti; Stock, Ann M.

2007-01-01

The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg2+. Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation. PMID:17545283

Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.

PubMed

Zhou, Suiping; Sorokina, Elena M; Harper, Sandra; Li, Haitao; Ralat, Luis; Dodia, Chandra; Speicher, David W; Feinstein, Sheldon I; Fisher, Aron B

2016-05-01

Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Trichomonas vaginalis metalloproteinase TvMP50 is a monomeric Aminopeptidase P-like enzyme.

PubMed

Arreola, Rodrigo; Villalpando, José Luis; Puente-Rivera, Jonathan; Morales-Montor, Jorge; Rudiño-Piñera, Enrique; Alvarez-Sánchez, María Elizbeth

2018-06-23

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.

Multiple hydrogen-bonded complexes based on 2-ureido-4[1H]-pyrimidinone: a theoretical study.

PubMed

Sun, Hao; Lee, Hui Hui; Blakey, Idriss; Dargaville, Bronwin; Chirila, Traian V; Whittaker, Andrew K; Smith, Sean C

2011-09-29

In the present work, the electronic structures and properties of a series of 2-ureido-4[1H]-pyrimidinone(UPy)-based monomers and dimers in various environments (vacuum, chloroform, and water) are studied by density functional theoretical methods. Most dimers prefer to form a DDAA-AADD (D, H-bond donor; A, H-bond acceptor) array in both vacuum and solvents. Topological analysis proved that intramolecular and intermolecular hydrogen bonds coexist in the dimers. Frequency and NBO calculations show that all the hydrogen bonds exhibit an obvious red shift in their stretching vibrational frequencies. Larger substituents at position 6 of the pyrimidinone ring with stronger electron-donating ability favor the total binding energy and free energy of dimerization. Calculations on the solvent effect show that dimerization is discouraged by the stronger polarity of the solvent. Further computations show that Dimer-1 may be formed in chloroform, but water molecules may interact with the donor or acceptor sites and hence disrupt the hydrogen bonds of Dimer-1. © 2011 American Chemical Society

Ligand regulation of a constitutively dimeric EGF receptor

NASA Astrophysics Data System (ADS)

Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

2015-06-01

Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers.

PubMed

Singh, Deo R; Pasquale, Elena B; Hristova, Kalina

2016-09-01

The EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands. Here, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays. We find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration. The experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer. This work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular nano-arches on silicon

NASA Astrophysics Data System (ADS)

Dobrin, S.

2007-08-01

The formation of molecular nano-arches on the Si(1 1 1)-7 × 7 surface was modeled using density functional theory (DFT). It has been suggested, based on the calculations, that the arches are formed by molecular dimers of chlorobenzene at near-monolayer coverages. Molecules of the dimer are covalently bound to two silicon adatoms and to each other thereby forming a molecular arch on the surface. The structure of the molecular dimer was calculated at the B3LYP/6-31G(d) level of theory. The dimers were found to be stable at room temperature, and to form a near-monolayer coverage, which has been observed in the experiment [X.H. Chen, Q. Kong, J.C. Polanyi, D. Rogers, S. So, Surf. Sci. 340 (1995) 224; Y. Cao, J.F. Deng, G.Q. Xu, J. Chem. Phys. 112 (2000) 4759].

Probing the Allosteric Modulator Binding Site of GluR2 with Thiazide Derivatives

PubMed Central

Ptak, Christopher P.; Ahmed, Ahmed H.; Oswald, Robert E.

2009-01-01

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission and are therapeutic targets for cognitive enhancement and treatment of schizophrenia. The binding domains of these tetrameric receptors consist of two dimers, and the dissociation of the dimer interface of the ligand-binding domain leads to desensitization in the continued presence of agonist. Positive allosteric modulators act by strengthening the dimer interface and reducing desensitization, thereby increasing steady-state activation. Removing the desensitized state for simplified analysis of receptor activation is commonly achieved using cyclothiazide (CTZ), the most potent modulator of the benzothiadiazide class, with the flip form of the AMPA receptor subtype. IDRA-21, the first benzothiadiazide to have an effect in behavioral tests, is an important lead compound in clinical trials for cognitive enhancement as it can cross the blood-brain barrier. Intermediate structures between CTZ and IDRA-21 show reduced potency suggesting that these two compounds have different contact points associated with binding. To understand how benzothiadiazides bind to the pocket bridging the dimer interface, we generated a series of crystal structures of the GluR2 ligand-binding domain complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) for comparison with an existing structure for cyclothiazide. The structures detail how changes in the substituents in the 3- and 7-positions of the hydrobenzothiadiazide ring shift the orientation of the drug in the binding site and, in some cases, change the stoichiometry of binding. All derivatives maintain a hydrogen bond with the Ser754 hydroxyl, affirming the partial selectivity of the benzothiadiazides for the flip form of AMPA receptors. PMID:19673491

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization.

PubMed

Allan, Rudi K; Mok, Danny; Ward, Bryan K; Ratajczak, Thomas

2006-03-17

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

A kinetico-mechanistic study on the C-H bond activation of primary benzylamines; cooperative and solid-state cyclopalladation on dimeric complexes.

PubMed

Font, Helena; Font-Bardia, Mercè; Gómez, Kerman; González, Gabriel; Granell, Jaume; Macho, Israel; Martínez, Manuel

2014-09-28

The cyclometallation reactions of dinuclear μ-acetato complexes of the type [Pd(AcO)(μ-AcO)L]2 (L = 4-RC6H4CH2NH2, R = H, Cl, F, CF3), a process found to occur readily even in the solid state, have been studied from a kinetico-mechanistic perspective. Data indicate that the dinuclear acetato bridged derivatives are excellent starting materials to activate carbon-hydrogen bonds in a facile way. In all cases the established concerted ambiphilic proton abstraction by a coordinated acetato ligand has been proved. The metallation has also been found to occur in a cooperative manner, with the metallation of the first palladium unit of the dimeric complex being rate determining; no intermediate mono-metallated compounds are observed in any of the processes. The kinetically favoured bis-cyclopalladated compound obtained after complete C-H bond activation does not correspond to the final isolated XRD-characterized complexes. This species, bearing the classical open-book dimeric form, has a much more complex structure than the final isolated compound, with different types of acetato ligands.

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure.

PubMed

Wulff, Philip; Thomas, Claudia; Sargent, Frank; Armstrong, Fraser A

2016-03-01

'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging*

PubMed Central

Subburaj, Yamunadevi; Ros, Uris; Hermann, Eduard; Tong, Rudi; García-Sáez, Ana J.

2015-01-01

α-Pore-forming toxins (α-PFTs) are ubiquitous defense tools that kill cells by opening pores in the target cell membrane. Despite their relevance in host/pathogen interactions, very little is known about the pore stoichiometry and assembly pathway leading to membrane permeabilization. Equinatoxin II (EqtII) is a model α-PFT from sea anemone that oligomerizes and forms pores in sphingomyelin-containing membranes. Here, we determined the spatiotemporal organization of EqtII in living cells by single molecule imaging. Surprisingly, we found that on the cell surface EqtII did not organize into a unique oligomeric form. Instead, it existed as a mixture of oligomeric species mostly including monomers, dimers, tetramers, and hexamers. Mathematical modeling based on our data supported a new model in which toxin clustering happened in seconds and proceeded via condensation of EqtII dimer units formed upon monomer association. Furthermore, altering the pathway of EqtII assembly strongly affected its toxic activity, which highlights the relevance of the assembly mechanism on toxicity. PMID:25525270

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

PubMed Central

Jaffe, Eileen K.; Shanmugam, Dhanasekaran; Gardberg, Anna; Dieterich, Shellie; Sankaran, Banumathi; Stewart, Lance J.; Myler, Peter J.; Roos, David S.

2011-01-01

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors. PMID:21383008

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor

PubMed Central

Ward, Richard J.; Pediani, John D.; Harikumar, Kaleeckal G.; Miller, Laurence J.

2017-01-01

Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein–protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant. PMID:28424368

Can Vitamin A be Improved to Prevent Blindness due to Age-Related Macular Degeneration, Stargardt Disease and Other Retinal Dystrophies?

PubMed

Saad, Leonide; Washington, Ilyas

2016-01-01

We discuss how an imperfect visual cycle results in the formation of vitamin A dimers, thought to be involved in the pathogenesis of various retinal diseases, and summarize how slowing vitamin A dimerization has been a therapeutic target of interest to prevent blindness. To elucidate the molecular mechanism of vitamin A dimerization, an alternative form of vitamin A, one that forms dimers more slowly yet maneuvers effortlessly through the visual cycle, was developed. Such a vitamin A, reinforced with deuterium (C20-D3-vitamin A), can be used as a non-disruptive tool to understand the contribution of vitamin A dimers to vision loss. Eventually, C20-D3-vitamin A could become a disease-modifying therapy to slow or stop vision loss associated with dry age-related macular degeneration (AMD), Stargardt disease and retinal diseases marked by such vitamin A dimers. Human clinical trials of C20-D3-vitamin A (ALK-001) are underway.

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures.

PubMed

Iglesias, A A; Andreo, C S

1990-09-24

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.

Lipoprotein lipase (LPL) strongly links native and oxidized low density lipoprotein particles to decorin-coated collagen. Roles for both dimeric and monomeric forms of LPL.

PubMed

Pentikäinen, M O; Oörni, K; Kovanen, P T

2000-02-25

Low density lipoprotein (LDL) and oxidized LDL are associated with collagen in the arterial intima, where the collagen is coated by the small proteoglycan decorin. When incubated in physiological ionic conditions, decorin-coated collagen bound only small amounts of native and oxidized LDL, the interaction being weak. When decorin-coated collagen was first allowed to bind lipoprotein lipase (LPL), binding of native and oxidized LDL increased dramatically (23- and 7-fold, respectively). This increase depended on strong interactions between LPL that was bound to the glycosaminoglycan chains of the collagen-bound decorin and native and oxidized LDL (kDa 12 and 5.9 nM, respectively). To distinguish between binding to monomeric (inactive) and dimeric (catalytically active) forms of LPL, affinity chromatography on heparin columns was conducted, which showed that native LDL bound to the monomeric LPL, whereas oxidized LDL, irrespective of the type of modification (Cu(2+), 2, 2'-azobis(2-amidinopropane)hydrochloride, hypochlorite, or soybean 15-lipoxygenase), bound preferably to dimeric LPL. However, catalytic activity of LPL was not required for binding to oxidized LDL. Finally, immunohistochemistry of atherosclerotic lesions of human coronary arteries revealed specific areas in which LDL, LPL, decorin, and collagen type I were present. The results suggest that LPL can retain LDL in atherosclerotic lesions along decorin-coated collagen fibers.

Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

PubMed Central

de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

2012-01-01

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs. PMID:22529931

Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

PubMed

de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

2012-01-01

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Caspase enzymology and activation mechanisms.

PubMed

Mace, Peter D; Riedl, Stefan J; Salvesen, Guy S

2014-01-01

Apical caspases 8, 9, and 10 are only active as dimers. These dimers are unstable, and to characterize their activity they need to be maintained in vitro in a dimeric state. We provide updated methods for those looking to characterize various aspects of caspase function. We describe full methods for those looking to activate caspases in vitro using kosmotropic reagents, an essential step in characterizing upstream (apical) caspases. We detail methods for fusion of caspase domains to engineered dimerization domains as an alternative method to trigger regulated dimerization of caspases. We also describe methods to determine caspase activity profiles in cells and provide methods for studying the ability of SMAC-mimetic reagents to release inhibition of caspases by IAPs. © 2014 Elsevier Inc. All rights reserved.

The hydroxyl functionality and a rigid proximal N are required for forming a novel non-covalent quinine-heme complex.

PubMed

Alumasa, John N; Gorka, Alexander P; Casabianca, Leah B; Comstock, Erica; de Dios, Angel C; Roepe, Paul D

2011-03-01

Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this important distinction, we study three newly synthesized and several commercially available QN analogues lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin (Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN (DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid-state NMR spectroscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation. Copyright © 2010 Elsevier Inc. All rights reserved.

99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand.

PubMed

Wang, Jianjun; Kim, Young-Seung; Liu, Shuang

2008-03-01

In this study, EDDA (ethylenediamine- N, N'-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC = 6-hydrazinonicotinamide; dimer = E[c(RGDfK)]2) and HYNIC-tetramer (tetramer = E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4 (-) in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity ( approximately 8.0 Ci/micromol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always <65%. The results from a mixed-ligand experiment show that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)]. The athymic nude mice bearing subcutaneous U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on the biodistribution characteristics and excretion kinetics of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. Surprisingly, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] had almost identical tumor uptake over the 2 h period. The use of EDDA as coligand to replace tricine/TPPTS (TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate) did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in noncancerous organs, such as the liver, kidneys, and lungs; but it did result in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster renal excretion. It was also found that the radiotracer tumor uptake decreases in a linear fashion as the tumor size increases. The smaller the tumors are, the higher the tumor uptake is regardless of the identity of radiotracer.

Alternative splicing produces transcripts coding for alpha and beta chains of a hetero-dimeric phosphagen kinase.

PubMed

Ellington, W Ross; Yamashita, Daisuke; Suzuki, Tomohiko

2004-06-09

Glycocyamine kinase (GK) catalyzes the reversible phosphorylation of glycocyamine (guanidinoacetate), a reaction central to cellular energy homeostasis in certain animals. GK is a member of the phosphagen kinase enzyme family and appears to have evolved from creatine kinase (CK) early in the evolution of multi-cellular animals. Prior work has shown that GK from the polychaete Neanthes (Nereis) diversicolor exits as a hetero-dimer in vivo and that the two polypeptide chains (termed alpha and beta) are coded for by unique transcripts. In the present study, we demonstrate that the GK from a congener Nereis virens is also hetero-dimeric and is coded for by alpha and beta transcripts, which are virtually identical to the corresponding forms in N. diversicolor. The GK gene from N. diversicolor was amplified by PCR. Sequencing of the PCR products showed that the alpha and beta chains are the result of alternative splicing of the GK primary mRNA transcript. These results also strongly suggest that this gene underwent an early tandem exon duplication event. Full-length cDNAs for N. virens GKalpha and GKbeta were individually ligated into expression vectors and the resulting constructs used to transform Escherichia coli expression hosts. Regardless of expression conditions, minimal GK activity was observed in both GKalpha and GKbeta constructs. Inclusion bodies for both were harvested, unfolded in urea and alpha chains, beta chains and mixtures of alpha and beta chains were refolded by sequential dialysis. Only modest amounts of GK activity were observed when alpha and beta were refolded individually. In contrast, when refolded the alpha and beta mixture yielded highly active hetero-dimers, as validated by size exclusion chromatography, electrophoresis and mass spectrometry, with a specific activity comparable to that of natural GK. The above evidence suggests that there is a preference for hetero-dimer formation in the GKs from these two polychaetes. The evolution of the alternate splicing and an additional exon in these GKs, producing alpha and beta transcripts, can be viewed as a possible compensation for a mutation(s) in the original gene, which most likely coded for a homo-dimeric protein.

Tubulin Dimer Reversible Dissociation

PubMed Central

Schuck, Peter; Sackett, Dan L.

2016-01-01

Tubulins are evolutionarily conserved proteins that reversibly polymerize and direct intracellular traffic. Of the tubulin family only αβ-tubulin forms stable dimers. We investigated the monomer-dimer equilibrium of rat brain αβ-tubulin using analytical ultracentrifugation and fluorescence anisotropy, observing tubulin in virtually fully monomeric and dimeric states. Monomeric tubulin was stable for a few hours and exchanged into preformed dimers, demonstrating reversibility of dimer dissociation. Global analysis combining sedimentation velocity and fluorescence anisotropy yielded Kd = 84 (54–123) nm. Dimer dissociation kinetics were measured by analyzing the shape of the sedimentation boundary and by the relaxation of fluorescence anisotropy following rapid dilution of labeled tubulin, yielding koff in the range 10−3–10−2 s−1. Thus, tubulin dimers reversibly dissociate with moderately fast kinetics. Monomer-monomer association is much less sensitive than dimer-dimer association to solution changes (GTP/GDP, urea, and trimethylamine oxide). PMID:26934918

Failure of matrix metalloproteinase-9 dimer induction by phorbol 12-myristate 13-acetate in normal human cell lines.

PubMed

Waheed Roomi, Mohd; Kalinovsky, Tatiana; Rath, Matthias; Niedzwiecki, Aleksandra

2015-06-01

Increasing experimental and clinical data has identified an association between increased levels of matrix metalloproteinase (MMP)-9 and shortened patient survival, cancer progression and metastasis. MMP-9 has a significant role in tumor cell invasion and metastasis, as it digests the basement membrane and components of the extracellular matrix. MMP-9 is secreted in either a monomeric or dimeric form. Although limited evidence exists concerning MMP-9 dimers, certain studies have demonstrated that the dimer is associated with aggressive tumor progression. This is believed to be due to the fact that cellular migration depends upon the MMP-9 dimer, and not the monomer. Our previous study revealed that cancer cell MMP-9 dimer secretion patterns could be divided into different categories, and that high MMP-9 and MMP-9 dimer secretion levels were correlated with the most aggressive cancer cell lines. It has been established that signal transduction pathways and cytokines, including those activated by phorbol 12-myristate 13-acetate (PMA), regulate the expression of MMPs. The aim of the present study was to analyze the expression patterns of MMP-2, MMP-9 and MMP-9 dimer in normal human cells from a number of tissues treated with PMA. Muscle, epithelial and connective tissues were selected for use in the present study, since adenosarcomas, carcinomas and sarcomas are derived from these tissue types, respectively. The cell lines were first cultured in 24-well tissue culture plates containing recommended media that was supplemented with 10% fetal bovine serum and antibiotics. When at confluency, the cells were washed and fresh medium was added. In addition, a parallel set of cultures was treated with PMA. Subsequent to a 24-h incubation period, the media were collected and analyzed using gelatinase zymography for the expression of MMP-2 and MMP-9 monomer and dimer forms. The results revealed that the cellular expression of MMP-2 and MMP-9 was dependent upon the primary tissue subtype. All cell lines, regardless of tissue origin, expressed MMP-2. PMA induced the expression of MMP-9 in muscle tissue, glandular epithelia and supportive connective tissue cell lines. By contrast, cell lines of endothelial origin and proper connective tissue were insensitive to treatment with PMA. MMP-9 dimer secretion was not observed in any of the cell lines, which indicated that cellular migration is not supported by these cells.

Oligomeric Properties of Survival Motor Neuron·Gemin2 Complexes*

PubMed Central

Gupta, Kushol; Martin, Renee; Sharp, Robert; Sarachan, Kathryn L.; Ninan, Nisha S.; Van Duyne, Gregory D.

2015-01-01

The survival motor neuron (SMN) protein forms the oligomeric core of a multiprotein complex required for the assembly of spliceosomal small nuclear ribonucleoproteins. Deletions and mutations in the SMN1 gene are associated with spinal muscular atrophy (SMA), a devastating neurodegenerative disease that is the leading heritable cause of infant mortality. Oligomerization of SMN is required for its function, and some SMA patient mutations disrupt the ability of SMN to self-associate. Here, we investigate the oligomeric nature of the SMN·Gemin2 complexes from humans and fission yeast (hSMN·Gemin2 and ySMN·Gemin2). We find that hSMN·Gemin2 forms oligomers spanning the dimer to octamer range. The YG box oligomerization domain of SMN is both necessary and sufficient to form these oligomers. ySMN·Gemin2 exists as a dimer-tetramer equilibrium with Kd = 1.0 ± 0.9 μm. A 1.9 Å crystal structure of the ySMN YG box confirms a high level of structural conservation with the human ortholog in this important region of SMN. Disulfide cross-linking experiments indicate that SMN tetramers are formed by self-association of stable, non-dissociating dimers. Thus, SMN tetramers do not form symmetric helical bundles such as those found in glycine zipper transmembrane oligomers. The dimer-tetramer nature of SMN complexes and the dimer of dimers organization of the SMN tetramer provide an important foundation for ongoing studies to understand the mechanism of SMN-assisted small nuclear ribonucleoprotein assembly and the underlying causes of SMA. PMID:26092730

Utilization of DR1 as true RARE in regulating the Ssm, a novel retinoic acid-target gene in the mouse testis.

PubMed

Han, Kyuyong; Song, Haengseok; Moon, Irene; Augustin, Robert; Moley, Kelle; Rogers, Melissa; Lim, Hyunjung

2007-03-01

Various nuclear receptors form dimers to activate target genes via specific response elements located within promoters or enhancers. Retinoid X receptor (RXR) serves as a dimerization partner for many nuclear receptors including retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR). Dimers show differential preference towards directly repeated response elements with 1-5 nucleotide spacing, and direct repeat 1 (DR1) is a promiscuous element which recruits RAR/RXR, RXR/RXR, and PPAR/RXR in vitro. In the present investigation, we report identification of a novel RAR/RXR target gene which is regulated by DR1s in the promoter region. This gene, namely spermatocyte-specific marker (Ssm), recruits all the three combinations of nuclear receptors in vitro, but in vivo regulation is observed by trans-retinoic acid-activated RAR/RXR dimer. Indeed, chromatin immunoprecipitation experiment demonstrates binding of RARbeta and RXRalpha in the promoter region of the Ssm. Interestingly, expression of Ssm is almost exclusively observed in spermatocytes in the adult mouse testis, where RA signaling is known to regulate developmental program of male germ cells. The results show that Ssm is a RAR/RXR target gene uniquely using DR1 and exhibits stage-specific expression in the mouse testis with potential function in later stages of spermatogenesis. This finding exemplifies usage of DR1s as retinoic acid response element (RARE) under a specific in vivo context.

Interaction of chemokine receptor CXCR4 in monomeric and dimeric state with its endogenous ligand CXCL12: coarse-grained simulations identify differences.

PubMed

Cutolo, Pasquale; Basdevant, Nathalie; Bernadat, Guillaume; Bachelerie, Françoise; Ha-Duong, Tâp

2017-02-01

Despite the recent resolutions of the crystal structure of the chemokine receptor CXCR4 in complex with small antagonists or viral chemokine, a description at the molecular level of the interactions between the full-length CXCR4 and its endogenous ligand, the chemokine CXCL12, in relationship with the receptor recognition and activation, is not yet completely elucidated. Moreover, since CXCR4 is able to form dimers, the question of whether the CXCR4-CXCL12 complex has a 1:1 or 2:1 preferential stoichiometry is still an open question. We present here results of coarse-grained protein-protein docking and molecular dynamics simulations of CXCL12 in association with CXCR4 in monomeric and dimeric states. Our proposed models for the 1:1 and 2:1 CXCR4-CXCL12 quaternary structures are consistent with recognition and activation motifs of both partners provided by the available site-directed mutagenesis data. Notably, we observed that in the 2:1 complex, the chemokine N-terminus makes more steady contacts with the receptor residues critical for binding and activation than in the 1:1 structure, suggesting that the 2:1 stoichiometry would favor the receptor signaling activity with respect to the 1:1 association.

Histone H1 chaperone activity of TAF-I is regulated by its subtype-dependent intramolecular interaction.

PubMed

Kajitani, Kaori; Kato, Kohsuke; Nagata, Kyosuke

2017-04-01

Linker histone H1 is involved in the regulation of gene activity through the maintenance of higher-order chromatin structure. Previously, we have shown that template activating factor-I (TAF-I or protein SET) is involved in linker histone H1 dynamics as a histone H1 chaperone. In human and murine cells, two TAF-I subtypes exist, namely TAF-Iα and TAF-Iβ. TAF-I has a highly acidic amino acid cluster in its C-terminal region and forms homo- or heterodimers through its dimerization domain. Both dimer formation and the C-terminal region of TAF-I are essential for the histone chaperone activity. TAF-Iα exhibits less histone chaperone activity compared with TAF-Iβ even though TAF-Iα and β differ only in their N-terminal regions. However, it is unclear how subtype-specific TAF-I activities are regulated. Here, we have shown that the N-terminal region of TAF-Iα autoinhibits its histone chaperone activity via intramolecular interaction with its C-terminal region. When the interaction between the N- and C-terminal regions of TAF-Iα is disrupted, TAF-Iα shows a histone chaperone activity similar to that of TAF-Iβ. Taken together, these results provide mechanistic insights into the concept that fine tuning of TAF-I histone H1 chaperone activity relies on the subtype compositions of the TAF-I dimer. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

PubMed

Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

2017-11-01

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

PubMed Central

Smulski, Cristian R.; Beyrath, Julien; Decossas, Marion; Chekkat, Neila; Wolff, Philippe; Estieu-Gionnet, Karine; Guichard, Gilles; Speiser, Daniel; Schneider, Pascal; Fournel, Sylvie

2013-01-01

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state. PMID:23463508

Evidence for dimer formation by an amphiphilic heptapeptide that mediates chloride and carboxyfluorescein release from liposomes

PubMed Central

Pajewski, Robert; Ferdani, Riccardo; Pajewska, Jolanta; Djedovič, Natasha; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

Heptapeptides having dioctadecyl, N-terminal hydrocarbon chains insert in phospholipid bilayer membranes and form pores through which at least chloride ions pass. Although amphiphilic, these compounds do not typically form vesicles themselves. They insert in the bilayers of phospholipid vesicles and mediate the release of carboxyfluorescein. Hill analysis indicates that at least two molecules of the amphiphile are involved in pore formation. In CD2Cl2, dimer formation is detected by NMR chemical shift changes. The anion release activity of individual anion transporters is increased by linking them covalently at the C-terminus or, even more, by linking them at the N-terminus. Evidence is presented that either linked molecule releases chloride from liposomes more effectively and rapidly than the individual transporter molecule at a comparable concentration. PMID:15703797

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

PubMed

Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

2016-03-15

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

Structure elucidation of dimeric transmembrane domains of bitopic proteins.

PubMed

Bocharov, Eduard V; Volynsky, Pavel E; Pavlov, Konstantin V; Efremov, Roman G; Arseniev, Alexander S

2010-01-01

The interaction between transmembrane helices is of great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism.

Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

PubMed Central

Mondal, Samiran; Begum, Nasim A.; Hu, Wenjun; Honjo, Tasuku

2016-01-01

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions. PMID:26929374

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

Baden, Elizabeth M.; Owen, Barbara A.L.; Peterson, Francis C.

Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the kl O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90 degrees from the kl O18/O8 dimer interface. The three non-conservativemore » mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than kl O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.« less

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

PubMed Central

2005-01-01

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

Fibulin 5 Forms a Compact Dimer in Physiological Solutions*

PubMed Central

Jones, Richard P. O.; Wang, Ming-Chuan; Jowitt, Thomas A.; Ridley, Caroline; Mellody, Kieran T.; Howard, Marjorie; Wang, Tao; Bishop, Paul N.; Lotery, Andrew J.; Kielty, Cay M.; Baldock, Clair; Trump, Dorothy

2009-01-01

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca2+ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin. PMID:19617354

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

PubMed

Ben Khalifa, N; Tyteca, D; Courtoy, P J; Renauld, J C; Constantinescu, S N; Octave, J N; Kienlen-Campard, P

2012-01-01

The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP. Copyright © 2012 S. Karger AG, Basel.

Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations

PubMed Central

Han, Jing; Pluhackova, Kristyna; Wassenaar, Tsjerk A.; Böckmann, Rainer A.

2015-01-01

Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin’s TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes. PMID:26287628

Kinetics and structural features of dimeric glutamine-dependent bacterial NAD+ synthetases suggest evolutionary adaptation to available metabolites.

PubMed

Santos, Adrian Richard Schenberger; Gerhardt, Edileusa Cristina Marques; Moure, Vivian Rotuno; Pedrosa, Fábio Oliveira; Souza, Emanuel Maltempi; Diamanti, Riccardo; Högbom, Martin; Huergo, Luciano Fernandes

2018-05-11

NADH (NAD + ) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD + also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD + biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD + synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE NH3 and octameric glutamine-dependent NadE Gln , and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE Gln in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE Gln enzymes may constitute evolutionary intermediates between dimeric NadE NH3 and octameric NadE Gln The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes. © 2018 Santos et al.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

PubMed

Comps-Agrar, Laëtitia; Dunshee, Diana Ronai; Eaton, Dan L; Sonoda, Junichiro

2015-10-02

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor βKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and βKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB*

PubMed Central

Creager-Allen, Rachel L.; Silversmith, Ruth E.; Bourret, Robert B.

2013-01-01

Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3−. Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ∼10-fold higher than for the monomer. In a test of the model, disruption of the known PhoBN dimerization interface by mutation led to markedly slower and noncooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA-binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation. PMID:23760278

Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation.

PubMed

Valle, Aisel; Pérez-Socas, Luis Benito; Canet, Liem; Hervis, Yadira de la Patria; de Armas-Guitart, German; Martins-de-Sa, Diogo; Lima, Jônatas Cunha Barbosa; Souza, Adolfo Carlos Barros; Barbosa, João Alexandre Ribeiro Gonçalves; de Freitas, Sonia Maria; Pazos, Isabel Fabiola

2018-04-26

The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.

Synthesis, characterization and biological activities of semicarbazones and their copper complexes.

PubMed

Venkatachalam, Taracad K; Bernhardt, Paul V; Noble, Chris J; Fletcher, Nicholas; Pierens, Gregory K; Thurecht, Kris J; Reutens, David C

2016-09-01

Substituted semicarbazones/thiosemicarbazones and their copper complexes have been prepared and several single crystal structures examined. The copper complexes of these semicarbazone/thiosemicarbazones were prepared and several crystal structures examined. The single crystal X-ray structure of the pyridyl-substituted semicarbazone showed two types of copper complexes, a monomer and a dimer. We also found that the p-nitrophenyl semicarbazone formed a conventional 'magic lantern' acetate-bridged dimer. Electron Paramagnetic Resonance (EPR) of several of the copper complexes was consistent with the results of single crystal X-ray crystallography. The EPR spectra of the p-nitrophenyl semicarbazone copper complex in dimethylsulfoxide (DMSO) showed the presence of two species, confirming the structural information. Since thiosemicarbazones and semicarbazones have been reported to exhibit anticancer activity, we examined the anticancer activity of several of the derivatives reported in the present study and interestingly only the thiosemicarbazone showed activity while the semicarbazones were not active indicating that introduction of sulphur atom alters the biological profile of these thiosemicarbazones. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamic asymmetry and the role of the conserved active-site thiol in rabbit muscle creatine kinase.

PubMed

Londergan, Casey H; Baskin, Rachel; Bischak, Connor G; Hoffman, Kevin W; Snead, David M; Reynoso, Christopher

2015-01-13

Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.

Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety

PubMed Central

He, Ran; Forman, Michael; Mott, Bryan T.; Venkatadri, Rajkumar; Posner, Gary H.

2013-01-01

We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838. PMID:23774439

Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway

PubMed Central

Todeschini, Adriane Regina; Dos Santos, Jose Nilson; Handa, Kazuko; Hakomori, Sen-itiroh

2008-01-01

Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through “cross-talk” of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway. PMID:18272501

Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway.

PubMed

Todeschini, Adriane Regina; Dos Santos, Jose Nilson; Handa, Kazuko; Hakomori, Sen-itiroh

2008-02-12

Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through "cross-talk" of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway.

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase.

PubMed

Kots, Ekaterina D; Lushchekina, Sofya V; Varfolomeev, Sergey D; Nemukhin, Alexander V

2017-08-28

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 μs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

Identifying paths of allosteric communication in the protein BirA through simulations

NASA Astrophysics Data System (ADS)

Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

Biotin ligase/repressor (BirA) is a bifunctional enzyme which adenylates biotin and transfers the product, biotinyl-5'-AMP (bio-5'-AMP) to biotin carboxyl carrier protein (BCCP). In the absence of BCCP, bio-5'-AMP promotes the dimerization of BirA. In dimer form, the BirA.bio-5'-AMP complex is able to bind to the biotin operator and prevents further synthesis of biotin. The bio-5'-AMP binds away from the dimer interface, so it is acting as an allosteric activator. We perform all-atom molecular dynamics simulations with BirA to look at fluctuations within the protein at equilibrium. We simulate apoBirA, liganded BirA, as well as two mutants, M211A and V219A. In agreement with experimental observations, several loops of the protein become stabilized for the liganded BirA when compared to the apo protein. In addition, changes in the dimer interface are observed for the M211A and V219A mutations, which are located in the ligand binding region. Using inter-residue correlation coefficients and pair energies a communication network through the protein is constructed. With this network we have identified paths which have the potential to be important in allosteric activation of BirA. These paths and the methods we use to identify them will be presented.

New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability

NASA Astrophysics Data System (ADS)

Maurice, Pascal; Baud, Stéphanie; Bocharova, Olga V.; Bocharov, Eduard V.; Kuznetsov, Andrey S.; Kawecki, Charlotte; Bocquet, Olivier; Romier, Beatrice; Gorisse, Laetitia; Ghirardi, Maxime; Duca, Laurent; Blaise, Sébastien; Martiny, Laurent; Dauchez, Manuel; Efremov, Roman G.; Debelle, Laurent

2016-12-01

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139-159 (TM1) and 316-333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

PubMed Central

2016-01-01

Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin.

PubMed

Varghese, Leila N; Defour, Jean-Philippe; Pecquet, Christian; Constantinescu, Stefan N

2017-01-01

A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

PubMed

Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

2016-09-20

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

D-dimer: An Overview of Hemostasis and Fibrinolysis, Assays, and Clinical Applications.

PubMed

Olson, John D

2015-01-01

D-dimer is the smallest fibrinolysis-specific degradation product found in the circulation. The origins, assays, and clinical use of D-dimer will be addressed. Hemostasis (platelet and vascular function, coagulation, fibrinolysis, hemostasis) is briefly reviewed. D-dimer assays are reviewed. The D-dimer is very sensitive to intravascular thrombus and may be markedly elevated in disseminated intravascular coagulation, acute aortic dissection, and pulmonary embolus. Because of its exquisite sensitivity, negative tests are useful in the exclusion venous thromboembolism. Elevations occur in normal pregnancy, rising two- to fourfold by delivery. D-dimer also rises with age, limiting its use in those >80 years old. There is a variable rise in D-dimer in active malignancy and indicates increased thrombosis risk in active disease. Elevated D-dimer following anticoagulation for a thrombotic event indicates increased risk of recurrent thrombosis. These and other issues are addressed. © 2015 Elsevier Inc. All rights reserved.

Molecular determinants of cadherin ideal bond formation: Conformation-dependent unbinding on a multidimensional landscape

PubMed Central

Manibog, Kristine; Sankar, Kannan; Kim, Sun-Ae; Zhang, Yunxiang; Jernigan, Robert L.; Sivasankar, Sanjeevi

2016-01-01

Classical cadherin cell–cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i.e., adhesive interactions that are insensitive to force). However, the biophysical mechanism for ideal bond formation is unknown. Here, we integrate single-molecule force measurements with coarse-grained and atomistic simulations to resolve the mechanistic basis for cadherin ideal bond formation. Using simulations, we predict the energy landscape for cadherin adhesion, the transition pathways for interconversion between X-dimers and strand-swap dimers, and the cadherin structures that form ideal bonds. Based on these predictions, we engineer cadherin mutants that promote or inhibit ideal bond formation and measure their force-dependent kinetics using single-molecule force-clamp measurements with an atomic force microscope. Our data establish that cadherins adopt an intermediate conformation as they shuttle between X-dimers and strand-swap dimers; pulling on this conformation induces a torsional motion perpendicular to the pulling direction that unbinds the proteins and forms force-independent ideal bonds. Torsional motion is blocked when cadherins associate laterally in a cis orientation, suggesting that ideal bonds may play a role in mechanically regulating cadherin clustering on cell surfaces. PMID:27621473

Structure of the Cytoplasmic Region of PelD, a Degenerate Diguanylate Cyclase Receptor That Regulates Exopolysaccharide Production in Pseudomonas aeruginosa*

PubMed Central

Whitney, John C.; Colvin, Kelly M.; Marmont, Lindsey S.; Robinson, Howard; Parsek, Matthew R.; Howell, P. Lynne

2012-01-01

High cellular concentrations of bis-(3′,5′)-cyclic dimeric guanosine mono-phosphate (c-di-GMP) regulate a diverse range of phenotypes in bacteria including biofilm development. The opportunistic pathogen Pseudomonas aeruginosa produces the PEL polysaccharide to form a biofilm at the air-liquid interface of standing cultures. Among the proteins required for PEL polysaccharide production, PelD has been identified as a membrane-bound c-di-GMP-specific receptor. In this work, we present the x-ray crystal structure of a soluble cytoplasmic region of PelD in its apo and c-di-GMP complexed forms. The structure of PelD reveals an N-terminal GAF domain and a C-terminal degenerate GGDEF domain, the latter of which binds dimeric c-di-GMP at an RXXD motif that normally serves as an allosteric inhibition site for active diguanylate cyclases. Using isothermal titration calorimetry, we demonstrate that PelD binds c-di-GMP with low micromolar affinity and that mutation of residues involved in binding not only decreases the affinity of this interaction but also abrogates PEL-specific phenotypes in vivo. Bioinformatics analysis of the juxtamembrane region of PelD suggests that it contains an α-helical stalk region that connects the soluble region to the transmembrane domains and that similarly to other GAF domain containing proteins, this region likely forms a coiled-coil motif that mediates dimerization. PelD with Alg44 and BcsA of the alginate and cellulose secretion systems, respectively, collectively constitute a group of c-di-GMP receptors that appear to regulate exopolysaccharide assembly at the protein level through activation of their associated glycosyl transferases. PMID:22605337

Surface expression of ω-transaminase in Escherichia coli.

PubMed

Gustavsson, Martin; Muraleedharan, Madhu Nair; Larsson, Gen

2014-04-01

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

Surface Expression of ω-Transaminase in Escherichia coli

PubMed Central

Gustavsson, Martin; Muraleedharan, Madhu Nair

2014-01-01

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity. PMID:24487538

Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

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

Yuan, Ping; Paterson, Reay G.; Leser, George P.

2012-09-06

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Heremore » we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase {beta}-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN{sub 0} and associated reduced virulence.« less

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

PubMed

Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J

2002-06-28

MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for seven other scFv molecules have shown that, while the contact residues for symmetric back-to-back dimer formation in MFE-23 are not fully conserved, in principle, back-to-back contacts can be formed in these too. This offers possibilities for the creation of other forms of scFv molecules. (c) 2002 Elsevier Science Ltd.

Ligand-mediated Galectin-1 endocytosis prevents intraneural H2O2 production promoting F-actin dynamics reactivation and axonal re-growth.

PubMed

Quintá, Héctor R; Wilson, Carlos; Blidner, Ada G; González-Billault, Christian; Pasquini, Laura A; Rabinovich, Gabriel A; Pasquini, Juana M

2016-09-01

Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel therapeutic modality for promoting nerve repair and preventing axonal loss. Copyright © 2016 Elsevier Inc. All rights reserved.

Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism.

PubMed

Zhang, H; Hu, G; Wang, H; Sciavolino, P; Iler, N; Shen, M M; Abate-Shen, C

1997-05-01

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeoproteins. Here we show that representative members of the Msx and Dlx homeoprotein families form homo- and heterodimeric complexes. We demonstrate that dimerization by Msx and Dlx proteins is mediated through their homeodomains and that the residues required for this interaction correspond to those necessary for DNA binding. Unlike most other known examples of homeoprotein interactions, association of Msx and Dlx proteins does not promote cooperative DNA binding; instead, dimerization and DNA binding are mutually exclusive activities. In particular, we show that Msx and Dlx proteins interact independently and noncooperatively with homeodomain DNA binding sites and that dimerization is specifically blocked by the presence of such DNA sites. We further demonstrate that the transcriptional properties of Msx and Dlx proteins display reciprocal inhibition. Specifically, Msx proteins act as transcriptional repressors and Dlx proteins act as activators, while in combination, Msx and Dlx proteins counteract each other's transcriptional activities. Finally, we show that the expression patterns of representative Msx and Dlx genes (Msx1, Msx2, Dlx2, and Dlx5) overlap in mouse embryogenesis during limb bud and craniofacial development, consistent with the potential for their protein products to interact in vivo. Based on these observations, we propose that functional antagonism through heterodimer formation provides a mechanism for regulating the transcriptional actions of Msx and Dlx homeoproteins in vivo.

Interaction investigations of HipA binding to HipB dimer and HipB dimer + DNA complex: a molecular dynamics simulation study.

PubMed

Li, Chaoqun; Wang, Yaru; Wang, Yan; Chen, Guangju

2013-11-01

We carried out molecular dynamics simulations and free energy calculations for a series of ternary and diplex models for the HipA protein, HipB dimer, and DNA molecule to address the mechanism of HipA sequestration and the binding order of events from apo HipB/HipA to 2HipA + HipB dimer + DNA complex. The results revealed that the combination of DNA with the HipB dimer is energetically favorable for the combination of HipB dimer with HipA protein. The binding of DNA to HipB dimer induces a long-range allosteric communication from the HipB2 -DNA interface to the HipA-HipB2 interface, which involves the closeness of α1 helices of HipB dimer to HipA protein and formations of extra hydrogen bonds in the HipA-HipB2 interface through the extension of α2/3 helices in the HipB dimer. These simulated results suggested that the DNA molecule, as a regulative media, modulates the HipB dimer conformation, consequently increasing the interactions of HipB dimer with the HipA proteins, which explains the mechanism of HipA sequestration reported by the previous experiment. Simultaneously, these simulations also explored that the thermodynamic binding order in a simulated physiological environment, that is, the HipB dimer first bind to DNA to form HipB dimer + DNA complex, then capturing strongly the HipA proteins to form a ternary complex, 2HipA + HipB dimer + DNA, for sequestrating HipA in the nucleoid. Copyright © 2013 John Wiley & Sons, Ltd.

Structural insights into the intertwined dimer of fyn SH2.

PubMed

Huculeci, Radu; Garcia-Pino, Abel; Buts, Lieven; Lenaerts, Tom; van Nuland, Nico

2015-12-01

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide-bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2's dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand. © 2015 The Protein Society.

Carboxyl group footprinting mass spectrometry and molecular dynamics identify key interactions in the HER2-HER3 receptor tyrosine kinase interface.

PubMed

Collier, Timothy S; Diraviyam, Karthikeyan; Monsey, John; Shen, Wei; Sept, David; Bose, Ron

2013-08-30

The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.

Molecular Simulation Uncovers the Conformational Space of the λ Cro Dimer in Solution

PubMed Central

Ahlstrom, Logan S.; Miyashita, Osamu

2011-01-01

The significant variation among solved structures of the λ Cro dimer suggests its flexibility. However, contacts in the crystal lattice could have stabilized a conformation which is unrepresentative of its dominant solution form. Here we report on the conformational space of the Cro dimer in solution using replica exchange molecular dynamics in explicit solvent. The simulated ensemble shows remarkable correlation with available x-ray structures. Network analysis and a free energy surface reveal the predominance of closed and semi-open dimers, with a modest barrier separating these two states. The fully open conformation lies higher in free energy, indicating that it requires stabilization by DNA or crystal contacts. Most NMR models are found to be unstable conformations in solution. Intersubunit salt bridging between Arg4 and Glu53 during simulation stabilizes closed conformations. Because a semi-open state is among the low-energy conformations sampled in simulation, we propose that Cro-DNA binding may not entail a large conformational change relative to the dominant dimer forms in solution. PMID:22098751

Dimer model for Tau proteins bound in microtubule bundles

NASA Astrophysics Data System (ADS)

Hall, Natalie; Kluber, Alexander; Hayre, N. Robert; Singh, Rajiv; Cox, Daniel

2013-03-01

The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant. Supported by US NSF Grant DMR 1207624.

Comparison of the catalytic activity for the Suzuki-Miyaura reaction of (η(5)-Cp)Pd(IPr)Cl with (η(3)-cinnamyl)Pd(IPr)(Cl) and (η(3)-1-t-Bu-indenyl)Pd(IPr)(Cl).

PubMed

Melvin, Patrick R; Hazari, Nilay; Lant, Hannah M C; Peczak, Ian L; Shah, Hemali P

2015-01-01

Complexes of the type (η(3)-allyl)Pd(L)(Cl) and (η(3)-indenyl)Pd(L)(Cl) are highly active precatalysts for the Suzuki-Miyaura reaction. Even though allyl and indenyl ligands are similar to cyclopentadienyl (Cp) ligands, there have been no detailed comparative studies exploring the activity of precatalysts of the type (η(5)-Cp)Pd(L)(Cl) for Suzuki-Miyaura reactions. Here, we compare the catalytic activity of (η(5)-Cp)Pd(IPr)(Cl) (IPr = 1,3-bis(2,6-diisopropylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene, Cp) with two commercially available catalysts (η(3)-cinnamyl)Pd(IPr)(Cl) (Cin) and (η(3)-1-t-Bu-indenyl)Pd(IPr)(Cl) ( (tBu) Ind). We show that Cp gives slightly better catalytic activity than Cin, but significantly inferior activity than (tBu) Ind. This order of activity is rationalized by comparing the rates at which the precatalysts are activated to the monoligated Pd(0) active species along with the tendency of the starting precatalysts to comproportionate with monoligated Pd(0) to form inactive Pd(I) dimers. As part of this work the Cp supported Pd(I) dimer (μ-Cp)(μ-Cl)Pd2(IPr)2 (Cp (Dim) ) was synthesized and crystallographically characterized. It does not readily disproportionate to form monoligated Pd(0) and consequently Cp (Dim) is a poor catalyst for the Suzuki-Miyaura reaction.

The influence of fatty acids on the GpA dimer interface by coarse-grained molecular dynamics simulation.

PubMed

Flinner, Nadine; Mirus, Oliver; Schleiff, Enrico

2014-08-15

The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA.

The Influence of Fatty Acids on the GpA Dimer Interface by Coarse-Grained Molecular Dynamics Simulation

PubMed Central

Flinner, Nadine; Mirus, Oliver; Schleiff, Enrico

2014-01-01

The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA. PMID:25196522

Structural Determinants Underlying Constitutive Dimerization of Unoccupied Human Follitropin Receptors

PubMed Central

Guan, Rongbin; Wu, Xueqing; Feng, Xiuyan; Zhang, Meilin; Hébert, Terence E.; Segaloff, Deborah L.

2009-01-01

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr110 in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269–277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr110 to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr110 of the ECD. PMID:19800402

Structural basis for the suppression of skin cancers by DNA polymerase [eta

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

Silverstein, Timothy D.; Johnson, Robert E.; Jain, Rinku

2010-09-13

DNA polymerase {eta} (Pol{eta}) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Pol{eta} (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Pol{eta} (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Pol{eta} to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in anmore » active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln55, Arg73 and Met74. Together, these features define the basis for Pol{eta}'s action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.« less

The Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus†

PubMed Central

Quinlan, R. Jason; Reinhart, Gregory D.

2008-01-01

Differences between the crystal structures of inhibitor-bound and uninihibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits containing two different extrinsic fluorophores simultaneously in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel Fluorescence Correlation Spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor-binding, as binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated. PMID:16981693

Participation of the extracellular domain in (pro)renin receptor dimerization

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

Suzuki-Nakagawa, Chiharu; Nishimura, Misa; Tsukamoto, Tomoko

Highlights: • The (pro)renin receptor [(P)RR] is a regulator of the renin–angiotensin system. • The region responsible for (P)RR dimerization was investigated. • (P)RR extracellular domain constructs were retained intracellularly. • The extracellular domain of (P)RR is responsible for its dimerization. • Novel insight into the regulatory mechanism of soluble (P)RR secretion is provided. - Abstract: The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but themore » region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.« less

Molecular dynamics simulations and modelling of the residue interaction networks in the BRAF kinase complexes with small molecule inhibitors: probing the allosteric effects of ligand-induced kinase dimerization and paradoxical activation.

PubMed

Verkhivker, G M

2016-10-20

Protein kinases are central to proper functioning of cellular networks and are an integral part of many signal transduction pathways. The family of protein kinases represents by far the largest and most important class of therapeutic targets in oncology. Dimerization-induced activation has emerged as a common mechanism of allosteric regulation in BRAF kinases, which play an important role in growth factor signalling and human diseases. Recent studies have revealed that most of the BRAF inhibitors can induce dimerization and paradoxically stimulate enzyme transactivation by conferring an active conformation in the second monomer of the kinase dimer. The emerging connections between inhibitor binding and BRAF kinase domain dimerization have suggested a molecular basis of the activation mechanism in which BRAF inhibitors may allosterically modulate the stability of the dimerization interface and affect the organization of residue interaction networks in BRAF kinase dimers. In this work, we integrated structural bioinformatics analysis, molecular dynamics and binding free energy simulations with the protein structure network analysis of the BRAF crystal structures to determine dynamic signatures of BRAF conformations in complexes with different types of inhibitors and probe the mechanisms of the inhibitor-induced dimerization and paradoxical activation. The results of this study highlight previously unexplored relationships between types of BRAF inhibitors, inhibitor-induced changes in the residue interaction networks and allosteric modulation of the kinase activity. This study suggests a mechanism by which BRAF inhibitors could promote or interfere with the paradoxical activation of BRAF kinases, which may be useful in informing discovery efforts to minimize the unanticipated adverse biological consequences of these therapeutic agents.

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

PubMed

Berry, Robert E; Yang, Fei; Shokhireva, Tatiana K; Amoia, Angela M; Garrett, Sarah A; Goren, Allena M; Korte, Stephanie R; Zhang, Hongjun; Weichsel, Andrzej; Montfort, William R; Walker, F Ann

2015-01-20

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.

PubMed

Maun, Henry R; Liu, Peter S; Franke, Yvonne; Eigenbrot, Charles; Forrest, William F; Schwartz, Lawrence B; Lazarus, Robert A

2018-04-16

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling.

PubMed

Farhat, Katja; Riekenberg, Sabine; Heine, Holger; Debarry, Jennifer; Lang, Roland; Mages, Jörg; Buwitt-Beckmann, Ute; Röschmann, Kristina; Jung, Günther; Wiesmüller, Karl-Heinz; Ulmer, Artur J

2008-03-01

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)(2)C-(VPGVG)(4)VPGKG, fibroblast-stimulating LP-1, and Pam(2)C-SK(4). Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.

Incorporation of hydroxy-cinnamaldehydes into lignins

Treesearch

John Ralph; Hoon Kim; Fachuang Lu; Sally A. Ralph; Larry L. Landucci; Takashi Ito; Shingo Kawai

1999-01-01

Peroxidase/H2O2-mediated radical coupling of hydroxycinnamaldehydes produced 81O14-, 8-5-, 818-, and 5-5dimers as had been documented earlier (although we found that the 815-dimer is produced in its cyclic phenylcoumaran form at neutral pH). Spectral data from dimers and oligomers has allowed a more substantive assignment of aldehyde components in lignins isolated from...

Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure*

PubMed Central

Pronker, Matti F.; Bos, Trusanne G. A. A.; Sharp, Thomas H.; Thies-Weesie, Dominique M. E.; Janssen, Bert J. C.

2015-01-01

Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the “V” is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal β-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1coil-Olf) that reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members. PMID:25903135

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

PubMed

Tomita, Toshio; Mizumachi, Yoshihiro; Chong, Kang; Ogawa, Kanako; Konishi, Norihide; Sugawara-Tomita, Noriko; Dohmae, Naoshi; Hashimoto, Yohichi; Takio, Koji

2004-12-24

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

NASA Astrophysics Data System (ADS)

Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

2014-12-01

Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA

PubMed Central

Berke, Ian C; Modis, Yorgo

2012-01-01

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16–18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5–RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry. PMID:22314235

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

PubMed Central

Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

2016-01-01

The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

21 CFR 176.120 - Alkyl ketene dimers.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., processing, preparing, treating, packaging, transporting, or holding food, subject to the provisions of this... paperboard. (c) The alkyl ketene dimers may be used in the form of an aqueous emulsion which may contain...

Synthesis and evaluation of hetero- and homo-dimers of ribosome-targeting antibiotics: Antimicrobial activity, in vitro inhibition of translation, and drug resistance

PubMed Central

Berkov-Zrihen, Yifat; Green, Keith D.; Labby, Kristin J.; Feldman, Mark; Garneau-Tsodikova, Sylvie; Fridman, Micha

2013-01-01

In this study, we describe the synthesis of a full set of homo- and hetero-dimers of three intact structures of different ribosome-targeting antibiotics: tobramycin, clindamycin, and chloramphenicol. Several aspects of the biological activity of the dimeric structures were evaluated including antimicrobial activity, inhibition of in vitro bacterial protein translation, and the effect of dimerization on the action of several bacterial resistance mechanisms that deactivate tobramycin and chloramphenicol. This study demonstrates that covalently linking two identical or different ribosome-targeting antibiotics may lead to (i) a broader spectrum of antimicrobial activity, (ii) improved inhibition of bacterial translation properties compared to that of the parent antibiotics, and (iii) reduction in the efficacy of some drug-modifying enzymes that confer high levels of resistance to the parent antibiotics from which the dimers were derived. PMID:23786357

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

PubMed Central

Snapp, Karen R.; Craig, Ron; Herron, Michael; Nelson, Robert D.; Stoolman, Lloyd M.; Kansas, Geoffrey S.

1998-01-01

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin. PMID:9660879

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation.

PubMed

Shepard, Eric M; Byer, Amanda S; Aggarwal, Priyanka; Betz, Jeremiah N; Scott, Anna G; Shisler, Krista A; Usselman, Robert J; Eaton, Gareth R; Eaton, Sandra S; Broderick, Joan B

2017-06-27

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H 2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S] 2+/+ and [2Fe-2S] 2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514-3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S] + and [4Fe-4S] + clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S] + and [2Fe-2S] + cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S] + and [4Fe-4S] + clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

PubMed Central

2017-01-01

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514–3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway. PMID:28525271

1-[(Anthracen-9-yl)carbon­yl]-2,7-di­meth­oxy­naphthalene: a chain-like structure composed of face-to-face type dimeric mol­ecular aggregates

PubMed Central

Siqingaowa; Tsumuki, Takehiro; Ogata, Kazuki; Yonezawa, Noriyuki; Okamoto, Akiko

2016-01-01

The asymmetric unit of the title compound, C27H20O3, contains two independent mol­ecules (A and B). The anthracene ring system is connected to the 2,7-di­meth­oxy­naphthalene core in a twisted manner, with dihedral angles of 86.38 (5) and 79.36 (8)°, respectively, for conformers A and B. In the crystal, face-to-face type dimeric mol­ecular aggregates of each conformer are observed. The dimer of conformer A is formed by two pairs of C—H⋯π inter­actions, and that of conformer B by a pair of (sp 2)C—H⋯O hydrogen bonds. The dimers of conformer A are linked to each other via a π–π stacking inter­action between the anthracene rings to form a chain along the b axis and the chains are aligned along the c axis, forming a sheet structure. The dimers of conformer B are connected to each other via a couple of C—H⋯π inter­actions to form a chain along the b axis. The chains are aligned along the c axis through (sp 2)C—H⋯O=C hydrogen bonds, forming a sheet parallel to the bc plane. The sheets of conformers A and B are alternately stacked along the a axis via two kinds of inter­molecular (sp 2)C—H⋯O=C hydrogen bonds. PMID:27980839

Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography.

PubMed

Andersen, J P; Vilsen, B; Nielsen, H; Møller, J V

1986-10-21

Sarcoplasmic reticulum Ca2+-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca2+-ATPase occurred within a few hours in the presence of less than or equal to 50 microM Ca2+. The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high Ca2+ concentration (500 microM), monomeric Ca2+-ATPase was stable for several hours. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca2+-ATPase was found to be 10(5)-10(6) M-1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca2+-ATPase, even above the critical micellar concentration of the detergent. Binding of Ca2+ and vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. These results suggest that formation of Ca2+-ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit.

One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions.

PubMed

Hvasanov, David; Nam, Ekaterina V; Peterson, Joshua R; Pornsaksit, Dithepon; Wiedenmann, Jörg; Marquis, Christopher P; Thordarson, Pall

2014-10-17

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Energetic Coupling between Ligand Binding and Dimerization in E. coli Phosphoglycerate Mutase

PubMed Central

Gardner, Nathan W.; Monroe, Lyman K.; Kihara, Daisuke; Park, Chiwook

2016-01-01

Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding in an exceptional quality. The homodimeric E. coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. In the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active-site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization. PMID:26919584

Structural Mechanism for the Temperature-Dependent Activation of the Hyperthermophilic Pf2001 Esterase.

PubMed

Varejão, Nathalia; De-Andrade, Rafael A; Almeida, Rodrigo V; Anobom, Cristiane D; Foguel, Debora; Reverter, David

2018-02-06

Lipases and esterases constitute a group of enzymes that catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70°C and 80°C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Yu, Bingke; Cheng, Hui-Chun; Brautigam, Chad A.

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data leadmore » to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.« less

A HeI photoelectron spectrum of the [Al(CH 3) 3] 2 dimer

NASA Astrophysics Data System (ADS)

Wang, Dianxun; Qian, Ximei; Zheng, Shijun; Shi, Yizhong

1997-10-01

The HeI photoelectron spectrum (PES) of the [Al(CH 3) 3] 2 dimer is recorded for the first time. To assign the PES bands, an ab initio SCF MO calculation for the dimer has also been performed. The four splitting peaks of the first PE band are respectively designated to electron ionization of the four frontier 8b u, 13a g, 7b g, and 7b u orbitals of the dimer. They originate from the recombination of the two HOMO (5e') of the two monomers in the forming of the dimer. That is to say, during the formation of the dimer from the two monomers, the reduction of molecular symmetry (from the C 3h symmetry of the monomer to the C 2h symmetry of the dimer) leads to the undegeneration of the degenerate orbitals.

Spectrin tetramer-dimer equilibrium and the stability of erythrocyte membrane skeletons

NASA Astrophysics Data System (ADS)

Liu, Shih-Chun; Palek, Jiri

1980-06-01

The inner side of the red-cell membrane is laminated by a two-dimensional network of membrane proteins which include spectrin, actin and some other components1-4. After extraction of lipids and integral proteins from the membrane, this membrane skeleton can be visualized as a ball-shaped network consisting of twisted fibres1-4 and globular protrusions4; however, the assembly of the individual proteins in the membrane skeleton is not well understood. Spectrin can be eluted from the membrane in the form of dimers and tetramers5-8. Electron microscopic study with low-angle shadowing technique shows that spectrin dimers are two parallel strands of twisted fibres presumably representing bands 1 and 2 of spectrin9. Spectrin tetramers presumably formed by head-to-head associations of two dimers are twice as long9. In solution, the spectrin dimer-tetramer equilibrium depends on temperature and salt concentration7,8; however, it is not known whether the same equilibrium exists in the membrane and whether it affects the physical properties of the membrane, such as its structural stability and deformability. We now demonstrate that spectrin dimers and tetramers are in a reversible equilibrium in the membrane and that in physiological conditions this equilibrium favours spectrin tetramers. Furthermore, we show that transformation of spectrin tetramers to dimers, as induced by ghost incubation in hypotonic conditions, diminishes the structural stability of the Triton-insoluble membrane skeletons.

Crosslinking Evidence for Motional Constraints within Chemoreceptor Trimers of Dimers

PubMed Central

Massazza, Diego A.; Parkinson, John S.; Studdert, Claudia A.

2011-01-01

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In E. coli, chemoreceptors show higher order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer of dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo crosslinking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed crosslinks, whereas reporters lying at different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernable effect on the crosslinking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced crosslinking at most of the reporter sites, indicating that individual dimers may move closer together under this condition. PMID:21174433

Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

PubMed Central

Smith, Alexander C.

2012-01-01

Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

Detectability of Noble Gases in Jovian Atmospheres Utilizing Dimer Spectral Structures

NASA Astrophysics Data System (ADS)

Kim, S. J.; Min, Y.; Kim, Y.; Lee, Y.; Trafton, L.; Miller, S.; McKellar, A. R. W.

1997-07-01

The detection of jovian hydrogen-hydrogen dimers through the clear telluric 2-micron window (Kim et al. 1995; Trafton et al. 1997) suggests possibility to detect noble gases in the form of dimer with hydrogen in jovian atmospheres. Since noble gases do not have spectral structures in the infrared, it has been difficult to derive their abundances in the atmospheres of jovian planets. If there is a significant component of noble gases other than helium in the jovian atmospheres, it might be detected through its dimer spectrum with hydrogen molecule. The relatively sharp spectral structures of hydrogen-argon and hydrogen-neon dimers compared with those of hydrogen-hydrogen dimers are useful for the detection, if adequate S/N is obtained. However, these dimer structures should be much weaker than the nearby hydrogen-hydrogen features because noble gases are expected to be minor constituents of these atmospheres. We will discuss the detectability of these dimers based on laboratory measurements (McKellar, 1994; 1996), and current technology of infrared observations.

A Hamiltonian replica exchange method for building protein-protein interfaces applied to a leucine zipper

NASA Astrophysics Data System (ADS)

Cukier, Robert I.

2011-01-01

Leucine zippers consist of alpha helical monomers dimerized (or oligomerized) into alpha superhelical structures known as coiled coils. Forming the correct interface of a dimer from its monomers requires an exploration of configuration space focused on the side chains of one monomer that must interdigitate with sites on the other monomer. The aim of this work is to generate good interfaces in short simulations starting from separated monomers. Methods are developed to accomplish this goal based on an extension of a previously introduced [Su and Cukier, J. Phys. Chem. B 113, 9595, (2009)] Hamiltonian temperature replica exchange method (HTREM), which scales the Hamiltonian in both potential and kinetic energies that was used for the simulation of dimer melting curves. The new method, HTREM_MS (MS designates mean square), focused on interface formation, adds restraints to the Hamiltonians for all but the physical system, which is characterized by the normal molecular dynamics force field at the desired temperature. The restraints in the nonphysical systems serve to prevent the monomers from separating too far, and have the dual aims of enhancing the sampling of close in configurations and breaking unwanted correlations in the restrained systems. The method is applied to a 31-residue truncation of the 33-residue leucine zipper (GCN4-p1) of the yeast transcriptional activator GCN4. The monomers are initially separated by a distance that is beyond their capture length. HTREM simulations show that the monomers oscillate between dimerlike and monomerlike configurations, but do not form a stable interface. HTREM_MS simulations result in the dimer interface being faithfully reconstructed on a 2 ns time scale. A small number of systems (one physical and two restrained with modified potentials and higher effective temperatures) are sufficient. An in silico mutant that should not dimerize because it lacks charged residues that provide electrostatic stabilization of the dimer does not with HTREM_MS, giving confidence in the method. The interface formation time scale is sufficiently short that using HTREM_MS as a screening tool to validate leucine zipper design methods may be feasible.

4-N, 4-S & 4-O Chloroquine Analogues: Influence of Side Chain Length and Quinolyl Nitrogen pKa on Activity vs. Chloroquine Resistant Malaria+, #

PubMed Central

Natarajan, Jayakumar K.; Alumasa, John; Yearick, Kimberly; Ekoue-Kovi, Kekeli A.; Casabianca, Leah B.; de Dios, Angel C.; Wolf, Christian; Roepe, Paul D.

2009-01-01

Using predictions from heme – quinoline antimalarial complex structures, previous modifications of chloroquine (CQ), and hypotheses for chloroquine resistance (CQR), we synthesize and assay CQ analogues that test structure – function principles. We vary side chain length for both monoethyl and diethyl 4N CQ derivatives. We alter the pKa of the quinolyl N by introducing alkylthio or alkoxy substituents into the 4 position, and vary side chain length for these analogues. We introduce an additional titratable amino group to the side chain of 4O analogues with promising CQR strain selectivity and increase activity while retaining selectivity. We solve atomic resolution structures for complexes formed between representative 4N, 4S and 4O derivatives vs. μ-oxo dimeric heme, measure binding constants for monomeric vs. dimeric heme, and quantify hemozoin (Hz) formation inhibition in vitro. The data provide additional insight for the design of CQ analogues with improved activity vs. CQR malaria. PMID:18512900

4-N-, 4-S-, and 4-O-chloroquine analogues: influence of side chain length and quinolyl nitrogen pKa on activity vs chloroquine resistant malaria.

PubMed

Natarajan, Jayakumar K; Alumasa, John N; Yearick, Kimberly; Ekoue-Kovi, Kekeli A; Casabianca, Leah B; de Dios, Angel C; Wolf, Christian; Roepe, Paul D

2008-06-26

Using predictions from heme-quinoline antimalarial complex structures, previous modifications of chloroquine (CQ), and hypotheses for chloroquine resistance (CQR), we synthesize and assay CQ analogues that test structure-function principles. We vary side chain length for both monoethyl and diethyl 4-N CQ derivatives. We alter the pKa of the quinolyl N by introducing alkylthio or alkoxy substituents into the 4 position and vary side chain length for these analogues. We introduce an additional titratable amino group to the side chain of 4-O analogues with promising CQR strain selectivity and increase activity while retaining selectivity. We solve atomic resolution structures for complexes formed between representative 4-N, 4-S, and 4-O derivatives vs mu-oxo dimeric heme, measure binding constants for monomeric vs dimeric heme, and quantify hemozoin (Hz) formation inhibition in vitro. The data provide additional insight for the design of CQ analogues with improved activity vs CQR malaria.

Surprising Impact of Remote Groups on the Folding-Unfolding and Dimer-Chain Equilibria of Bifunctionl H-Bonding Unimers

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

Liu, Rui; Cheng, Shuang; Baker, Erin Shammel

2016-01-28

Oligoamide 1, consisting of two H-bonding units linked by a trimethylene linker, was previously found to form a very stable, folded dimer. In this work, replacing the side chains and end groups of 1 led to derivatives that show the surprising impact of end groups on the folding and dimer-chain equilibria of the resultant molecules.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-05-11

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed Central

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-01-01

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process. Images PMID:1645868

Synthesis of the iron phthalocyaninate radical cation μ-nitrido dimer and its interaction with hydrogen peroxide

NASA Astrophysics Data System (ADS)

Grishina, E. S.; Makarova, A. S.; Kudrik, E. V.; Makarov, S. V.; Koifman, O. I.

2016-03-01

The iron phthalocyaninate μ-nitrido dimer radical cation, as well as the μ-nitrido dimer complexes of iron phthalocyaninate, was found to have high catalytic activity in the oxidation of organic compounds. It was concluded that this compound is of interest as a model of active intermediates—catalase and oxidase enzymes.

Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

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

Goffinont, S.; Davidkova, M.; Spotheim-Maurizot, M., E-mail: spotheim@cnrs-orleans.fr

2009-08-21

The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro {gamma}-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucinemore » residues of the tetramerization domain.« less

Theoretical investigation on the 2e/12c bond and second hyperpolarizability of azaphenalenyl radical dimers: strength and effect of dimerization.

PubMed

Zhong, Rong-Lin; Xu, Hong-Liang; Sun, Shi-Ling; Qiu, Yong-Qing; Zhao, Liang; Su, Zhong-Min

2013-09-28

An increasing number of chemists have focused on the investigations of two-electron/multicenter bond (2e/mc) that was first introduced to describe the structure of radical dimers. In this work, the dimerization of two isoelectronic radicals, triazaphenalenyl (TAP) and hexaazaphenalenyl (HAP) has been investigated in theory. Results show TAP2 is a stable dimer with stronger 2e/12c bond and larger interaction energy, while HAP2 is a less stable dimer with larger diradical character. Interestingly, the ultraviolet-visible absorption spectra suggest that the dimerization induces a longer wavelength absorption in visible area, which is dependent on the strength of dimerization. Significantly, the amplitude of second hyperpolarizability (γ(yyyy)) of HAP2 is 1.36 × 10(6) a.u. that is larger than 7.79 × 10(4) a.u. of TAP2 because of the larger diradical character of HAP2. Therefore, the results indicate that the strength of radical dimerization can be effectively detected by comparing the magnitude of third order non-linear optical response, which is beneficial for further theoretical and experimental studies on the properties of complexes formed by radical dimerization.

Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning

PubMed Central

Rondard, Philippe; Huang, Siluo; Monnier, Carine; Tu, Haijun; Blanchard, Bertrand; Oueslati, Nadia; Malhaire, Fanny; Li, Ying; Trinquet, Eric; Labesse, Gilles; Pin, Jean-Philippe; Liu, Jianfeng

2008-01-01

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABAB1 and GABAB2. GABAB1 binds agonists, whereas GABAB2 is required for trafficking GABAB1 to the cell surface, increasing agonist affinity to GABAB1, and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABAB1 VFT leads to GABAB2 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABAB VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABAB2, including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation. PMID:18388862

Structural History of Human SRGAP2 Proteins

PubMed Central

Sporny, Michael; Guez-Haddad, Julia; Kreusch, Annett; Shakartzi, Sivan; Neznansky, Avi; Cross, Alice; Isupov, Michail N.; Qualmann, Britta; Kessels, Michael M.

2017-01-01

Abstract In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4–2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A’s inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history. PMID:28333212

Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

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

Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu

Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

Pancake π–π Bonding Goes Double: Unexpected 4e/All-Sites Bonding in Boron- and Nitrogen-Doped Phenalenyls

DOE PAGES

Tian, Yong-Hui; Sumpter, Bobby G.; Du, Shiyu; ...

2015-06-03

Phenalenyl is an important neutral pi-radical due to its capability to form unconventional pancake pi-pi bonding interactions, whereas its analogues with graphitic boron (B) or nitrogen (N)-doping have been regarded as closed-shell systems and therefore received much less attention. By using high-level quantum chemistry calculations, we also show that the B- and N-doped closed-shell phenalenyls unexpectedly form open-shell singlet pi-dimers with diradicaloid character featuring 2e/all-sites double pi-pi bonding. Moreover, by proper substitutions, the doped phenalenyl derivatives can be made open-shell species that form closed shell singlet pi-dimers bound by stronger 4e/all-sites double pi-pi bonding. Moreover, covalent pi-pi bonding overlap ismore » distributed on all of the atomic sites giving robust and genuine pancake-shaped pi-dimers which, depending on the number of electrons available in the bonding interactions, are equally or more stable than the pi-dimers of the pristine phenalenyl.« less

G-Quadruplex Induction by the Hairpin Pyrrole-Imidazole Polyamide Dimer.

PubMed

Obata, Shunsuke; Asamitsu, Sefan; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2018-02-06

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole-imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy.

Dimeric arrangement and structure of the membrane-bound acetylcholine receptor studied by electron microscopy.

PubMed Central

Zingsheim, H P; Neugebauer, D C; Frank, J; Hänicke, W; Barrantes, F J

1982-01-01

The acetylcholine receptor protein (AChR) from the electric organ of Torpedo marmorata is studied in its membrane-bound form by electron microscopy and single-particle image averaging. About half the molecule protrudes from the membrane surface by approximately 5 nm. The low-resolution 3-D structure of this hydrated portion, including its handedness, can be deduced from averaged axial and lateral projections and from freeze-etched membrane surfaces. In native membrane fragments, a dimeric form of the AChR is observed and the relative orientation of the AChR monomers within the dimer is established. The dimers disappear upon disulfide reduction of the membrane preparations, whereas the average axial projections of the AChR monomer remain unaffected. Since the existence of disulfide bonds linking AChR monomers between their respective delta-subunits is well documented, the approximate position of the delta-subunit within the low-resolution structure of the AChR molecule can be deduced from the structure of the dimers. Images Fig. 1. Fig. 2. Fig. 3. PMID:7188351

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

NASA Astrophysics Data System (ADS)

Wijeratne, Sithara S.; Li, Jingqiang; Yeh, Hui-Chun; Nolasco, Leticia; Zhou, Zhou; Bergeron, Angela; Frey, Eric W.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

2016-01-01

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100 dynes/cm2 for 3 min at 37°C ). We conclude that under the shear conditions used (100 dynes/cm2 for 3 min at 37°C ) , VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

Experimental and theoretical IR study of methyl thioglycolate, CH3OC(O)CH2SH, in different phases: Evidence of a dimer formation

NASA Astrophysics Data System (ADS)

Bava, Yanina B.; Tamone, Luciana M.; Juncal, Luciana C.; Seng, Samantha; Tobón, Yeny A.; Sobanska, Sophie; Picone, A. Lorena; Romano, Rosana M.

2017-07-01

The IR spectrum of methyl thioglycolate (MTG) was studied in three different phases, and interpreted with the aid of DFT calculations. The gas phase IR spectrum was explainable by the presence of the most stable conformer (syn-gauche-(-)gauche) only, while the IR spectrum of the liquid reveals strong intermolecular interactions, coincident with the formation of a dimeric form. The matrix-isolated spectra allow the identification of the second conformer (syn-gauche-gauche), in addition to the most stable form. The MTG dimer was also isolated by increasing the proportion of MTG in the matrix. The theoretical most stable structure of the dimer, which calculated IR spectrum agrees very well with the experimental one, is stabilized by a double interaction of the lone pair of the O atom of each of the Cdbnd O groups with the antibonding orbitals σ* (Ssbnd H).

Studies on the Effect of Sub-zero Temperatures on the Formation of Extremely Low Volatility Dimer Esters in Secondary Organic Aerosol from Alpha-Pinene

NASA Astrophysics Data System (ADS)

Kristensen, Kasper; Normann Jensen, Louise; Bilde, Merete

2016-04-01

The oxidation of volatile organic compounds (VOC) is considered a major source of secondary organic aerosols (SOA) in the atmosphere. Recently, extremely low volatility organic compounds, or ELVOC, formed from the oxidation of VOCs have been shown to play a crucial role in new particle formation (Ehn et al., 2014). In addition, higher molecular weight dimer esters originating from the oxidation of the biogenic VOC alpha-pinene have been observed in both laboratory-generated and ambient SOA (Kristensen et al., 2013). The low volatility of the dimer esters along with an observed rapid formation makes these high molecular weight compounds likely candidates involved in new particle formation from the oxidation of alpha-pinene. Furthermore, laboratory experiments show that the dimer esters only form in the presence of ozone, thus may be used as tracers for the ozone-initiated oxidation of alpha-pinene, and are therefore indicative of enhanced anthropogenic activities. In this work, we present the results of a series of oxidation experiments performed in the newly constructed cold-room smog chamber at Aarhus University. This unique and state-of-the-art Teflon chamber allows for atmospheric simulations of the oxidation VOCs and subsequent SOA formation at temperatures down to -16 °C. In this study, ozonolysis and photochemical oxidations of alpha-pinene are performed at temperatures ranging from +20 to -16 °C. Chemical characterization of the formed SOA is performed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The results show significant differences in the chemical composition related to the experiment temperature. In particularly, the concentration of the high molecular weight dimer esters showed to be highly affected by temperature. Interestingly, preliminary results show higher formation of dimer esters related to increased SOA formation rate, thus indicating that these particle-phase ELVOCs may be linked with new particle formation. Ehn, M., Thornton, J. A., Kleist, E., Sipila, M., Junninen, H., Pullinen, I., Springer, M., Rubach, F., Tillmann, R., Lee, B., Lopez-Hilfiker, F., Andres, S., Acir, I. H., Rissanen, M., Jokinen, T., Schobesberger, S., Kangasluoma, J., Kontkanen, J., Nieminen, T., Kurten, T., Nielsen, L. B., Jorgensen, S., Kjaergaard, H. G., Canagaratna, M., Dal Maso, M., Berndt, T., Petaja, T., Wahner, A., Kerminen, V. M., Kulmala, M., Worsnop, D. R., Wildt, J., and Mentel, T. F.: A large source of low-volatility secondary organic aerosol, Nature, 506, 476-+, 10.1038/nature13032, 2014. Kristensen, K., Enggrob, K. L., King, S. M., Worton, D. R., Platt, S. M., Mortensen, R., Rosenoern, T., Surratt, J. D., Bilde, M., Goldstein, A. H., and Glasius, M.: Formation and occurrence of dimer esters of pinene oxidation products in atmospheric aerosols, Atmospheric Chemistry and Physics, 13, 3763-3776, 2013.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed

Darlix, J L; Gabus, C; Allain, B

1992-12-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed Central

Darlix, J L; Gabus, C; Allain, B

1992-01-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein. Images PMID:1331519

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

PubMed Central

2011-01-01

Background The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. Results The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. Conclusions The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds. PMID:21486470

Conformational Changes in the Epidermal Growth Factor Receptor: Role of the Transmembrane Domain Investigated by Coarse-Grained MetaDynamics Free Energy Calculations

PubMed Central

2016-01-01

The epidermal growth factor receptor (EGFR) is a dimeric membrane protein that regulates key aspects of cellular function. Activation of the EGFR is linked to changes in the conformation of the transmembrane (TM) domain, brought about by changes in interactions of the TM helices of the membrane lipid bilayer. Using an advanced computational approach that combines Coarse-Grained molecular dynamics and well-tempered MetaDynamics (CG-MetaD), we characterize the large-scale motions of the TM helices, simulating multiple association and dissociation events between the helices in membrane, thus leading to a free energy landscape of the dimerization process. The lowest energy state of the TM domain is a right-handed dimer structure in which the TM helices interact through the N-terminal small-X3-small sequence motif. In addition to this state, which is thought to correspond to the active form of the receptor, we have identified further low-energy states that allow us to integrate with a high level of detail a range of previous experimental observations. These conformations may lead to the active state via two possible activation pathways, which involve pivoting and rotational motions of the helices, respectively. Molecular dynamics also reveals correlation between the conformational changes of the TM domains and of the intracellular juxtamembrane domains, paving the way for a comprehensive understanding of EGFR signaling at the cell membrane. PMID:27459426

Conformational Changes in the Epidermal Growth Factor Receptor: Role of the Transmembrane Domain Investigated by Coarse-Grained MetaDynamics Free Energy Calculations.

PubMed

Lelimousin, Mickaël; Limongelli, Vittorio; Sansom, Mark S P

2016-08-24

The epidermal growth factor receptor (EGFR) is a dimeric membrane protein that regulates key aspects of cellular function. Activation of the EGFR is linked to changes in the conformation of the transmembrane (TM) domain, brought about by changes in interactions of the TM helices of the membrane lipid bilayer. Using an advanced computational approach that combines Coarse-Grained molecular dynamics and well-tempered MetaDynamics (CG-MetaD), we characterize the large-scale motions of the TM helices, simulating multiple association and dissociation events between the helices in membrane, thus leading to a free energy landscape of the dimerization process. The lowest energy state of the TM domain is a right-handed dimer structure in which the TM helices interact through the N-terminal small-X3-small sequence motif. In addition to this state, which is thought to correspond to the active form of the receptor, we have identified further low-energy states that allow us to integrate with a high level of detail a range of previous experimental observations. These conformations may lead to the active state via two possible activation pathways, which involve pivoting and rotational motions of the helices, respectively. Molecular dynamics also reveals correlation between the conformational changes of the TM domains and of the intracellular juxtamembrane domains, paving the way for a comprehensive understanding of EGFR signaling at the cell membrane.

The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme

PubMed Central

Lartigue, Audrey; Burlat, Bénédicte; Coutard, Bruno; Chaspoul, Florence; Claverie, Jean-Michel

2014-01-01

ABSTRACT Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. IMPORTANCE Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs. PMID:25355875

Heterogenous fluorescence decay of (4→6)-and (4→8)-linked dimers of (+)-catechin and (-) epicatechin as a result of rotational isomerism.

Treesearch

Wolfgang R. Bergmann; Mary D. Barkley; Richard W. Hemingway; Wayne Mattice

1987-01-01

The time-resolved fluorescence of (+)-catechin and ( -)-epicatechin decays as a single exponential. In contrast dimers formed from (+)-catechin and (-)-epicatechin have more complex decays unless rotation about the interflavan bond is constrained by the introduction of a new ring. The fluorescence decay in unconstrained dimers is adequately described by the sum of two...

Fibrinolytic activity in cerebrospinal fluid of dogs with different neurological disorders.

PubMed

de la Fuente, C; Monreal, L; Cerón, J; Pastor, J; Viu, J; Añor, S

2012-01-01

Fibrinolytic activity in cerebrospinal fluid (CSF) is activated in humans by different pathologic processes. To investigate fibrinolytic activity in the CSF of dogs with neurological disorders by measuring CSF D-dimer concentrations. One hundred and sixty-nine dogs with neurological disorders, 7 dogs with systemic inflammatory diseases without central nervous system involvement (SID), and 7 healthy Beagles were included in the study. Dogs with neurological disorders included 11 with steroid-responsive meningitis-arteritis (SRMA), 37 with other inflammatory neurological diseases (INF), 38 with neoplasia affecting the central nervous system (NEO), 28 with spinal compressive disorders (SCC), 15 with idiopathic epilepsy (IE), and 40 with noninflammatory neurological disorders (NON-INF). Prospective observational study. D-dimers and C-reactive protein (CRP) were simultaneously measured in paired CSF and blood samples. D-dimers and CRP were detected in 79/183 (43%) and in 182/183 (99.5%) CSF samples, respectively. All dogs with IE, SID, and controls had undetectable concentrations of D-dimers in the CSF. CSF D-dimer concentrations were significantly (P < .001) higher in dogs with SRMA than in dogs with other diseases and controls. CSF CRP concentration in dogs with SRMA was significantly (P < .001) higher than in dogs of other groups and controls, except for the SID group. No correlation was found between blood and CSF D-dimer concentrations. Intrathecal fibrinolytic activity seems to be activated in some canine neurological disorders, and it is high in severe meningeal inflammatory diseases. CSF D-dimer concentrations may be considered a diagnostic marker for SRMA. Copyright © 2012 by the American College of Veterinary Internal Medicine.

The biochemistry and immunology of non-canonical forms of HLA-B27.

PubMed

Shaw, Jacqueline; Hatano, Hiroko; Kollnberger, Simon

2014-01-01

HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 is expressed at the cell surface of antigen presenting cells (APC) both as canonical β2m-associated and non-canonical β2m-free heavy chain (FHC) forms which include B27 dimers (termed B272). B27 FHC forms arise in an endosomal compartment from recycling β2m-associated B27. Formation of cell surface FHC dimers is critically dependent on an unpaired reactive cysteine 67 in the α1 helix of the class I heavy chain. HLA-B27 also form redox-inducible β2m-associated dimers on exosomes and apoptosing cells. By contrast with cell surface expressed cysteine 67-dependent heavy chain dimers these dimers are dependent on a cytoplasmic cysteine 325 for their formation. HLA-B27 binds to immunoregulatory receptors including members of the Killer cell Immunoglobulin-like (KIR) and Leukocyte Immunoglobulin-like receptor family. B27 FHC bind to different but overlapping sets of these immunoreceptors compared to classical β2m-associated HLA-B27. B27 FHC bind more strongly to KIR3DL2 and LILRB2 immune receptor than other β2m-associated HLA-class I ligands. Genetic studies have implicated genes which control production of the important proinflammatory cytokine IL-17 in the pathogenesis of spondyloarthritis. Cell surface HLA-B27 FHC binding to these immune receptors or acting through other mechanisms could impact on the pathogenesis of spondyloarthritis by promoting immune cell production of IL-17. Here we review the literature on these non-canonical forms of HLA-B27 and the immune receptors they bind to and discuss the possible relevance of these interactions to the pathogenesis of spondyloarthropathy. Copyright © 2013 Elsevier Ltd. All rights reserved.

X-ray crystallographic analysis of the sulfur carrier protein SoxY from Chlorobium limicola f. thiosulfatophilum reveals a tetrameric structure

PubMed Central

Stout, Jan; Van Driessche, Gonzalez; Savvides, Savvas N.; Van Beeumen, Jozef

2007-01-01

Dissimilatory oxidation of thiosulfate in the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum is carried out by the ubiquitous sulfur-oxidizing (Sox) multi-enzyme system. In this system, SoxY plays a key role, functioning as the sulfur substrate-binding protein that offers its sulfur substrate, which is covalently bound to a conserved C-terminal cysteine, to another oxidizing Sox enzyme. Here, we report the crystal structures of a stand-alone SoxY protein of C. limicola f. thiosulfatophilum, solved at 2.15 Å and 2.40 Å resolution using X-ray diffraction data collected at 100 K and room temperature, respectively. The structure reveals a monomeric Ig-like protein, with an N-terminal α-helix, that oligomerizes into a tetramer via conserved contact regions between the monomers. The tetramer can be described as a dimer of dimers that exhibits one large hydrophobic contact region in each dimer and two small hydrophilic interface patches in the tetramer. At the tetramer interface patch, two conserved redox-active C-terminal cysteines form an intersubunit disulfide bridge. Intriguingly, SoxY exhibits a dimer/tetramer equilibrium that is dependent on the redox state of the cysteines and on the type of sulfur substrate component bound to them. Taken together, the dimer/tetramer equilibrium, the specific interactions between the subunits in the tetramer, and the significant conservation level of the interfaces strongly indicate that these SoxY oligomers are biologically relevant. PMID:17327392

Electric-field-induced assembly and propulsion of chiral colloidal clusters.

PubMed

Ma, Fuduo; Wang, Sijia; Wu, David T; Wu, Ning

2015-05-19

Chiral molecules with opposite handedness exhibit distinct physical, chemical, or biological properties. They pose challenges as well as opportunities in understanding the phase behavior of soft matter, designing enantioselective catalysts, and manufacturing single-handed pharmaceuticals. Microscopic particles, arranged in a chiral configuration, could also exhibit unusual optical, electric, or magnetic responses. Here we report a simple method to assemble achiral building blocks, i.e., the asymmetric colloidal dimers, into a family of chiral clusters. Under alternating current electric fields, two to four lying dimers associate closely with a central standing dimer and form both right- and left-handed clusters on a conducting substrate. The cluster configuration is primarily determined by the induced dipolar interactions between constituent dimers. Our theoretical model reveals that in-plane dipolar repulsion between petals in the cluster favors the achiral configuration, whereas out-of-plane attraction between the central dimer and surrounding petals favors a chiral arrangement. It is the competition between these two interactions that dictates the final configuration. The theoretical chirality phase diagram is found to be in excellent agreement with experimental observations. We further demonstrate that the broken symmetry in chiral clusters induces an unbalanced electrohydrodynamic flow surrounding them. As a result, they rotate in opposite directions according to their handedness. Both the assembly and propulsion mechanisms revealed here can be potentially applied to other types of asymmetric particles. Such kinds of chiral colloids will be useful for fabricating metamaterials, making model systems for both chiral molecules and active matter, or building propellers for microscale transport.

Comparison of the catalytic activity for the Suzuki–Miyaura reaction of (η5-Cp)Pd(IPr)Cl with (η3-cinnamyl)Pd(IPr)(Cl) and (η3-1-t-Bu-indenyl)Pd(IPr)(Cl)

PubMed Central

Melvin, Patrick R; Lant, Hannah M C; Peczak, Ian L; Shah, Hemali P

2015-01-01

Summary Complexes of the type (η3-allyl)Pd(L)(Cl) and (η3-indenyl)Pd(L)(Cl) are highly active precatalysts for the Suzuki–Miyaura reaction. Even though allyl and indenyl ligands are similar to cyclopentadienyl (Cp) ligands, there have been no detailed comparative studies exploring the activity of precatalysts of the type (η5-Cp)Pd(L)(Cl) for Suzuki–Miyaura reactions. Here, we compare the catalytic activity of (η5-Cp)Pd(IPr)(Cl) (IPr = 1,3-bis(2,6-diisopropylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene, Cp) with two commercially available catalysts (η3-cinnamyl)Pd(IPr)(Cl) (Cin) and (η3-1-t-Bu-indenyl)Pd(IPr)(Cl) (tBu Ind). We show that Cp gives slightly better catalytic activity than Cin, but significantly inferior activity than tBu Ind. This order of activity is rationalized by comparing the rates at which the precatalysts are activated to the monoligated Pd(0) active species along with the tendency of the starting precatalysts to comproportionate with monoligated Pd(0) to form inactive Pd(I) dimers. As part of this work the Cp supported Pd(I) dimer (μ-Cp)(μ-Cl)Pd2(IPr)2 (Cp Dim) was synthesized and crystallographically characterized. It does not readily disproportionate to form monoligated Pd(0) and consequently Cp Dim is a poor catalyst for the Suzuki–Miyaura reaction. PMID:26732227

IDENTIFICATION OF NF-KAPPAB IN THE MARINE FISH STENOTOMUS CHRYSOPS AND EXAMINATION OF ITS ACTIVATION BY ARYL HYDROCARBON RECEPTOR AGONISTS. (R827102)

EPA Science Inventory

Abstract

Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor Maturation of hepatic lipase. Formation of functional enzyme in the endoplasmic reticulum is the rate-limiting step in its secretion.

PubMed

Ben-Zeev, Osnat; Doolittle, Mark H

2004-02-13

Among three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t(1/2) = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme.

Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

PubMed Central

2015-01-01

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer–dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and 1H{15N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The “closed loop” form of the A–B and G–H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer. PMID:25489673

Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

DOE PAGES

Berry, Robert E.; Yang, Fei; Shokhireva, Tatiana K.; ...

2014-12-09

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a K d of ~8 μM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer–dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0more » and 1H{ 15N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The “closed loop” form of the A–B and G–H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. Lastly, the homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.« less

Energy landscapes of the monomer and dimer of the Alzheimer's peptide A β (1 -28 )

NASA Astrophysics Data System (ADS)

Dong, Xiao; Chen, Wei; Mousseau, Normand; Derreumaux, Philippe

2008-03-01

The cytoxicity of Alzheimer's disease has been linked to the self-assembly of the 40 /42 amino acid of the amyloid-β (A β ) peptide into oligomers. To understand the assembly process, it is important to characterize the very first steps of aggregation at an atomic level of detail. Here, we focus on the N-terminal fragment 1-28, known to form fibrils in vitro. Circular dichroism and NMR experiments indicate that the monomer of A β (1 -28 ) is α -helical in a membranelike environment and random coil in aqueous solution. Using the activation-relaxation technique coupled with the OPEP coarse grained force field, we determine the structures of the monomer and of the dimer of A β (1 -28 ) . In agreement with experiments, we find that the monomer is predominantly random coil in character, but displays a non-negligible β -strand probability in the N-terminal region. Dimerization impacts the structure of each chain and leads to an ensemble of intertwined conformations with little β -strand content in the region Leu17-Ala21. All these structural characteristics are inconsistent with the amyloid fibril structure and indicate that the dimer has to undergo significant rearrangement en route to fibril formation.

The use of serum free light chain dimerization patterns assist in the diagnosis of AL amyloidosis.

PubMed

Gatt, Moshe E; Kaplan, Batia; Yogev, Dean; Slyusarevsky, Elana; Pogrebijski, Galina; Golderman, Sizilia; Kukuy, Olga; Livneh, Avi

2018-05-16

The discrimination between benign and malignant forms of plasma cell dyscrasia (PCD) is often difficult. Free light chain monomer-dimer pattern analysis (FLC-MDPA) may assist in solving this dilemma and distinguish between AL amyloidosis and benign PCD. Serum samples of patients with AL amyloidosis and benign PCD were analysed in a blinded manner. Quantitative Western blotting was performed to estimate dimerization and clonality indices, and thereby determine the source of the tested samples, as derived either from benign or malignant PCD. The findings obtained by the FLC-MDPA were compared with the actual diagnosis. Of 37 samples from patients with active AL amyloidosis, 34 (91·9%) fulfilled dimerization criteria for diagnosis of AL amyloidosis. Of the 45 samples from patients with benign PCD, 10 (21·2%) tested falsely positive or gave an inconclusive result. Thus, the sensitivity of the analysis was 92·5% with a remarkable negative predictive value of 91·9%. In addition, of 20 patients who were in complete or very good partial remission, only one tested positive. By multivariate analysis, FLC-MDPA was the best independent marker predicting AL amyloidosis (odds ratio of 84). The FLC-MDPA offers a highly effective tool in the diagnostic assessment of patients with PCD. © 2018 John Wiley & Sons Ltd.

Crystallization of multiple forms of bovine seminal ribonuclease in the liganded and unliganded state

NASA Astrophysics Data System (ADS)

Sica, F.; Adinolfi, S.; Berisio, R.; De Lorenzo, C.; Mazzarella, L.; Piccoli, R.; Vitagliano, L.; Zagari, A.

1999-01-01

Bovine seminal ribonuclease (BS-RNase) is an intriguing homodimeric enzyme which exists as two conformational isomers, characterized by distinct catalytic and biological properties, referred to as M×M and M=M. Reduction of inter-chain disulfide bridges produces a stable monomeric derivative (M) which is still active. This paper reports the screening and optimization of crystallization conditions for growing single diffraction-quality crystals for the various BS-RNase forms. The crystallization trials were performed using both the vapor diffusion and microbatch methods. The M×M dimer was crystallized in the free form from polyethylene glycol (PEG) 4000 at pH 8.5 and as a complex with the substrate analog uridylyl(2'- 5')guanosine (UpG) from an unbuffered ammonium sulfate (AS) solution. These two crystal types diffract X-rays to 2.5 and 1.9 Å resolution, respectively. Two different crystal types were obtained both for the M=M dimer and for the monomeric derivative. (M=M)a crystals, grown from PEG 4000 (8% w/v) at pH 5.6, diffract X-rays to 4.0 Å. At higher PEG concentration (15% w/v) a different crystal type was obtained, (M=M)b, which showed a better diffraction limit (2.5 Å). For the monomer, type (M)a and (M)b crystals, diffracting X-rays to 2.5 Å resolution, were obtained from AS at pH 6.5 and from PEG 4000 at pH 8.5, respectively. A comparison with previously crystallized forms of the dimer M×M and its complexes with uridylyl(2'-5')adenosine and 2'-deoxycytidylyl(3'-5')-2'-deoxyadenosine is also presented. The three-dimensional structure analysis of (M×M)·UpG and (M=M)b is in progress.

Structural Basis for a Reciprocating Mechanism of Negative Cooperativity in Dimeric Phosphagen Kinase Activity

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

Wu, X.; Ye, S; Guo, S

Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 {angstrom}, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widelymore » than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase. - Wu, X., Ye, S., Guo, S., Yan, W., Bartlam, M., Rao, Z. Structural basis for a reciprocating mechanism of negative cooperativity in dimeric phosphagen kinase activity.« less

Fluxional σ-Bonds of the 2,5,8-Trimethylphenalenyl Dimer: Direct Observation of the Sixfold σ-Bond Shift via a π-Dimer.

PubMed

Uchida, Kazuyuki; Mou, Zhongyu; Kertesz, Miklos; Kubo, Takashi

2016-04-06

Direct evidence for σ-bond fluxionality in a phenalenyl σ-dimer was successfully obtained by a detailed investigation of the solution-state dynamics of 2,5,8-trimethylphenalenyl (TMPLY) using both experimental and theoretical approaches. TMPLY formed three diamagnetic dimers, namely, the σ-dimer (RR/SS), σ-dimer (RS), and π-dimer, which were fully characterized by (1)H NMR spectroscopy and electronic absorption measurements. The experimental findings gave the first quantitative insights into the essential preference of these competitive and unusual dimerization modes. The spectroscopic analyses suggested that the σ-dimer (RR/SS) is the most stable in terms of energy, whereas the others are metastable; the energy differences between these three isomers are less than 1 kcal mol(-1). Furthermore, the intriguing dynamics of the TMPLY dimers in the solution state were fully revealed by means of (1)H-(1)H exchange spectroscopy (EXSY) measurements and variable-temperature (1)H NMR studies. Surprisingly, the σ-dimer (RR/SS) demonstrated a sixfold σ-bond shift between the six sets of α-carbon pairs. This unusual σ-bond fluxionality is ascribed to the presence of a direct interconversion pathway between the σ-dimer (RR/SS) and the π-dimer, which was unambiguously corroborated by the EXSY measurements. The proposed mechanism of the sixfold σ-bond shift based on the experimental findings was well-supported by theoretical calculations.

Synthesis of novel ring-contracted artemisinin dimers with potent anticancer activities.

PubMed

Zhang, Ning; Yu, Zhimei; Yang, Xiaohong; Hu, Ping; He, Yun

2018-04-25

Artemisinin is a potential anticancer agent with an interesting trioxane sesquiterpene structure. In order to improve the biological activity and metabolic stability of artemisinin, a series of novel ring-contracted artemisinin dimers were synthesized. These dimers were evaluated by MTT assay against six cancer cell lines. Most of the dimmers exhibited improved antiproliferative activities over artemisinin. Especially, compound 8b showed the most pronounced anti-cancer activity for PC12 cancer cells with an IC 50 value of 1.56 μM. Thus, PC12 cancer cells were used to further investigate the mechanism of antiproliferation for this series of compounds. Compound 8b arrested cell cycle at G1 phase and induced cell apoptosis via up-regulation of Bad, Bax, caspase-3 and caspase-9 protein expressions while inhibiting the expression of Bcl-xL. The present studies are the first to synthesize the ring-contracted artemisinin as dimers and show that these dimers have potent anti-tumor activities against several cancer cell lines. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Glycolaldehyde Formation via the Dimerization of the Formyl Radical

NASA Astrophysics Data System (ADS)

Woods, Paul M.; Slater, Ben; Raza, Zamaan; Viti, Serena; Brown, Wendy A.; Burke, Daren J.

2013-11-01

Glycolaldehyde, the simplest monosaccharide sugar, has recently been detected in low- and high-mass star-forming cores. Following our previous investigation into glycolaldehyde formation, we now consider a further mechanism for the formation of glycolaldehyde that involves the dimerization of the formyl radical, HCO. Quantum mechanical investigation of the HCO dimerization process upon an ice surface is predicted to be barrierless and therefore fast. In an astrophysical context, we show that this mechanism can be very efficient in star-forming cores. It is limited by the availability of the formyl radical, but models suggest that only very small amounts of CO are required to be converted to HCO to meet the observational constraints.

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

PubMed Central

Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

PubMed

Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

Activation helix orientation of the estrogen receptor is mediated by receptor dimerization: evidence from molecular dynamics simulations.

PubMed

Fratev, Filip

2015-05-28

In recent years, the nuclear receptors (NR) dynamics have been studied extensively by various approaches. However, the transition path of helix 12 (H12) to an agonist or an antagonist conformation and the exchange pathway between these states is not clear yet. A number of accelerated molecular dynamics (aMD) runs were performed on both an ERα monomer and a homodimer with a total length of 2.2 μs. We have been able to sample reasonably well the H12 conformational landscape to reproduce precisely both the agonist and the antagonist conformations, starting from an unfolded position, and to describe the transition path between them, even in the presence of an agonist ligand. These conformations were the most prevalent, suggesting that the extended H12 state is not likely to exist and that the natural ERα H12 position might exist in both the agonist and antagonist states. Remarkably, the H12 transition occurs and is regulated only in a dimer form and the proper agonist or antagonist H12 conformation can be achieved solely in one of the dimer subunits. These results clearly demonstrate that clusters of the two well-known H12 states exist by themselves in the protein free energy landscape, i.e. they are not constituted directly by the ligands, and dimerization favors the switch between them. Conversely, in a monomer, no transitions have been observed. Thus, the dimer formation helps the constitution of populations of discrete H12 conformational states and reshapes the conformational landscape. Further analyses have shown that these observations can be explained by specific interface and long range protein-protein interactions, resulting in conformational fluctuations in helices 5 and 11. Based on these results, a new ERα activation/deactivation mechanism and a sequence of binding events during receptor activity modulation have been suggested according to which ligands control the H12 conformation via alterations of the inter-dimer interactions. These findings agree with the HDX and fluorescence experiments and provide an explanation on a structural basis of these data, demonstrating that the dynamics of H12 are not altered greatly upon ligand binding and large fluctuations at the end of H11 are present.

Regulation of Oligomeric Organization of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Observed by Spatial Intensity Distribution Analysis*

PubMed Central

Ward, Richard J.; Pediani, John D.; Godin, Antoine G.; Milligan, Graeme

2015-01-01

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor. PMID:25825490

A mechanism for the activation of the Na/H exchanger NHE-1 by cytoplasmic acidification and mitogens

PubMed Central

Lacroix, Jérôme; Poët, Mallorie; Maehrel, Céline; Counillon, Laurent

2004-01-01

Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na+/H+ exchanger NHE-1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton-sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE-1 activation is best described by a Monod–Wyman–Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low-affinity form of NHE-1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton-sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high-affinity form. PMID:14710192

Simulation-Based Validation of the p53 Transcriptional Activity with Hybrid Functional Petri Net.

PubMed

Doi, Atsushi; Nagasaki, Masao; Matsuno, Hiroshi; Miyano, Satoru

2011-01-01

MDM2 and p19ARF are essential proteins in cancer pathways forming a complex with protein p53 to control the transcriptional activity of protein p53. It is confirmed that protein p53 loses its transcriptional activity by forming the functional dimer with protein MDM2. However, it is still unclear that protein p53 keeps its transcriptional activity when it forms the trimer with proteins MDM2 and p19ARF. We have observed mutual behaviors among genes p53, MDM2, p19ARF and their products on a computational model with hybrid functional Petri net (HFPN) which is constructed based on information described in the literature. The simulation results suggested that protein p53 should have the transcriptional activity in the forms of the trimer of proteins p53, MDM2, and p19ARF. This paper also discusses the advantages of HFPN based modeling method in terms of pathway description for simulations.

Predictors of 30-day mortality and the risk of recurrent systemic thromboembolism in cancer patients suffering acute ischemic stroke.

PubMed

Nam, Ki-Woong; Kim, Chi Kyung; Kim, Tae Jung; An, Sang Joon; Oh, Kyungmi; Mo, Heejung; Kang, Min Kyoung; Han, Moon-Ku; Demchuk, Andrew M; Ko, Sang-Bae; Yoon, Byung-Woo

2017-01-01

Stroke in cancer patients is not rare but is a devastating event with high mortality. However, the predictors of mortality in stroke patients with cancer have not been well addressed. D-dimer could be a useful predictor because it can reflect both thromboembolic events and advanced stages of cancer. In this study, we evaluate the possibility of D-dimer as a predictor of 30-day mortality in stroke patients with active cancer. We included 210 ischemic stroke patients with active cancer. The 30-day mortality data were collected by reviewing medical records. We also collected follow-up D-dimer levels in 106 (50%) participants to evaluate the effects of treatment response on D-dimer levels. Of the 210 participants, 30-day mortality occurred in 28 (13%) patients. Higher initial NIHSS scores, D-dimer levels, and CRP levels as well as frequent cryptogenic mechanism, systemic metastasis, multiple vascular territory lesion, hemorrhagic transformation, and larger infarct volume were related to 30-day mortality. In the multivariate analysis, D-dimer [adjusted OR (aOR) = 2.19; 95% CI, 1.46-3.28, P < 0.001] predicted 30-day mortality after adjusting for confounders. The initial NIHSS score (aOR = 1.07; 95% CI, 1.00-1.14, P = 0.043) and hemorrhagic transformation (aOR = 3.02; 95% CI, 1.10-8.29, P = 0.032) were also significant independent of D-dimer levels. In the analysis of D-dimer changes after treatment, the mortality group showed no significant decrease in D-dimer levels, despite treatment, while the survivor group showed the opposite response. D-dimer levels may predict 30-day mortality in acute ischemic stroke patients with active cancer.

Predictors of 30-day mortality and the risk of recurrent systemic thromboembolism in cancer patients suffering acute ischemic stroke

PubMed Central

Kim, Tae Jung; An, Sang Joon; Oh, Kyungmi; Mo, Heejung; Kang, Min Kyoung; Han, Moon-Ku; Demchuk, Andrew M.; Ko, Sang-Bae; Yoon, Byung-Woo

2017-01-01

Background Stroke in cancer patients is not rare but is a devastating event with high mortality. However, the predictors of mortality in stroke patients with cancer have not been well addressed. D-dimer could be a useful predictor because it can reflect both thromboembolic events and advanced stages of cancer. Aim In this study, we evaluate the possibility of D-dimer as a predictor of 30-day mortality in stroke patients with active cancer. Methods We included 210 ischemic stroke patients with active cancer. The 30-day mortality data were collected by reviewing medical records. We also collected follow-up D-dimer levels in 106 (50%) participants to evaluate the effects of treatment response on D-dimer levels. Results Of the 210 participants, 30-day mortality occurred in 28 (13%) patients. Higher initial NIHSS scores, D-dimer levels, and CRP levels as well as frequent cryptogenic mechanism, systemic metastasis, multiple vascular territory lesion, hemorrhagic transformation, and larger infarct volume were related to 30-day mortality. In the multivariate analysis, D-dimer [adjusted OR (aOR) = 2.19; 95% CI, 1.46–3.28, P < 0.001] predicted 30-day mortality after adjusting for confounders. The initial NIHSS score (aOR = 1.07; 95% CI, 1.00–1.14, P = 0.043) and hemorrhagic transformation (aOR = 3.02; 95% CI, 1.10–8.29, P = 0.032) were also significant independent of D-dimer levels. In the analysis of D-dimer changes after treatment, the mortality group showed no significant decrease in D-dimer levels, despite treatment, while the survivor group showed the opposite response. Conclusions D-dimer levels may predict 30-day mortality in acute ischemic stroke patients with active cancer. PMID:28282388

DNA's Encounter with Ultraviolet Light: An Instinct for Self-Preservation?

PubMed

Barlev, Adam; Sen, Dipankar

2018-02-20

Photochemical modification is the major class of environmental damage suffered by DNA, the genetic material of all free-living organisms. Photolyases are enzymes that carry out direct photochemical repair (photoreactivation) of covalent pyrimidine dimers formed in DNA from exposure to ultraviolet light. The discovery of catalytic RNAs in the 1980s led to the "RNA world hypothesis", which posits that early in evolution RNA or a similar polymer served both genetic and catalytic functions. Intrigued by the RNA world hypothesis, we set out to test whether a catalytic RNA (or a surrogate, a catalytic DNA) with photolyase activity could be contemplated. In vitro selection from a random-sequence DNA pool yielded two DNA enzymes (DNAzymes): Sero1C, which requires serotonin as an obligate cofactor, and UV1C, which is cofactor-independent and optimally uses light of 300-310 nm wavelength to repair cyclobutane thymine dimers within a gapped DNA substrate. Both Sero1C and UV1C show multiple turnover kinetics, and UV1C repairs its substrate with a quantum yield of ∼0.05, on the same order as the quantum yields of certain classes of photolyase enzymes. Intensive study of UV1C has revealed that its catalytic core consists of a guanine quadruplex (G-quadruplex) positioned proximally to the bound substrate's thymine dimer. We hypothesize that electron transfer from photoexcited guanines within UV1C's G-quadruplex is responsible for substrate photoreactivation, analogous to electron transfer to pyrimidine dimers within a DNA substrate from photoexcited flavin cofactors located within natural photolyase enzymes. Though the analogy to evolution is necessarily limited, a comparison of the properties of UV1C and Sero1C, which arose out of the same in vitro selection experiment, reveals that although the two DNAzymes comparably accelerate the rate of thymine dimer repair, Sero1C has a substantially broader substrate repertoire, as it can repair many more kinds of pyrimidine dimers than UV1C. Therefore, the co-opting of an amino acid-like cofactor by a nucleic acid enzyme in this case contributes functional versatility rather than a greater rate enhancement. In recent work on UV1C, we have succeeded in shifting its action spectrum from the UVB into the blue region of the spectrum and determined that although it catalyzes both repair and de novo formation of thymine dimers, UV1C is primarily a catalyst for thymine dimer repair. Our work on photolyase DNAzymes has stimulated broader questions about whether analogous, purely nucleotide-based photoreactivation also occurs in double-helical DNA, the dominant form of DNA in living cells. Recently, a number of different groups have reported that this kind of repair is indeed operational in DNA duplexes, i.e., that there exist nucleotide sequences that actively protect, by way of photoreactivation (rather than by simply preventing their formation), pyrimidine dimers located proximal to them. Nucleotide-based photoreactivation thus appears to be a salient, if unanticipated, property of DNA and RNA. The phenomenon also offers pointers in the direction of how in primordial evolution-in an RNA world-early nucleic acids may have protected themselves from structural and functional damage wrought by ultraviolet light.

Efficient renaturation of inclusion body proteins denatured by SDS.

PubMed

He, Chuan; Ohnishi, Kouhei

2017-09-02

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Diethyl [(4-bromo­phen­yl)(5-chloro-2-hydroxy­anilino)meth­yl]phospho­nate

PubMed Central

Babu, V. H. H. Surendra; Krishnaiah, M.; Prasad, G. Syam; C. Suresh Reddy; Kant, Rajni

2009-01-01

In the title compound, C17H20BrClNO4P, inter­molecular C—H⋯O and N—H⋯O hydrogen bonds form centrosymmetric R 2 2(10) dimers linked through O—H⋯O inter­molecular hydrogen bonds, which form centrosymmetric R 2 2(16) dimers. All these hydrogen bonds form chains along [010]. In addition, the crystal structure is stabilized by weak C—H⋯Br hydrogen bonds. The very weak intramolecular N—H⋯O interaction forms a five-membered ring. PMID:21578446

A novel dimeric thymosin beta 4 with enhanced activities accelerates the rate of wound healing

PubMed Central

Xu, Tian-Jiao; Wang, Qi; Ma, Xiao-Wen; Zhang, Zhen; Zhang, Wei; Xue, Xiao-Chang; Zhang, Cun; Hao, Qiang; Li, Wei-Na; Zhang, Ying-Qi; Li, Meng

2013-01-01

Objective Thymosin beta 4 (Tβ4) is a peptide with 43 amino acids that is critical for repair and remodeling tissues on the skin, eye, heart, and neural system following injury. To fully realize its utility as a treatment for disease caused by injury, the authors constructed a cost-effective novel Tβ4 dimer and demonstrated that it was better able to accelerate tissue repair than native Tβ4. Methods A prokaryotic vector harboring two complete Tβ4 genes with a short linker was constructed and expressed in Escherichia coli. A pilot-scale fermentation (10 L) was performed to produce engineered bacteria and the Tβ4 dimer was purified by one-step hydrophobic interaction chromatography. The activities of the Tβ4 dimer to promote endothelial cell proliferation, migration, and sprouting were assessed by tetramethylbenzidine (methylthiazol tetrazolium), trans-well, scratch, and tube formation assays. The ability to accelerate dermal healing was assessed on rats. Results After fermentation, the Tβ4 dimer accounted for about 30% of all the bacteria proteins. The purity of the Tβ4 dimer reached 98% after hydrophobic interaction chromatography purification. An average of 562.4 mg/L Tβ4 dimer was acquired using a 10 L fermenter. In each assay, the dimeric Tβ4 exhibited enhanced activities compared with native Tβ4. Notably, the ability of the dimeric Tβ4 to promote cell migration was almost two times higher than that of Tβ4. The rate of dermal healing in the dimeric Tβ4-treated rats was approximately 1 day faster than with native Tβ4-treated rats. Conclusion The dimeric Tβ4 exhibited enhanced activity on wound healing than native Tβ4, and the purification process was simple and cost-effective. This data could be of significant benefit for the high pain and morbidity associated with chronic wounds disease. A better strategy to develop Tβ4 as a treatment for other diseases caused by injuries such as heart attack, neurotrophic keratitis, and multiple sclerosis was also described. PMID:24109178

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

PubMed

Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V; Burgers, Peter M; Zhang, Xiaodong

2016-06-24

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Refined structure of dimeric diphtheria toxin at 2.0 A resolution.

PubMed Central

Bennett, M. J.; Choe, S.; Eisenberg, D.

1994-01-01

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed. PMID:7833807

Interactions with the Bifunctional Interface of the Transcriptional Coactivator DCoH1 Are Kinetically Regulated

DOE PAGES

Wang, Dongli; Coco, Matthew W.; Rose, Robert B.

2014-12-23

Pterin-4a-carbinolamine dehydratase (PCD) is a highly conserved enzyme that evolved a second, unrelated function in mammals, as a transcriptional coactivator. As a coactivator, PCD is known as DCoH or dimerization cofactor of the transcription factor HNF-1. These two activities are associated with a change in oligomeric state: from two dimers interacting as an enzyme in the cytoplasm to a dimer interacting with a dimer of HNF-1 in the nucleus. The same interface of DCoH forms both complexes. To determine how DCoH partitions between its two functions, we studied in this paper the folding and stability of the DCoH homotetramer. Wemore » show that the DCoH1 homotetramer is kinetically trapped, meaning once it forms it will not dissociate to interact with HNF-1. In contrast, DCoH2, a paralog of DCoH1, unfolds within hours. A simple mutation in the interface of DCoH2 from Ser-51 to Thr, as found in DCoH1, increases the kinetic stability by 9 orders of magnitude, to τ½ ~2 million years. This suggests that the DCoH1·HNF-1 complex must co-fold to interact. We conclude that simple mutations can dramatically affect the dissociation kinetics of a complex. Residue 51 represents a “kinetic hot spot” instead of a “thermodynamic hot spot.” Kinetic regulation allows PCD to adopt two distinct functions. Finally, mutations in DCoH1 associated with diabetes affect both functions of DCoH1, perhaps by disrupting the balance between the two DCoH complexes.« less

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E*

PubMed Central

Siddiqui, Shoib; Schwarz, Flavio; Springer, Stevan; Khedri, Zahra; Yu, Hai; Deng, Lingquan; Verhagen, Andrea; Naito-Matsui, Yuko; Jiang, Weiping; Kim, Daniel; Zhou, Jie; Ding, Beibei; Chen, Xi; Varki, Nissi; Varki, Ajit

2017-01-01

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer. PMID:27920204

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation*

PubMed Central

Macdonald-Obermann, Jennifer L.; Pike, Linda J.

2014-01-01

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. PMID:25086039

Crystal structure of cbbF from Zymomonas mobilis and its functional implication

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

Hwang, Hyo-Jeong; Park, Suk-Youl; Kim, Jeong-Sun, E-mail: jsunkim@chonnam.ac.kr

2014-02-28

Highlights: • The crystal structure of one cbbF from Zymomonas mobilis was revealed. • Scores of residues form two secondary structures with a non-polar protruded residue. • It exists as a dimeric form in solution. - Abstract: A phosphate group at the C1-atom of inositol-monophosphate (IMP) and fructose-1,6-bisphosphate (FBP) is hydrolyzed by a phosphatase IMPase and FBPase in a metal-dependent way, respectively. The two enzymes are almost indiscernible from each other because of their highly similar sequences and structures. Metal ions are bound to residues on the β1- and β2-strands and one mobile loop. However, FBP has another phosphate andmore » FBPases exist as a higher oligomeric state, which may discriminate FBPases from IMPases. There are three genes annotated as FBPases in Zymomonas mobilis, termed also cbbF (ZmcbbF). The revealed crystal structure of one ZmcbbF shows a globular structure formed by five stacked layers. Twenty-five residues in the middle of the sequence form an α-helix and a β-strand, which occupy one side of the catalytic site. A non-polar Leu residue among them is protruded to the active site, pointing out unfavorable access of a bulky charged group to this side. In vitro assays have shown its dimeric form in solution. Interestingly, two β-strands of β1 and β2 are disordered in the ZmcbbF structure. These data indicate that ZmcbbF might structurally belong to IMPase, and imply that its active site would be reorganized in a yet unreported way.« less

Insights into Strand Exchange in BTB Domain Dimers from the Crystal Structures of FAZF and Miz1

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

Stogios, Peter J.; Cuesta-Seijo, Jose Antonio; Chen, Lu

2010-09-22

The BTB domain is a widely distributed protein-protein interaction motif that is often found at the N-terminus of zinc finger transcription factors. Previous crystal structures of BTB domains have revealed tightly interwound homodimers, with the N-terminus from one chain forming a two-stranded anti-parallel {beta}-sheet with a strand from the other chain. We have solved the crystal structures of the BTB domains from Fanconi anemia zinc finger (FAZF) and Miz1 (Myc-interacting zinc finger 1) to resolutions of 2.0 {angstrom} and 2.6 {angstrom}, respectively. Unlike previous examples of BTB domain structures, the FAZF BTB domain is a nonswapped dimer, with each N-terminalmore » {beta}-strand associated with its own chain. As a result, the dimerization interface in the FAZF BTB domain is about half as large as in the domain-swapped dimers. The Miz1 BTB domain resembles a typical swapped BTB dimer, although it has a shorter N-terminus that is not able to form the interchain sheet. Using cysteine cross-linking, we confirmed that the promyelocytic leukemia zinc finger (PLZF) BTB dimer is strand exchanged in solution, while the FAZF BTB dimer is not. A phylogenic tree of the BTB fold based on both sequence and structural features shows that the common ancestor of the BTB domain in BTB-ZF (bric a brac, tramtrack, broad-complex zinc finger) proteins was a domain-swapped dimer. The differences in the N-termini seen in the FAZF and Miz1 BTB domains appear to be more recent developments in the structural evolution of the domain.« less

Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer.

PubMed

Bhattarai, Nisha; Gc, Jeevan B; Gerstman, Bernard S; Stahelin, Robert V; Chapagain, Prem P

2017-04-26

Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane.

Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure

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

Korasick, David A.; Singh, Harkewal; Pemberton, Travis A.

2017-08-01

Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case (Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significancemore » of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain-swapped dimer as the core structural and functional unit of type A PutAs.« less

Structure of an electric double layer containing a 2:2 valency dimer electrolyte

DOE PAGES

Silvestre-Alcantara, Whasington; Henderson, Douglas; Wu, Jianzhong; ...

2014-12-05

In this study, the structure of a planar electric double layer formed by a 2:2 valency dimer electrolyte in the vicinity of a uniformly charged planar hard electrode is investigated using density functional theory and Monte Carlo simulations. The dimer electrolyte consists of a mixture of charged divalent dimers and charged divalent monomers in a dielectric continuum. A dimer is constructed by two tangentially tethered rigid spheres, one of which is divalent and positively charged and the other neutral, whereas the monomer is a divalent and negatively charged rigid sphere. The density functional theory reproduces well the simulation results formore » (i) the singlet distributions of the various ion species with respect to the electrode, and (ii) the mean electrostatic potential. Lastly, comparison with earlier results for a 2:1/1:2 dimer electrolyte shows that the double layer structure is similar when the counterion has the same valency.« less

Dimeric Matrine-Type Alkaloids from the Roots of Sophora flavescens and Their Anti-Hepatitis B Virus Activities.

PubMed

Zhang, Yu-Bo; Zhan, Li-Qin; Li, Guo-Qiang; Wang, Feng; Wang, Ying; Li, Yao-Lan; Ye, Wen-Cai; Wang, Guo-Cai

2016-08-05

Six unusual matrine-type alkaloid dimers, flavesines A-F (1-6, respectively), together with three proposed biosynthetic intermediates (7-9) were isolated from the roots of Sophora flavescens. Compounds 1-5 were the first natural matrine-type alkaloid dimers, and compound 6 represented an unprecedented dimerization pattern constructed by matrine and (-)-cytisine. Their structures were elucidated by NMR, MS, single-crystal X-ray diffraction, and a chemical method. The hypothetical biogenetic pathways of 1-6 were also proposed. Compounds 1-9 exhibited inhibitory activities against hepatitis B virus.

The residual repair capacity of xeroderma pigmentosum complementation group C fibroblasts is highly specific for transcriptionally active DNA.

PubMed Central

Venema, J; van Hoffen, A; Natarajan, A T; van Zeeland, A A; Mullenders, L H

1990-01-01

We have measured removal of pyrimidine dimers in defined DNA sequences in confluent and actively growing normal human and xeroderma pigmentosum complementation group C (XP-C) fibroblasts exposed to 10 J/m2 UV-irradiation. In normal fibroblasts 45% and 90% of the dimers are removed from the transcriptionally active adenosine deaminase (ADA) gene within 4 and 24 hours after irradiation respectively. Equal repair efficiencies are found in fragments located entirely within the transcription unit or partly in the 3' flanking region of the ADA gene. The rate and extent of dimer removal from the dihydrofolate reductase (DHFR) gene is very similar to that of the ADA gene. Repair of the transcriptionally inactive 754 locus is less efficient: 18% and 52% of the dimers are removed within 4 and 24 hours respectively. In spite of the limited overall repair capacity, confluent XP-C fibroblasts efficiently remove dimers from the ADA and DHFR genes: about 90% and 50% within 24 hours respectively. The 3' end of the ADA gene is repaired as efficiently as in normal human fibroblasts, but less efficient repair occurs in DNA fragments located in the DHFR gene and at the 5' end of the ADA gene. Repair of the inactive 754 locus does not exceed the very slow rate of dimer removal from the genome overall. Confluent and actively growing XP-C cells show similar efficiencies of repair of the ADA, DHFR and 754 genes. Our findings suggest the existence of two independently operating pathways directed towards repair of pyrimidine dimers in either active or inactive chromatin. XP-C cells have lost the capacity to repair inactive chromatin, but are still able to repair active chromatin. Images PMID:2308842

The Dimer Interface of the Membrane Type 1 Matrix Metalloproteinase Hemopexin Domain

PubMed Central

Tochowicz, Anna; Goettig, Peter; Evans, Richard; Visse, Robert; Shitomi, Yasuyuki; Palmisano, Ralf; Ito, Noriko; Richter, Klaus; Maskos, Klaus; Franke, Daniel; Svergun, Dmitri; Nagase, Hideaki; Bode, Wolfram; Itoh, Yoshifumi

2011-01-01

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion. PMID:21193411

The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions.

PubMed

Tochowicz, Anna; Goettig, Peter; Evans, Richard; Visse, Robert; Shitomi, Yasuyuki; Palmisano, Ralf; Ito, Noriko; Richter, Klaus; Maskos, Klaus; Franke, Daniel; Svergun, Dmitri; Nagase, Hideaki; Bode, Wolfram; Itoh, Yoshifumi

2011-03-04

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Artemisinin dimer anti-cancer activity correlates with heme-catalyzed ROS generation and ER stress induction

PubMed Central

Stockwin, Luke H.; Han, Bingnan; Yu, Sherry X.; Hollingshead, Melinda G.; ElSohly, Mahmoud A.; Gul, Waseem; Slade, Desmond; Galal, Ahmed M.; Newton, Dianne L.

2009-01-01

Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anti-cancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the anti-oxidant L-NAC, the iron chelator desferroxamine, and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity while inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage; iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian ER-resident SERCA Ca2+-ATPases. A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity. PMID:19533749

Molecular cloning and biochemical characterization of rabbit factor XI.

PubMed Central

Sinha, Dipali; Marcinkiewicz, Mariola; Gailani, David; Walsh, Peter N

2002-01-01

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160 kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80 kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process. PMID:12084014

The Hinge Region as a Key Regulatory Element of Androgen Receptor Dimerization, DNA Binding, and Transactivation

DTIC Science & Technology

2005-05-01

an impaired activity (see report of 2003). We obtained an EGFP fusion from Dr. Karen Knudsen (Ohio University, Cincinatti) in which a Gly-Ala linker ... Smad3 after its acetylation. The mutation of this lysine to glutamine or threonine (mimics acetylation), when expressed in DU145 cells promoted cell...forms. A Gly-Ala linker between the two proteins is necessary, since a direct fusion protein was largely impaired in its activity (not shown). 6. The

Effect of the achondroplasia mutation on FGFR3 dimerization and FGFR3 structural response to fgf1 and fgf2: A quantitative FRET study in osmotically derived plasma membrane vesicles

PubMed Central

Sarabipour, Sarvenaz; Hristova, Kalina

2016-01-01

The G380R mutation in the transmembrane domain of FGFR3 is a germline mutation responsible for most cases of Achondroplasia, a common form of human dwarfism. Here we use quantitative Föster Resonance Energy Transfer (FRET) and osmotically derived plasma membrane vesicles to study the effect of the achondroplasia mutation on the early stages of FGFR3 signaling in response to the ligands fgf1 and fgf2. Using a methodology that allows us to capture structural changes on the cytoplasmic side of the membrane in response to ligand binding to the extracellular domain of FGFR3, we observe no measurable effects of the G380R mutation on FGFR3 ligand-bound dimer configurations. Instead, the most notable effect of the achondroplasia mutation is increased propensity for FGFR3 dimerization in the absence of ligand. This work reveals new information about the molecular events that underlie the achondroplasia phenotype, and highlights differences in FGFR3 activation due to different single amino-acid pathogenic mutations. PMID:27040652

N-Benzyl­pyridin-2-amine

PubMed Central

Wang, Jun; Dai, Chuntao; Nie, Jianhua

2010-01-01

In the crystal of the title compound, C12H12N2, inter­molecular N—H⋯N hydrogen bonds form rings of graph-set motif R 2 2(8) and C—H⋯π inter­actions further consolidate the dimers. Neighbouring dimers are further connected into a three-dimensional network by C—H⋯π inter­actions. The benzyl and pyridyl rings form a dihedral angle of 67.2 (1)° PMID:21589385

Self-association and domain rearrangements between complement C3 and C3u provide insight into the activation mechanism of C3.

PubMed

Li, Keying; Gor, Jayesh; Perkins, Stephen J

2010-10-01

Component C3 is the central protein of the complement system. During complement activation, the thioester group in C3 is slowly hydrolysed to form C3u, then the presence of C3u enables the rapid conversion of C3 into functionally active C3b. C3u shows functional similarities to C3b. To clarify this mechanism, the self-association properties and solution structures of C3 and C3u were determined using analytical ultracentrifugation and X-ray scattering. Sedimentation coefficients identified two different dimerization events in both proteins. A fast dimerization was observed in 50 mM NaCl but not in 137 mM NaCl. Low amounts of a slow dimerization was observed for C3u and C3 in both buffers. The X-ray radius of gyration RG values were unchanged for both C3 and C3u in 137 mM NaCl, but depend on concentration in 50 mM NaCl. The C3 crystal structure gave good X-ray fits for C3 in 137 mM NaCl. By randomization of the TED (thioester-containing domain)/CUB (for complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains in the C3b crystal structure, X-ray fits showed that the TED/CUB domains in C3u are extended and differ from the more compact arrangement of C3b. This TED/CUB conformation is intermediate between those of C3 and C3b. The greater exposure of the TED domain in C3u (which possesses the hydrolysed reactive thioester) accounts for the greater self-association of C3u in low-salt conditions. This conformational variability of the TED/CUB domains would facilitate their interactions with a broad range of antigenic surfaces. The second dimerization of C3 and C3u may correspond to a dimer observed in one of the crystal structures of C3b.

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

Kuz'mina, L. G., E-mail: kuzmina@igic.ras.ru; Kucherepa, N. S.; Syrbu, S. A.

The crystal and molecular structure of p-(decaoxybenzylidene)-p'-toluidine C{sub 10}H{sub 21}O-C{sub 6}H{sub 4}-CH=N-C{sub 6}H{sub 4}-CH{sub 3} is studied. The molecule is nearly planar. In the crystal packing, loose regions formed by aliphatic fragments of molecules alternate with pseudostacks of aromatic fragments of molecules that are related by the centers of symmetry. The stacks are built of dimers, in which molecules are linked by {pi}-stacking interactions between benzene rings. There are no weak directional interactions between dimers in a stack. The presence of a single structure-forming element in the crystal, namely, the {pi}-stacking interactions in the dimers, along with the similarity ofmore » the crystal packing to that of the C{sub 8}H{sub 17}O-homologue, which forms a nematic mesophase on melting, indicate that the crystals under study should exhibit nematic properties.« less

Enzymatic Reaction with Unnatural Substrates: DNA Photolyase (Escherichia coli) Recognizes and Reverses Thymine [2+2] Dimers in the DNA Strand of a DNA/PNA Hybrid Duplex

NASA Astrophysics Data System (ADS)

Ramaiah, Danaboyina; Kan, Yongzhi; Koch, Troels; Orum, Henrik; Schuster, Gary B.

1998-10-01

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson--Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1).

PubMed

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W; Prestegard, James H

2016-09-16

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC'C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC'C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

PubMed Central

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

Sequential cancer immunotherapy: targeted activity of dimeric TNF and IL-8

PubMed Central

Adrian, Nicole; Siebenborn, Uta; Fadle, Natalie; Plesko, Margarita; Fischer, Eliane; Wüest, Thomas; Stenner, Frank; Mertens, Joachim C.; Knuth, Alexander; Ritter, Gerd; Old, Lloyd J.; Renner, Christoph

2009-01-01

Polymorphonuclear neutrophils (PMNs) are potent effectors of inflammation and their attempts to respond to cancer are suggested by their systemic, regional and intratumoral activation. We previously reported on the recruitment of CD11b+ leukocytes due to tumor site-specific enrichment of TNF activity after intravenous administration of a dimeric TNF immunokine with specificity for fibroblast activation protein (FAP). However, TNF-induced chemo-attraction and extravasation of PMNs from blood into the tumor is a multistep process essentially mediated by interleukin 8. With the aim to amplify the TNF-induced and IL-8-mediated chemotactic response, we generated immunocytokines by N-terminal fusion of a human anti-FAP scFv fragment with human IL-8 (IL-872) and its N-terminally truncated form IL-83-72. Due to the dramatic difference in chemotaxis induction in vitro, we favored the mature chemokine fused to the anti-FAP scFv for further investigation in vivo. BALB/c nu/nu mice were simultaneously xenografted with FAP-positive or -negative tumors and extended chemo-attraction of PMNs was only detectable in FAP-expressing tissue after intravenous administration of the anti-FAP scFv-IL-872 construct. As TNF-activated PMNs are likewise producers and primary targets for IL-8, we investigated the therapeutic efficacy of co-administration of both effectors: Sequential application of scFv-IL-872 and dimeric IgG1-TNF fusion proteins significantly enhanced anti-tumor activity when compared either to a single effector treatment regimen or sequential application of non-targeted cytokines, indicating that the tumor-restricted sequential application of IL-872 and TNF is a promising approach for cancer therapy. PMID:19267427

Molecular dynamics studies of the 3D structure and planar ligand binding of a quadruplex dimer.

PubMed

Li, Ming-Hui; Luo, Quan; Xue, Xiang-Gui; Li, Ze-Sheng

2011-03-01

G-rich sequences can fold into a four-stranded structure called a G-quadruplex, and sequences with short loops are able to aggregate to form stable quadruplex multimers. Few studies have characterized the properties of this variety of quadruplex multimers. Using molecular modeling and molecular dynamics simulations, the present study investigated a dimeric G-quadruplex structure formed from a simple sequence of d(GGGTGGGTGGGTGGGT) (G1), and its interactions with a planar ligand of a perylene derivative (Tel03). A series of analytical methods, including free energy calculations and principal components analysis (PCA), was used. The results show that a dimer structure with stacked parallel monomer structures is maintained well during the entire simulation. Tel03 can bind to the dimer efficiently through end stacking, and the binding mode of the ligand stacked with the 3'-terminal thymine base is most favorable. PCA showed that the dominant motions in the free dimer occur on the loop regions, and the presence of the ligand reduces the flexibility of the loops. Our investigation will assist in understanding the geometric structure of stacked G-quadruplex multimers and may be helpful as a platform for rational drug design.

Receptor signaling: when dimerization is not enough.

PubMed

Jiang, G; Hunter, T

Activation of receptors that signal via tyrosine kinase domains has been thought to involve receptor dimerization and transphosphorylation of juxtaposed catalytic domains. Recent results suggest things might be more complex - specific intersubunit conformational changes within a dimer can also be important.

Dehalogenation of Chlorinated Hydroxybiphenyls by Fungal Laccase

PubMed Central

Schultz, Asgard; Jonas, Ulrike; Hammer, Elke; Schauer, Frieder

2001-01-01

We have investigated the transformation of chlorinated hydroxybiphenyls by laccase produced by Pycnoporus cinnabarinus. The compounds used were transformed to sparingly water-soluble colored precipitates which were identified by gas chromatography-mass spectrometry as oligomerization products of the chlorinated hydroxybiphenyls. During oligomerization of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl, dechlorinated C—C-linked dimers were formed, demonstrating the dehalogenation ability of laccase. In addition to these nonhalogenated dimers, both monohalogenated and dihalogenated dimers were identified. PMID:11526052

Stereochemical investigations of a novel class of chiral phosphapalladacycle complexes derived from 1-[(2,5-dimethyl)phenyl]ethyldiphenylphosphine.

PubMed

Ng, Joseph Kok-Peng; Li, Yongxin; Tan, Geok-Kheng; Koh, Lip-Lin; Vittal, Jagadese J; Leung, Pak-Hing

2005-12-26

The phosphapalladacycle derived from 1-(2',5'-dimethylphenyl)ethyldiphenylphosphine has been prepared in the optically active and racemic forms. The phosphine was synthesized as a racemate by the treatment of 1-chloro-1-(2',5'-dimethylphenyl)ethane with sodium diphenylphosphide in THF. The racemic phosphapalladacycle was subsequently obtained as the chloro-bridged dimer by the treatment of the phosphine with palladium(II) acetate followed by anion metathesis with lithium chloride. Alternatively, the phosphine could be optically resolved via metal complexation using (R,R)-bis(mu-chloro)bis{1-[1-(N,N-dimethylamino)ethyl]naphthyl-C(2),N}dipalladium(II) as the resolving agent. An efficient separation of the resulting diastereomeric complexes was achieved by silica gel chromatography. The obtained optically resolved diastereomers were next subject to chemoselective removal of the (R)-N,N-(dimethylamino)-1-(1-naphthyl)ethylaminate auxiliary by treatment with concentrated hydrochloric acid. This process yielded the binuclear dimer complexes containing the resolved eta(1)-P ligand. Cyclopalladation of the coordinated phosphine could next be performed by treatment of its eta(1)-P binuclear dimer with silver(I) hexafluorophosphate(V) in a dichloromethane/water mixture followed by treatment with lithium chloride, giving rise to a pair of optically pure enantiomeric dimers with [alpha](D) -322 and +319 degrees in CH(2)Cl(2). Despite the possibilities of the phosphine to attain a five- membered structure by ortho-palladation or a six-membered ring formation by aliphatic C-H bond activation, only the former was observed. X-ray crystallographic data of the meso dimer and an acetylacetonate derivative indicated that the phosphapalladacycle alpha-C methyl substituent was axially located. The 2-D (1)H-(1)H ROESY spectrum of the acetylacetonate derivative further revealed that the phosphapalladacycle was conformationally rigid in CDCl(3).

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase

PubMed Central

Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N.; Braus, Gerhard H.

2002-01-01

The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212–226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212→Asp–Phe-28→Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase. PMID:11997452

Alignment and Imaging of the CS2 Dimer Inside Helium Nanodroplets

NASA Astrophysics Data System (ADS)

Pickering, James D.; Shepperson, Benjamin; Hübschmann, Bjarke A. K.; Thorning, Frederik; Stapelfeldt, Henrik

2018-03-01

The carbon disulphide (CS2) dimer is formed inside He nanodroplets and identified using fs laser-induced Coulomb explosion, by observing the CS2+ ion recoil velocity. It is then shown that a 160 ps moderately intense laser pulse can align the dimer in advantageous spatial orientations which allow us to determine the cross-shaped structure of the dimer by analysis of the correlations between the emission angles of the nascent CS2+ and S+ ions, following the explosion process. Our method will enable fs time-resolved structural imaging of weakly bound molecular complexes during conformational isomerization, including formation of exciplexes.

Estimation of quantum yields of weak fluorescence from eosin Y dimers formed in aqueous solutions.

PubMed

Enoki, Masami; Katoh, Ryuzi

2018-05-17

We studied the weak fluorescence from the dimer of eosin Y (EY) in aqueous solutions. We used a newly developed ultrathin optical cell with a thickness ranging from of the order of microns to several hundreds of microns to successfully measure the fluorescence spectra of highly concentrated aqueous solutions of EY without artifacts caused by the reabsorption of fluorescence. The spectra we obtained were similar to the fluorescence spectrum of the EY monomer; almost no fluorescence was observed from the EY dimer. By a careful comparison of the spectra of solutions at low and high concentrations of EY, we succeeded in extracting the fluorescence spectrum of the EY dimer. The fluorescence quantum yield of the EY dimer was estimated to be 0.005.

Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

PubMed Central

Juskaite, Victoria; Corcoran, David S; Leitinger, Birgit

2017-01-01

The collagen-binding receptor tyrosine kinase DDR1 (discoidin domain receptor 1) is a drug target for a wide range of human diseases, but the molecular mechanism of DDR1 activation is poorly defined. Here we co-expressed different types of signalling-incompetent DDR1 mutants (‘receiver’) with functional DDR1 (‘donor’) and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor function, we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver. The receiver ectodomain is not required, but phosphorylation in trans is abolished by mutation of the transmembrane domain. Finally, we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters. Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers. DOI: http://dx.doi.org/10.7554/eLife.25716.001 PMID:28590245

Crystal structure of benzyl (E)-2-(3,4-di-meth-oxy-benzyl-idene)hydrazine-1-carbodi-thio-ate.

PubMed

Tan, Yew-Fung; Break, Mohammed Khaled Bin; Tahir, M Ibrahim M; Khoo, Teng-Jin

2015-02-01

The title compound, C17H18N2O2S2, synthesized via a condensation reaction between S-benzyl di-thio-carbazate and 3,4-di-meth-oxy-benzaldehyde, crystallized with two independent mol-ecules (A and B) in the asymmetric unit. Both mol-ecules have an L-shape but differ in the orientation of the benzyl ring with respect to the 3,4-di-meth-oxy-benzyl-idine ring, this dihedral angle is 65.59 (8)° in mol-ecule A and 73.10 (8)° in mol-ecule B. In the crystal, the A and B mol-ecules are linked via pairs of N-H⋯S hydrogen bonds, forming dimers with an R 2 (2)(8) ring motif. The dimers are linked via pairs of C-H⋯O hydrogen bonds, giving inversion dimers of dimers. These units are linked by C-H⋯π inter-actions, forming ribbons propagating in the [100] direction.

On the self-association potential of transmembrane tight junction proteins.

PubMed

Blasig, I E; Winkler, L; Lassowski, B; Mueller, S L; Zuleger, N; Krause, E; Krause, G; Gast, K; Kolbe, M; Piontek, J

2006-02-01

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

Oxidation and formation of deposit precursors in hydrocarbon fuels

NASA Technical Reports Server (NTRS)

Buttrill, S. E., Jr.; Mayo, F. R.; Lan, B.; St.john, G. A.; Dulin, D.

1982-01-01

A practical fuel, home heating oil no. 2 (Fuel C), and the pure hydrocarbon, n-dodecane, were subjected to mild oxidation at 130 C and the resulting oxygenated reaction products, deposit precursors, were analyzed using field ionization mass spectrometry. Results for fuel C indicated that, as oxidation was initially extended, certain oxygenated reaction products of increasing molecular weights in the form of monomers, dimers and some trimers were produced. Further oxidation time increase resulted in further increase in monomers but a marked decrease in dimers and trimers. This suggests that these larger molecular weight products have proceeded to form deposit and separated from the fuel mixture. Results for a dodecane indicated that yields for dimers and trimers were very low. Dimers were produced as a result of interaction between oxygenated products with each other rather than with another fuel molecule. This occurred even though fuel molecule concentration was 50 times, or more greater than that for these oxygenated reaction products.

An X-ray structural study of pyruvate dehydrogenase kinase: A eukaryotic serine kinase with a prokaryotic histidine-kinase fold

NASA Astrophysics Data System (ADS)

Steussy, Calvin Nicklaus, Jr.

2001-07-01

Pyruvate Dehydrogenase Kinase is an enzyme that controls the flow of glucose through the eukaryotic cell and contributes to the pathology of diabetes mellitus. Early work on this kinase demonstrated that it has an amino acid sequence much like bacterial histidine kinases, but an activity similar to that of modern serine/threonine kinases. This project utilized the techniques of X-ray crystallography to determine molecular structure of pyruvate dehydrogenase kinase, isozyme 2. The structure was phased using selenium substituted for sulfur in methionine residues, and data at multiple wavelengths was collected at the National Synchrotron Light Source, Brookhaven National Laboratories. PDK 2 was found to fold into a two-domain monomer that forms a dimer through two beta sheets in the C-terminal domain. The N-terminal domain is an alpha-helical bundle while the C-terminal domain is an alpha/beta sandwich. The fold of the C-terminal domain is very similar to that of the prokaryotic histidine kinases, indicating that they share a common ancestor. The catalytic mechanism, however, has evolved to use general base catalysis to activate the serine substrate, rather than the direct nucleophilic attack by the imidazole sidechain used in the prokaryotic kinases. Thus, the structure of the protein echoes its prokaryotic ancestor, while the chemical mechanism has adapted to a serine substrate. The electrostatic surface of PDK2 leads to the suggestion that the lipoyl domain of the pyruvate dehydrogenase kinase, an important associated structure, may bind in the cleft formed between the N- and C-terminal domains. In addition, a network of hydrogen bonds directly connects the nucleotide binding pocket to the dimer interface, suggesting that there may be some interaction between dimer formation and ATP binding or ADP release.

Dihydroxo-bridged dimeric Cu(II) system containing sandwiched non-coordinating phenylacetate anion: Crystal structure, spectroscopic, anti-bacterial, anti-fungal and DNA-binding studies of [(phen)(H2O)Cu(OH)2Cu(H2O)(phen)]2L.6H2O: (HL = phenylacetic acid; phen = 1,10-phenanthroline)

NASA Astrophysics Data System (ADS)

Iqbal, Muhammad; Ali, Saqib; Tahir, Muhammad Nawaz; Shah, Naseer Ali

2017-09-01

This paper reports the synthesis, X-ray crystal structure, DNA-binding, antibacterial and antifungal studies of a rare dihydroxo-bridged dinuclear copper(II) complex including 1,10-phenanthroline (Phen) ligands and phenylacetate (L) anions, [Cu2(Phen)2(OH)2(H2O)2].2L.6H2O. Structural data revealed distorted square-pyramidal geometry for each copper(II) atom with the basal plane formed by the two nitrogen atoms of the phenantroline ligand and the oxygen atoms of two bridging hydroxyl groups. The apical positions are filled by the oxygen atom from a water molecule. This forms a centrosymmetric cationic dimer where the uncoordinated phenylacetate ligands serve to balance the electrical charge. The dimers interact by means of hydrogen bonds aided by the coordinated as well as uncoordinated water molecules and phenyl-acetate moieties in the crystal lattice. The binding ability of the complex with salmon sperm DNA was determined using cyclic voltammetry and absorption spectroscopy yielding binding constants 2.426 × 104 M-1 and 1.399 × 104 M-1, respectively. The complex was screened against two Gram-positive (Micrococcus luteus and Bacillus subtilis) and one Gram-negative (Escherichia coli) bacterial strains exhibiting significant activity against all the three strains. The complex exhibited significant, moderate and no activity against fungal strains Mucor piriformis, Helminthosporium solani and Aspergillus Niger, respectively. These preliminary tests indicate the competence of the complex towards the development of a potent biological drug.

High Molecular Weight Dimer Esters in α-Pinene Secondary Organic Aerosol

NASA Astrophysics Data System (ADS)

Kristensen, Kasper; Cui, Tianqu; Zhang, Haofei; Gold, Avram; Glasius, Marianne; Surratt, Jason D.

2014-05-01

Monoterpenes, such as α-pinene, constitute an important group of biogenic volatile organic compounds (BVOC). Once emitted into the atmosphere α-pinene is removed by oxidization by the hydroxyl radical (OH), reactions with ozone (O3), and with nitrate radicals (NO3) resulting in the formation of first-generation oxidation products, such as semi-volatile carboxylic acids. In addition, higher molecular weight dimer esters originating from the oxidation of α-pinene have been observed in both laboratory-generated and ambient secondary organic aerosols (SOA). While recent studies suggest that the dimers are formed through esterification between carboxylic acids in the particle phase, the formation mechanism of the dimer esters is still ambiguous. In this work, we present the results of a series of smog chamber experiments to assess the formation of dimer esters formed from the oxidation of α-pinene. Experiments were conducted in the University of North Carolina (UNC) dual outdoor smog chamber facility to investigate the effect of oxidant species (OH versus O3), relative humidity (RH), and seed aerosol acidity in order to obtain a better understanding of the conditions leading to the formation of the dimer esters and how these parameters may affect the formation and chemical composition of SOA. The chemical composition of α-pinene SOA was investigated by ultra-performance liquid chromatography/electrospray ionization high-resolution quadrupole time-of-flight mass spectrometry (UPLC/ESI-HR-Q-TOFMS), and a total of eight carboxylic acids and four dimer esters were identified, constituting between 8 and 12 % of the total α-pinene SOA mass.

Cerium(IV)-mediated oxidation of flavonol with relevance to flavonol 2,4-dioxygenase. Direct evidence for spin delocalization in the flavonoxy radical.

PubMed

Kaizer, József; Ganszky, Ildikó; Speier, Gábor; Rockenbauer, Antal; Korecz, László; Giorgi, Michel; Réglier, Marius; Antonczak, Serge

2007-06-01

The cerium(IV)-mediated oxidation of 3-hydroxy-4'-methylflavone (1) proceeds by H-atom abstraction forming the flavonoxy radical (7), and the subsequent combination of its resonance forms leads to the 3-hydroxy-4'-methylflavone dehydro dimer (9). The above system serves as direct evidence for the intermediacy of the flavonoxy radical, its spin delocalization, and also indirect evidence for valence tautomerism as a key step on the substrate activation both in the quercetinase and its biomimic model system.

Progress on New Hepatitis C Virus Targets: NS2 and NS5A

NASA Astrophysics Data System (ADS)

Marcotrigiano, Joseph

Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

PubMed

Peters, B P; Krzesicki, R F; Hartle, R J; Perini, F; Ruddon, R W

1984-12-25

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

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

Huang, Yongjian; Bharill, Shashank; Karandur, Deepti

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if thismore » is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.« less

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

DOE PAGES

Huang, Yongjian; Bharill, Shashank; Karandur, Deepti; ...

2016-03-28

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if thismore » is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.« less

Structure reveals regulatory mechanisms of a MaoC-like hydratase from Phytophthora capsici involved in biosynthesis of polyhydroxyalkanoates (PHAs).

PubMed

Wang, Huizheng; Zhang, Kai; Zhu, Jie; Song, Weiwei; Zhao, Li; Zhang, Xiuguo

2013-01-01

Polyhydroxyalkanoates (PHAs) have attracted increasing attention as "green plastic" due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R)-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic. We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R)-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC. The data in our study reveal the regulatory mechanism of an (R)-hydratase, providing information on enzyme engineering to produce low cost PHAs.

Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles*

PubMed Central

Nagpal, Latika; Haque, Mohammad M.; Saha, Amit; Mukherjee, Nirmalya; Ghosh, Arnab; Ranu, Brindaban C.; Stuehr, Dennis J.; Panda, Koustubh

2013-01-01

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases. PMID:23696643

Electric-field–induced assembly and propulsion of chiral colloidal clusters

PubMed Central

Ma, Fuduo; Wang, Sijia; Wu, David T.; Wu, Ning

2015-01-01

Chiral molecules with opposite handedness exhibit distinct physical, chemical, or biological properties. They pose challenges as well as opportunities in understanding the phase behavior of soft matter, designing enantioselective catalysts, and manufacturing single-handed pharmaceuticals. Microscopic particles, arranged in a chiral configuration, could also exhibit unusual optical, electric, or magnetic responses. Here we report a simple method to assemble achiral building blocks, i.e., the asymmetric colloidal dimers, into a family of chiral clusters. Under alternating current electric fields, two to four lying dimers associate closely with a central standing dimer and form both right- and left-handed clusters on a conducting substrate. The cluster configuration is primarily determined by the induced dipolar interactions between constituent dimers. Our theoretical model reveals that in-plane dipolar repulsion between petals in the cluster favors the achiral configuration, whereas out-of-plane attraction between the central dimer and surrounding petals favors a chiral arrangement. It is the competition between these two interactions that dictates the final configuration. The theoretical chirality phase diagram is found to be in excellent agreement with experimental observations. We further demonstrate that the broken symmetry in chiral clusters induces an unbalanced electrohydrodynamic flow surrounding them. As a result, they rotate in opposite directions according to their handedness. Both the assembly and propulsion mechanisms revealed here can be potentially applied to other types of asymmetric particles. Such kinds of chiral colloids will be useful for fabricating metamaterials, making model systems for both chiral molecules and active matter, or building propellers for microscale transport. PMID:25941383

Design of novel antimicrobial peptide dimer analogues with enhanced antimicrobial activity in vitro and in vivo by intermolecular triazole bridge strategy.

PubMed

Liu, Beijun; Huang, Haifeng; Yang, Zhibin; Liu, Beiyin; Gou, Sanhu; Zhong, Chao; Han, Xiufeng; Zhang, Yun; Ni, Jingman; Wang, Rui

2017-02-01

Currently, antimicrobial peptides have attracted considerable attention because of their broad-sprectum activity and low prognostic to induce antibiotic resistance. In our study, for the first time, a series of side-chain hybrid dimer peptides J-AA (Anoplin-Anoplin), J-RR (RW-RW), and J-AR (Anoplin-RW) based on the wasp peptide Anoplin and the arginine- and tryptophan-rich hexapeptide RW were designed and synthesized by click chemistry, with the intent to improve the antimicrobial efficacy of peptides against bacterial pathogens. The results showed that all dimer analogues exhibited up to a 4-16 fold increase in antimicrobial activity compared to the parental peptides against bacterial strains. Furthermore, the antimicrobial activity was confirmed by time-killing kinetics assay with two strains which showed that these dimer analogues at 1, 2×MIC were rapidly bactericidal and reduced the initial inoculum significantly during the first 2-6h. Notably, dimer peptides showed synergy and additivity effects when used in combination with conventional antibiotics rifampin or penicillin respectively against the multidrug-resistant strains. In the Escherichia coli-infected mouse model, all of hybrid dimer analogues had significantly lower degree of bacterial load than the untreated control group when injected once i.p. at 5mg/kg. In addition, the infected mice by methicillin-resistant (MRSA) strain could be effectively treated with J-RR. All of dimer analogues had membrane-active action mode. And the membrane-dependent mode of action signifies that peptides functions freely and without regard to conventional resistant mechanisms. Circular dichroism analyses of all dimer analogues showed a general predominance of α-helix conformation in 50% trifluoroethanol (TFE). Additionally, the acute toxicities study indicated that J-RR or J-AR did not show the signs of toxicity when adult mice exposed to concentration up to 120mg/kg. The 50% lethal dose (LD 50 ) of J-AA was 53.6mg/kg. In conclusion, to design and synthesize side chain-hybrid dimer analogues via click chemistry may offer a new strategy for antibacterial therapeutic option. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular modelling investigations on the possibility of phenanthrene dimers to be the primary nuclei of soot

NASA Astrophysics Data System (ADS)

Wei, Mingrui; Wu, Sheng; Li, Fan; Zhang, Dongju; Zhang, Tingting; Guo, Guanlun

2017-11-01

Pyrene dimerisation was successfully used to model the beginning of soot nucleation in some simulation models. However, the quantum mechanics (QM) calculations proved that the binding energy of a PAH dimer with three six-member rings was similar to that of a pyrene dimer. Meanwhile, the high concentration of phenanthrene at flame conditions indicated high probability of collisions among them. The small difference of the binding energy and high concentration indicated that PAHs structurally smaller than pyrene also could be involved in soot inception. Hence, binary collisions of phenanthrene were simulated to find out whether phenanthrene dimers can serve as soot primary nuclei or not by using non-equilibrium molecular dynamics (MD). Three temperatures, six collision orientations and 155 initial translational velocities (ITVs) were considered. The results indicated that the number of dimers with lifetime over 10 ps which can serve as soot nuclei decreased from 52 at 1000 K to 17 at 1600 K, and further to 6 at 2400 K, which means that low temperature was more favourable for phenanthrene to form soot nuclei. Meanwhile, no soot nuclei were formed at the high velocity region (HVR), compared to 43 and 9 at low and middle velocity regions (LVR and MVR), respectively, when temperature was 1000 K. Also, no soot nuclei were formed at HVR when the temperature was raised to 1600 K and 2400 K. This indicated that HVR was unfavourable for phenanthrene to form soot nuclei. The results computationally further illustrated that small PAHs such as phenanthrene could serve as soot primary nuclei, since they have similar mole fractions in some flames. This may be useful for future soot simulation models.

Dynamics and kinetics of reversible homo-molecular dimerization of polycyclic aromatic hydrocarbons

NASA Astrophysics Data System (ADS)

Mao, Qian; Ren, Yihua; Luo, K. H.; van Duin, Adri C. T.

2017-12-01

Physical dimerization of polycyclic aromatic hydrocarbons (PAHs) has been investigated via molecular dynamics (MD) simulation with the ReaxFF reactive force field that is developed to bridge the gap between the quantum mechanism and classical MD. Dynamics and kinetics of homo-molecular PAH collision under different temperatures, impact parameters, and orientations are studied at an atomic level, which is of great value to understand and model the PAH dimerization. In the collision process, the enhancement factors of homo-molecular dimerizations are quantified and found to be larger at lower temperatures or with smaller PAH instead of size independent. Within the capture radius, the lifetime of the formed PAH dimer decreases as the impact parameter increases. Temperature and PAH characteristic dependent forward and reverse rate constants of homo-molecular PAH dimerization are derived from MD simulations, on the basis of which a reversible model is developed. This model can predict the tendency of PAH dimerization as validated by pyrene dimerization experiments [H. Sabbah et al., J. Phys. Chem. Lett. 1(19), 2962 (2010)]. Results from this study indicate that the physical dimerization cannot be an important source under the typical flame temperatures and PAH concentrations, which implies a more significant role played by the chemical route.

Two new dimeric naphthoquinones with neuraminidase inhibitory activity from Lithospermum erythrorhizon.

PubMed

Yang, Yanqin; Zhao, Dapeng; Yuan, Kailong; Zhou, Guojun; Wang, Yu; Xiao, Yanmeng; Wang, Chenxu; Xu, Jingwei; Yang, Wei

2015-01-01

The crude methanol extract of roots of Lithospermum erythrorhizon was subjected to successive chromatographic fractionation which afforded two new dimeric naphthoquinone derivatives shikometabolin E (2) and shikometabolin F (3) as well as one known compound shikometabolin A (1). The structures of compounds 1-3 were elucidated by using UV, MS, 1D and 2D NMR spectroscopic analysis. The two new dimeric naphthoquinone derivatives showed significant neuraminidase inhibitory activities.

Dimerization of the keto tautomer of acetohydroxamic acid—infrared matrix isolation and theoretical study

NASA Astrophysics Data System (ADS)

Sałdyka, Magdalena; Mielke, Zofia

2005-05-01

Dimerization of the keto tautomer of acetohydroxamic acid has been studied using FTIR matrix isolation spectroscopy and DFT(B3LYP)/6-31+G(d,p) calculations. Analysis of CH 3CONHOH/Ar matrix spectra indicates formation of two dimers in which two intramolecular CO···H sbnd ON bonds within two interacting acetohydroxamic acid molecules are retained. A chain dimer I is stabilized by the intermolecular CO···H sbnd N hydrogen bond, whereas the cyclic dimer II is stabilized by two intermolecular N sbnd H···O(H)N bonds. Twelve vibrations were identified for dimer I and six vibrations for dimer II; the observed frequency shifts show a good agreement with the calculated ones for the structures I and II. Both dimers have comparable binding energies ( ΔEZPECPI, II = -7.02, -6.34 kcal mol -1) being less stable than calculated structures III and IV ( ΔEZPECPIII, IV = -9.50, -8.87 kcal mol -1) in which one or two intramolecular hydrogen bonds are disrupted. In the most stable 10-membered cyclic dimer III, two intermolecular CO···H sbnd ON hydrogen bonds are formed at expense of intramolecular hydrogen bonds of the same type. The formation of the less stable (AHA) 2 dimers in the studied matrixes indicates that the formation of (AHA) 2 is kinetically and not thermodynamically controlled.

Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability.

PubMed

Cox, Julia M; Davis, Caroline A; Chan, Chikio; Jourden, Michael J; Jorjorian, Andrea D; Brym, Melissa J; Snider, Mark J; Borders, Charles L; Edmiston, Paul L

2003-02-25

Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for this quaternary structure are unclear, given that there is no evidence of active site communication and more primitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimer interface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directed mutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, and R147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatography and analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared to be an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants, R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although k(cat) was 60% of the wild-type enzyme. Unlike the R147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibrium mechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatine binding first. Substrate binding affinities were also significantly lower for the R147A mutant, but k(cat) was 11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showed that increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomeric mutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that protein stability is significantly decreased in the monomeric mutants compared to wild-type. Denaturation experiments measuring lambda(max) of the intrinsic fluorescence as a function of guanidine hydrochloride concentration helped confirm the quaternary structures and indicated that the general unfolding pathway of all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is not a prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.

PGL germ granule assembly protein is a base-specific, single-stranded RNase

PubMed Central

Aoki, Scott T.; Kershner, Aaron M.; Bingman, Craig A.; Wickens, Marvin; Kimble, Judith

2016-01-01

Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. Using nematode “P-granules” as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, single-stranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds. PMID:26787882

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

2014-09-01

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

Density functional perturbational orbital theory of spin polarization in electronic systems. II. Transition metal dimer complexes.

PubMed

Seo, Dong-Kyun

2007-11-14

We present a theoretical scheme for a semiquantitative analysis of electronic structures of magnetic transition metal dimer complexes within spin density functional theory (DFT). Based on the spin polarization perturbational orbital theory [D.-K. Seo, J. Chem. Phys. 125, 154105 (2006)], explicit spin-dependent expressions of the spin orbital energies and coefficients are derived, which allows to understand how spin orbitals form and change their energies and shapes when two magnetic sites are coupled either ferromagnetically or antiferromagnetically. Upon employment of the concept of magnetic orbitals in the active-electron approximation, a general mathematical formula is obtained for the magnetic coupling constant J from the analytical expression for the electronic energy difference between low-spin broken-symmetry and high-spin states. The origin of the potential exchange and kinetic exchange terms based on the one-electron picture is also elucidated. In addition, we provide a general account of the DFT analysis of the magnetic exchange interactions in compounds for which the active-electron approximation is not appropriate.

Structural features of the KPI domain control APP dimerization, trafficking, and processing.

PubMed

Ben Khalifa, Naouel; Tyteca, Donatienne; Marinangeli, Claudia; Depuydt, Mathieu; Collet, Jean-François; Courtoy, Pierre J; Renauld, Jean-Christophe; Constantinescu, Stefan; Octave, Jean-Noël; Kienlen-Campard, Pascal

2012-02-01

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.

The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

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

Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin

2010-07-20

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar tomore » the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.« less

Antioxidant potential of curcumin-related compounds studied by chemiluminescence kinetics, chain-breaking efficiencies, scavenging activity (ORAC) and DFT calculations.

PubMed

Slavova-Kazakova, Adriana K; Angelova, Silvia E; Veprintsev, Timur L; Denev, Petko; Fabbri, Davide; Dettori, Maria Antonietta; Kratchanova, Maria; Naumov, Vladimir V; Trofimov, Aleksei V; Vasil'ev, Rostislav F; Delogu, Giovanna; Kancheva, Vessela D

2015-01-01

This study compares the ability to scavenge different peroxyl radicals and to act as chain-breaking antioxidants of monomers related to curcumin (1): dehydrozingerone (2), zingerone (3), (2Z,5E)-ethyl 2-hydroxy-6-(4-hydroxy-3-methoxyphenyl)-4-oxohexa-2,5-dienoate (4), ferulic acid (5) and their corresponding C 2-symmetric dimers 6-9. Four models were applied: model 1 - chemiluminescence (CL) of a hydrocarbon substrate used for determination of the rate constants (k A) of the reactions of the antioxidants with peroxyl radicals; model 2 - lipid autoxidation (lipidAO) used for assessing the chain-breaking antioxidant efficiency and reactivity; model 3 - oxygen radical absorbance capacity (ORAC), which yields the activity against peroxyl radicals generated by an azoinitiator; model 4 - density functional theory (DFT) calculations at UB3LYP/6-31+G(d,p) level, applied to explain the structure-activity relationship. Dimers showed 2-2.5-fold higher values of k A than their monomers. Model 2 gives information about the effects of the side chains and revealed much higher antioxidant activity for monomers and dimers with α,β-unsaturated side chains. Curcumin and 6 in fact are dimers of the same monomer 2. We conclude that the type of linkage between the two "halves" by which the molecule is made up does not exert influence on the antioxidant efficiency and reactivity of these two dimers. The dimers and the monomers demonstrated higher activity than Trolox (10) in aqueous medium (model 3). A comparison of the studied compounds with DL-α-tocopherol (11), Trolox and curcumin is made. All dimers are characterized through lower bond dissociation enthalpies (BDEs) than their monomers (model 4), which qualitatively supports the experimental results.

A Monte Carlo simulation study of associated liquid crystals

NASA Astrophysics Data System (ADS)

Berardi, R.; Fehervari, M.; Zannoni, C.

We have performed a Monte Carlo simulation study of a system of ellipsoidal particles with donor-acceptor sites modelling complementary hydrogen-bonding groups in real molecules. We have considered elongated Gay-Berne particles with terminal interaction sites allowing particles to associate and form dimers. The changes in the phase transitions and in the molecular organization and the interplay between orientational ordering and dimer formation are discussed. Particle flip and dimer moves have been used to increase the convergency rate of the Monte Carlo (MC) Markov chain.

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity.

PubMed

Torres, Eduardo; Aburto, Jorge

2005-05-15

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.

Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor.

PubMed

Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

2016-07-25

The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

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

Herman, Michael F.; Currier, Robert P.; Peery, Travis B.

Intermolecular coupling of dipole moments is studied for a model system consisting of two diatomic molecules (AB monomers) arranged co-linearly and which can form non-covalently bound dimers. The dipolar coupling is a function of the bond length in each molecule as well as of the distance between the centers-of-mass of the two molecules. The calculations show that intermolecular coupling of the vibrations results in an isotope-dependent modification of the AB-AB intermolecular potential. This in turn alters the energies of the low-lying bound states of the dimers, producing isotope-dependent changes in the AB-AB dimer partition function. Explicit inclusion of intermolecular vibrationalmore » coupling then changes the predicted gas-dimer isotopic fractionation. In addition, a mass dependence in the intermolecular potential can also result in changes in the number of bound dimer states in an equilibrium mixture. This in turn leads to a significant dimer population shift in the model monomer-dimer equilibrium system considered here. Finally, the results suggest that intermolecular coupling terms should be considered when probing the origins of isotopic fractionation.« less

A test of the significance of intermolecular vibrational coupling in isotopic fractionation

DOE PAGES

Herman, Michael F.; Currier, Robert P.; Peery, Travis B.; ...

2017-07-15

Intermolecular coupling of dipole moments is studied for a model system consisting of two diatomic molecules (AB monomers) arranged co-linearly and which can form non-covalently bound dimers. The dipolar coupling is a function of the bond length in each molecule as well as of the distance between the centers-of-mass of the two molecules. The calculations show that intermolecular coupling of the vibrations results in an isotope-dependent modification of the AB-AB intermolecular potential. This in turn alters the energies of the low-lying bound states of the dimers, producing isotope-dependent changes in the AB-AB dimer partition function. Explicit inclusion of intermolecular vibrationalmore » coupling then changes the predicted gas-dimer isotopic fractionation. In addition, a mass dependence in the intermolecular potential can also result in changes in the number of bound dimer states in an equilibrium mixture. This in turn leads to a significant dimer population shift in the model monomer-dimer equilibrium system considered here. Finally, the results suggest that intermolecular coupling terms should be considered when probing the origins of isotopic fractionation.« less

Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution.

PubMed

Jaskólski, M; Miller, M; Rao, J K; Leis, J; Wlodawer, A

1990-06-26

The structure of Rous sarcoma virus protease has been solved by multiple isomorphous replacement in the crystal form belonging to space group P3(1)21, with unit-cell parameters a = 88.95 A and c = 78.90 A. The enzyme belongs to the family of aspartic proteases with two identical subunits composing the active homodimer. The noncrystallographic dyad relating these two subunits was identified after preliminary tracing in the MIR map and was used for phase improvement by electron-density averaging. Structure refinement resulted in a model that included 1772 protein atoms and 252 water molecules, with an R factor of 0.144 for data extending to 2 A. The secondary structure of a retroviral protease molecule closely resembles that of a single domain in pepsin-like aspartic proteases and consists of several beta-strands and of one well-defined and one distorted alpha-helix. The dimer interface is composed of the N- and C-terminal chains from both subunits which are intertwined to form a well-ordered four-stranded antiparallel beta-sheet. In each monomer, the catalytic triad (Asp-Ser-Gly) is located in a loop that forms a part of the psi-structure characteristic to all aspartic proteases. The position of a water molecule between the active-site aspartate residues and the general scheme of H bonding within the active site bear close resemblance to those in pepsin-like aspartic proteases and therefore suggest a similar enzymatic mechanism. The binding cleft over the active site is covered by two flap arms, one from each monomer, which are partially disordered. The retroviral protease dimer has been compared with several enzymes of cellular origin, with chains aligning to an rms deviation of 1.90 A or better.

Molecular studies on structural changes and oligomerisation of violaxanthin de-epoxidase associated with the pH-dependent activation.

PubMed

Hallin, Erik Ingmar; Hasan, Mahmudul; Guo, Kuo; Åkerlund, Hans-Erik

2016-07-01

Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.

Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane

PubMed Central

Gao, Xiu-Gong; Maldonado, Ernesto; Pérez-Montfort, Ruy; Garza-Ramos, Georgina; de Gómez-Puyou, Marietta Tuena; Gómez-Puyou, Armando; Rodríguez-Romero, Adela

1999-01-01

To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2-Å resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 Å from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design. PMID:10468562

Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane.

PubMed

Gao, X G; Maldonado, E; Pérez-Montfort, R; Garza-Ramos, G; de Gómez-Puyou, M T; Gómez-Puyou, A; Rodríguez-Romero, A

1999-08-31

To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2-A resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 A from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

PubMed Central

Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

2013-01-01

Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into the viscous bilayer, thereby increasing the diffusional friction. Ensemble measurements of PH domain affinity for PIP3 on plasma membrane-like bilayers reveals that dimeric WT PH domain possesses a one-order of magnitude higher target membrane affinity than the previously characterized monomeric PH domains, consistent with a dimerization-triggered, allosterically-enhanced affinity for one PIP3 molecule (a much larger affinity enhancement would be expected for dimerization-triggered binding to two PIP3 molecules). The monomeric T513E PDK1 PH domain, like other monomeric PH domains, exhibits a PIP3 affinity and bound state lifetime that are each a full order of magnitude lower than dimeric WT PH domain, which is predicted to facilitate release of activated, monomeric PDK1 to cytoplasm. Overall, the study yields the first molecular picture of PH domain regulation via electrostatic control of dimer-monomer conversion. PMID:23745598

The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

PubMed

Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

2013-07-16

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows deeper insertion of the protein into the viscous bilayer, thereby increasing the diffusional friction. Ensemble measurements of PH domain affinity for PIP3 on plasma membrane-like bilayers reveal that the dimeric WT PH domain possesses a one order of magnitude higher target membrane affinity than the previously characterized monomeric PH domains, consistent with a dimerization-triggered, allosterically enhanced affinity for one PIP3 molecule (a much larger affinity enhancement would be expected for dimerization-triggered binding to two PIP3 molecules). The monomeric T513E PDK1 PH domain, like other monomeric PH domains, exhibits a PIP3 affinity and bound state lifetime that are each 1 order of magnitude lower than those of the dimeric WT PH domain, which is predicted to facilitate release of activated, monomeric PDK1 to the cytoplasm. Overall, the study yields the first molecular picture of PH domain regulation via electrostatic control of dimer-monomer conversion.

Ortho and para hydrogen dimers on G/SiC(0001): combined STM and DFT study.

PubMed

Merino, P; Švec, M; Martínez, J I; Mutombo, P; Gonzalez, C; Martín-Gago, J A; de Andres, P L; Jelinek, P

2015-01-01

The hydrogen (H) dimer structures formed upon room-temperature H adsorption on single layer graphene (SLG) grown on SiC(0001) are addressed using a combined theoretical-experimental approach. Our study includes density functional theory (DFT) calculations for the full (6√3 × 6√3)R30° unit cell of the SLG/SiC(0001) substrate and atomically resolved scanning tunneling microscopy images determining simultaneously the graphene lattice and the internal structure of the H adsorbates. We show that H atoms normally group in chemisorbed coupled structures of different sizes and orientations. We make an atomic scale determination of the most stable experimental geometries, the small dimers and ellipsoid-shaped features, and we assign them to hydrogen adsorbed in para dimers and ortho dimers configuration, respectively, through comparison with the theory.

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

PubMed Central

Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

2013-01-01

Fluorescent probes for the detection of a double-stranded DNA were prepared by labeling a triplex-forming DNA oligonucleotide with a thiazole orange (TO) dimer unit. They belong to ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes which we have previously reported. The excitonic interaction between the two TO molecules was expected to effectively suppress the background fluorescence of the probes. The applicability of the ECHO probes for the detection of double-stranded DNA was confirmed by examining the thermal stability and photophysical and kinetic properties of the DNA triplexes formed by the ECHO probes. PMID:23445822

Dimer motion on a periodic substrate: spontaneous symmetry breaking and absolute negative mobility.

PubMed

Speer, David; Eichhorn, Ralf; Evstigneev, Mykhaylo; Reimann, Peter

2012-06-01

We consider two coupled particles moving along a periodic substrate potential with negligible inertia effects (overdamped limit). Even when the particles are identical and the substrate spatially symmetric, a sinusoidal external driving of appropriate amplitude and frequency may lead to spontaneous symmetry breaking in the form of a permanent directed motion of the dimer. Thermal noise restores ergodicity and thus zero net velocity, but entails arbitrarily fast diffusion of the dimer for sufficiently weak noise. Moreover, upon application of a static bias force, the dimer exhibits a motion opposite to that force (absolute negative mobility). The key requirement for all these effects is a nonconvex interaction potential of the two particles.

Solution Structure of the Soluble Receptor for Advanced Glycation End Products (sRAGE)*

PubMed Central

Sárkány, Zsuzsa; Ikonen, Teemu P.; Ferreira-da-Silva, Frederico; Saraiva, Maria João; Svergun, Dmitri; Damas, Ana Margarida

2011-01-01

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor involved in various human diseases, as it binds to numerous molecules and proteins that modulate the activity of other proteins. Elucidating the three-dimensional structure of this receptor is therefore most important for understanding its function during activation and cellular signaling. The major alternative splice product of RAGE comprises its extracellular region that occurs as a soluble protein (sRAGE). Although the structures of sRAGE domains were available, their assembly into the functional full-length protein remained unknown. We observed that the protein has concentration-dependent oligomerization behavior, and this is also mediated by the presence of Ca2+ ions. Moreover, using synchrotron small angle x-ray scattering, the solution structure of human sRAGE was determined in the monomeric and dimeric forms. The model for the monomer displays a J-like shape, whereas the dimer is formed through the association of the two N-terminal domains and has an elongated structure. These results provide insights into the assembly of the RAGE homodimer, which is essential for signal transduction, and the sRAGE:RAGE heterodimer that leads to blockage of the receptor signaling, paving the way for the design of therapeutic strategies for a large number of different pathologies. PMID:21865159

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

PubMed

Chadda, R; Robertson, J L

2016-01-01

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F - ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl - /H + antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known. © 2016 Elsevier Inc. All rights reserved.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance.

PubMed

Kabe, Yasuaki; Nakane, Takanori; Koike, Ikko; Yamamoto, Tatsuya; Sugiura, Yuki; Harada, Erisa; Sugase, Kenji; Shimamura, Tatsuro; Ohmura, Mitsuyo; Muraoka, Kazumi; Yamamoto, Ayumi; Uchida, Takeshi; Iwata, So; Yamaguchi, Yuki; Krayukhina, Elena; Noda, Masanori; Handa, Hiroshi; Ishimori, Koichiro; Uchiyama, Susumu; Kobayashi, Takuya; Suematsu, Makoto

2016-03-18

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

PubMed Central

Kabe, Yasuaki; Nakane, Takanori; Koike, Ikko; Yamamoto, Tatsuya; Sugiura, Yuki; Harada, Erisa; Sugase, Kenji; Shimamura, Tatsuro; Ohmura, Mitsuyo; Muraoka, Kazumi; Yamamoto, Ayumi; Uchida, Takeshi; Iwata, So; Yamaguchi, Yuki; Krayukhina, Elena; Noda, Masanori; Handa, Hiroshi; Ishimori, Koichiro; Uchiyama, Susumu; Kobayashi, Takuya; Suematsu, Makoto

2016-01-01

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. PMID:26988023

Photomodulation of the nucleating activity of a photocleavable crosslinked actin dimer.

PubMed

Marriott, G; Miyata, H; Kinosita, K

1992-04-01

The ability to generate substrate concentration jumps through photo-deprotection of amine, carboxyl and phosphate groups has been an important development for investigations of protein activity in complex systems. To broaden the versatility and applications of photo-deprotection techniques for the photomodulation of protein activity we describe the synthesis and characterisation of a reagent for generating free thiol from thioether groups and a related photocleavable, heterobifunctional crosslinking reagent. Chemical and spectroscopic studies of a model thiol protected derivative were used to show some features of thiol group photodeprotection. To demonstrate how the photocleavable crosslinking reagent may be used to modulate the activity of proteins we investigated the effect of light on the nucleating activity of crosslinked actin dimer; thus following near-ultraviolet irradiation of the actin dimer the crosslink was cleaved, presumeably at the thioether bond, resulting in the concomitant dissociation of dimer, loss of nucleating activity and creation of a concentration jump of polymerisable G-actin monomer. On the basis of this initial study we discuss applications and limitations of these reagents for the photomodulation of protein activity in vitro and in vivo.

Hydrogen Recombination and Dimer Formation on Graphite from Ab Initio Molecular Dynamics Simulations.

PubMed

Casolo, S; Tantardini, G F; Martinazzo, R

2016-07-14

We studied Eley-Rideal molecular hydrogen formation on graphite using ab initio molecular dynamics, in the energy range relevant for the chemistry of the interstellar medium and for terrestrial experiments employing cold plasma (0.02-1 eV). We found substantial projectile steering effects that prevent dimer formation at low energies, thereby ruling out any catalytic synthetic pathways that form hydrogen molecules. Ortho and para dimers do form efficiently thanks to preferential sticking, but only at energies that are too high to be relevant for the chemistry of the interstellar medium. Computed reaction cross sections and ro-vibrational product populations are in good agreement with available experimental data and capable of generating adsorbate configurations similar to those observed with scanning tunneling microscopy techniques.

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

A brain-penetrant RAF dimer antagonist for the noncanonical BRAF oncoprotein of pediatric low-grade astrocytomas.

PubMed

Sun, Yu; Alberta, John A; Pilarz, Catherine; Calligaris, David; Chadwick, Emily J; Ramkissoon, Shakti H; Ramkissoon, Lori A; Garcia, Veronica Matia; Mazzola, Emanuele; Goumnerova, Liliana; Kane, Michael; Yao, Zhan; Kieran, Mark W; Ligon, Keith L; Hahn, William C; Garraway, Levi A; Rosen, Neal; Gray, Nathanael S; Agar, Nathalie Y; Buhrlage, Sara J; Segal, Rosalind A; Stiles, Charles D

2017-06-01

Activating mutations or structural rearrangements in BRAF are identified in roughly 75% of all pediatric low-grade astrocytomas (PLGAs). However, first-generation RAF inhibitors approved for adult melanoma have poor blood-brain penetrance and are only effective on tumors that express the canonical BRAFV600E oncoprotein, which functions as a monomer. These drugs (type I antagonists that target the "DFG-in" conformation of the kinase) fail to block signaling via KIAA1549:BRAF, a truncation/fusion BRAF oncoprotein which functions as a dimer and is found in the most common form of PLGA. A panel of small molecule RAF inhibitors (including type II inhibitors, targeting the "DFG-out" conformation of the kinase) was screened for drugs showing efficacy on murine models of PLGA and on authentic human PLGA cells expressing KIAA1549:BRAF. We identify a type II RAF inhibitor that serves as an equipotent antagonist of BRAFV600E, KIAA1549:BRAF, and other noncanonical BRAF oncoproteins that function as dimers. This drug (MLN2480, also known as TAK-580) has good brain penetrance and is active on authentic human PLGA cells in brain organotypic cultures. MLN2480 may be an effective therapeutic for BRAF mutant pediatric astrocytomas. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Crystal structures of the F and pSLT plasmid TraJ N-terminal regions reveal similar homodimeric PAS folds with functional interchangeability

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

Lu, Jun; Wu, Ruiying; Adkins, Joshua N.

2014-09-16

In the F-family of conjugative plasmids, TraJ is an essential transcriptional activator of the tra operon that encodes most of the proteins required for conjugation. Here we report for the first time the X-ray crystal structures of the TraJ N-terminal regions from the prototypic F plasmid (TraJF11-130) and from the Salmonella virulence plasmid pSLT (TraJpSLT 1-128). Both proteins form similar homodimeric Per-ARNT-Sim (PAS) fold structures. Mutational analysis reveals that the observed dimeric interface is critical for TraJF transcriptional activation, indicating that dimerization of TraJ is required for its in vivo function. An artificial ligand (oxidized dithiothreitol) occupies a cavity inmore » the TraJF dimer interface, while a smaller cavity in corresponding region of the TraJpSLT structure lacks a ligand. Gas chromatography/mass spectrometry-electron ionization analysis of dithiothreitol-free TraJF suggests indole may be the natural TraJ ligand; however, disruption of the indole biosynthetic pathway does not affect TraJF function. Heterologous PAS domains from pSLT and R100 TraJ can functionally replace the TraJF PAS domain, suggesting that TraJ allelic specificity is mediated by the region C-terminal to the PAS domain.« less

The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

PubMed

Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

2016-05-13

Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

PubMed Central

Cunningham, Orla; Andolfo, Annapaola; Santovito, Maria Lisa; Iuzzolino, Lucia; Blasi, Francesco; Sidenius, Nicolai

2003-01-01

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion. PMID:14609946

Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

NASA Astrophysics Data System (ADS)

Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

2016-07-01

Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

Insights into the transmembrane helix associations of kit ligand by molecular dynamics simulation and TOXCAT.

PubMed

Chai, Mengya; Liu, Bo; Sun, Fude; Wei, Peng; Chen, Peng; Xu, Lida; Luo, Shi-Zhong

2017-07-01

Kit ligand (KITL) plays important roles in cell proliferation, differentiation, and survival via interaction with its receptor Kit. The previous studies demonstrated that KITL formed a noncovalent homodimer through transmembrane (TM) domain; however, the undergoing mechanism of transmembrane association that determines KITL TM dimerization is still not clear. Herein, molecular dynamics (MD) simulation strategy and TOXCAT assay were combined to characterize the dimerization interface and structure of KITL TM in details. KITL TM formed a more energetically favorable noncovalent dimer through a conserved SxxxGxxxG motif in the MD simulation. Furthermore, the TOXCAT results demonstrated that KITL TM self-associated strongly in the bilayer membrane environment. Mutating any one of the small residues Ser11, Gly15 or Gly19 to Ile disrupted KITL TM dimerization dramatically, which further validated our MD simulation results. In addition, our results showed that Tyr22 could help to stabilize the TM interactions via interacting with the phosphoric group in the bilayer membrane. Pro7 did not induce helix kinks or swivel angles in KITL TM, but it was related with the pitch of the turn around this residue so as to affect the dimer formation. Combining the results of computer modeling and experimental mutagenesis studies on the KITL TM provide new insights for the transmembrane helix association of KITL dimerization. Proteins 2017; 85:1362-1370. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

Li, Cui-Ping; Nie, Li; College of Materials Science and Engineering, Nanjing Tech University, Nanjing 210009

A new salt [H{sub 2}DABCO][Pt(mnt){sub 2}]{sub 2} (1) (mnt{sup 2-}=maleonitriledithiolate and H{sub 2}DABCO{sup 2+} is diprotonated 1,4-diazabicyclo[2.2.2]octane) has been synthesized; its crystal structure, magnetic and near-IR absorption properties have been investigated. Two different [Pt(mnt){sub 2}]{sup -} anions form the strong π-dimers, labeled as Pt(1)-dimer and Pt(2)-dimer, with quite shorter Pt…Pt and S…S distances and molecular plane-to-plane distance (<3.5 Å) within a dimer. The [Pt(mnt){sub 2}]{sub 2}{sup 2-} π-dimers are connected through the cations in the strong H-bond manner to form three-dimensional H-bond supramolecular crystal. The salt shows weak paramagnetism in 1.99–300 K and this is due to the existence ofmore » strong antiferromagnetic coupling within a π-dimer. In addition, a small thermal hysteresis loop is observed at ca. 120 K, indicating that a phase transition probably occurs that is further confirmed by variable-temperature IR spectra. Another fascinating functionality of 1 is the intense near-IR absorption in the region of 750–2500 nm, and this near-IR absorption feature makes it to be a promising optical material. - Graphical abstract: A H-bond supramolecular crystal of [H{sub 2}DABCO][Pt(mnt){sub 2}]{sub 2} shows a magnetic phase transition at ca. 120 K with sizable thermal hysteresis loop and intense near-IR absorption in the region of 750–2500 nm.« less

Changes at the KinA PAS-A Dimerization Interface Influence Histidine Kinase Function

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

Lee, James; Tomchick, Diana R.; Brautigam, Chad A.

2008-11-12

The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatorymore » role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using {beta}-sheet/{beta}-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.« less

The H2A-H2B dimeric kinetic intermediate is stabilized by widespread hydrophobic burial with few fully native interactions.

PubMed

Guyett, Paul J; Gloss, Lisa M

2012-01-20

The H2A-H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within ∼5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I₂ and I₂*, the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I₂ ensemble folds to the native heterodimer, N₂, through an observable, first-order kinetic phase. To determine the regions of structure in the I₂ ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I₂, the transition state and N₂; however, only residues in the hydrophobic core of the dimer interface perturbed the I₂* population. Destabilization of I₂* correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Φ values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I₂ and N₂, but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I₂. These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I₂, but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N₂, more native-like interactions are developed and nonnative interactions are rearranged. Copyright © 2011 Elsevier Ltd. All rights reserved.

The human dopamine transporter forms a tetramer in the plasma membrane: cross-linking of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs.

PubMed

Hastrup, Hanne; Sen, Namita; Javitch, Jonathan A

2003-11-14

Using cysteine cross-linking, we demonstrated previously that the dopamine transporter (DAT) is at least a homodimer, with the extracellular end of transmembrane segment (TM) 6 at a symmetrical dimer interface. We have now explored the possibility that DAT exists as a higher order oligomer in the plasma membrane. Cysteine cross-linking of wild type DAT resulted in bands on SDS-PAGE consistent with dimer, trimer, and tetramer, suggesting that DAT forms a tetramer in the plasma membrane. A cysteine-depleted DAT (CD-DAT) into which only Cys243 or Cys306 was reintroduced was cross-linked to dimer, suggesting that these endogenous cysteines in TM4 and TM6, respectively, were cross-linked at a symmetrical dimer interface. Reintroduction of both Cys243 and Cys306 into CD-DAT led to a pattern of cross-linking indistinguishable from that of wild type, with dimer, trimer, and tetramer bands. This indicated that the TM4 interface and the TM6 interface are distinct and further suggested that DAT may exist in the plasma membrane as a dimer of dimers, with two symmetrical homodimer interfaces. The cocaine analog MFZ 2-12 and other DAT inhibitors, including benztropine and mazindol, protected Cys243 against cross-linking. In contrast, two substrates of DAT, dopamine and tyramine, did not significantly impact cross-linking. We propose that the impairment of cross-linking produced by the inhibitors results from a conformational change at the TM4 interface, further demonstrating that these compounds are not neutral blockers but by themselves have effects on the structure of the transporter.

Probing amyloid fibril formation of the NFGAIL peptide by computer simulations

NASA Astrophysics Data System (ADS)

Melquiond, Adrien; Gelly, Jean-Christophe; Mousseau, Normand; Derreumaux, Philippe

2007-02-01

Amyloid fibril formation, as observed in Alzheimer's disease and type II diabetes, is currently described by a nucleation-condensation mechanism, but the details of the process preceding the formation of the nucleus are still lacking. In this study, using an activation-relaxation technique coupled to a generic energy model, we explore the aggregation pathways of 12 chains of the hexapeptide NFGAIL. The simulations show, starting from a preformed parallel dimer and ten disordered chains, that the peptides form essentially amorphous oligomers or more rarely ordered β-sheet structures where the peptides adopt a parallel orientation within the sheets. Comparison between the simulations indicates that a dimer is not a sufficient seed for avoiding amorphous aggregates and that there is a critical threshold in the number of connections between the chains above which exploration of amorphous aggregates is preferred.

Hierarchical Cluster Formation in Concentrated Monoclonal Antibody Formulations

NASA Astrophysics Data System (ADS)

Godfrin, P. Douglas; Zarzar, Jonathan; Zarraga, Isidro Dan; Porcar, Lionel; Falus, Peter; Wagner, Norman; Liu, Yun

Reversible cluster formation has been identified as an underlying cause of large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations. As high solution viscosity prevents the use of subcutaneous injection as a delivery method for some mAbs, a fundamental understanding of the interactions responsible for high viscosities in concentrated mAb solutions is of significant relevance to mAb applications in human health care as well as of intellectual interest. Here, we present a detailed investigation of a well-studied IgG1 based mAb to relate the short time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. Using a combination of experimental techniques, it is found that upon adding Na2SO4, these antibodies dimerize in solution. Proteins form strongly bounded reversible dimers at dilute concentrations that, when concentrated, interact with each other to form loosely bounded, large, transient clusters. The combined effect of forming strongly bounded dimers and a large transient network is a significant increase in the solution viscosity. Strongly bounded, reversible dimers may exist in many IgG1 based mAb systems such that these results contribute to a more comprehensive understanding of the physical mechanisms producing high viscosities in concentrated protein solutions.

Dimer self-organization of impurity ytterbium ions in synthetic forsterite single crystals

NASA Astrophysics Data System (ADS)

Tarasov, V. F.; Sukhanov, A. A.; Dudnikova, V. B.; Zharikov, E. V.; Lis, D. A.; Subbotin, K. A.

2017-07-01

Paramagnetic centers formed by impurity Yb3+ ions in synthetic forsterite (Mg2SiO4) grown by the Czochralski technique are studied by X-band CW and pulsed EPR spectroscopy. These centers are single ions substituting magnesium in two different crystallographic positions denoted M1 and M2, and dimer associates formed by two Yb3+ ions in nearby positions M1. It is established that there is a pronounced mechanism favoring self-organization of ytterbium ions in dimer associates during the crystal growth, and the mechanism of the spin-spin coupling between ytterbium ions in the associate has predominantly a dipole-dipole character, which makes it possible to control the energy of the spin-spin interaction by changing the orientation of the external magnetic field. The structural computer simulation of cluster ytterbium centers in forsterite crystals is carried out by the method of interatomic potentials using the GULP 4.0.1 code (General Utility Lattice Program). It is established that the formation of dimer associates in the form of a chain parallel to the crystallographic axis consisting of two ytterbium ions with a magnesium vacancy between them is the most energetically favorable for ytterbium ions substituting magnesium in the position M1.

Protective V127 prion variant prevents prion disease by interrupting the formation of dimer and fibril from molecular dynamics simulations.

PubMed

Zhou, Shuangyan; Shi, Danfeng; Liu, Xuewei; Liu, Huanxiang; Yao, Xiaojun

2016-02-24

Recent studies uncovered a novel protective prion protein variant: V127 variant, which was reported intrinsically resistant to prion conversion and propagation. However, the structural basis of its protective effect is still unknown. To uncover the origin of the protective role of V127 variant, molecular dynamics simulations were performed to explore the influence of G127V mutation on two key processes of prion propagation: dimerization and fibril formation. The simulation results indicate V127 variant is unfavorable to form dimer by reducing the main-chain H-bond interactions. The simulations of formed fibrils consisting of β1 strand prove V127 variant will make the formed fibril become unstable and disorder. The weaker interaction energies between layers and reduced H-bonds number for V127 variant reveal this mutation is unfavorable to the formation of stable fibril. Consequently, we find V127 variant is not only unfavorable to the formation of dimer but also unfavorable to the formation of stable core and fibril, which can explain the mechanism on the protective role of V127 variant from the molecular level. Our findings can deepen the understanding of prion disease and may guide the design of peptide mimetics or small molecule to mimic the protective effect of V127 variant.

Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test.

PubMed

Takeyoshi, Masahiro; Iida, Kenji; Suzuki, Keiko; Yamazaki, Shunsuke

2008-05-01

Allergic contact dermatitis is the serious unwanted effect arising from the use of consumer products such as cosmetics. Isoeugenol is a fragrance chemical with spicy, carnation-like scent, is used in many kinds of cosmetics and is a well-known moderate human sensitizer. It was previously reported that the dimerization of eugenol yielded two types of dimer possessing different sensitization potencies. This study reports the differences in skin sensitization potencies for isoeugenol and two types of dimer, beta-O-4-dilignol and dehydrodiisoeugenol (DIEG), as evaluated by the non-radioisotopic local lymph node assay (non-RI LLNA) and guinea pig maximization test. In the guinea pig maximization test, isoeugenol, beta-O-4-dilignol and DIEG were classified as extreme, weak and moderate sensitizers, respectively. As for the results of non-RI LLNA, the EC3 for isoeugenol, beta-O-4-dilignol and DIEG were calculated as 12.7%, >30% and 9.4%, respectively. The two types of isoeugenol dimer showed different sensitizing activities similar to the case for eugenol dimers. A reduction of sensitization potency achieved by dimerization may lead to developing safer cosmetic ingredients. Isoeugenol dimers are not currently used for fragrance chemicals. However, the dimerization of isoeugenol may yield a promising candidate as a cosmetic ingredient with low sensitization risk. The data may also provide useful information for the structure-activity relationship (SAR) in skin sensitization. Copyright (c) 2007 John Wiley & Sons, Ltd.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

PubMed Central

Needham, Sarah R.; Roberts, Selene K.; Arkhipov, Anton; Mysore, Venkatesh P.; Tynan, Christopher J.; Zanetti-Domingues, Laura C.; Kim, Eric T.; Losasso, Valeria; Korovesis, Dimitrios; Hirsch, Michael; Rolfe, Daniel J.; Clarke, David T.; Winn, Martyn D.; Lajevardipour, Alireza; Clayton, Andrew H. A.; Pike, Linda J.; Perani, Michela; Parker, Peter J.; Shan, Yibing; Shaw, David E.; Martin-Fernandez, Marisa L.

2016-01-01

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling. PMID:27796308

The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistically typical 2-Cys subfamily without any formation of toroid-shaped decamers

PubMed Central

Smeets, Aude; Loumaye, Eléonore; Clippe, André; Rees, Jean-François; Knoops, Bernard; Declercq, Jean-Paul

2008-01-01

The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 Å resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone. PMID:18359859

Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

PubMed

Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

2015-03-01

In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

PubMed Central

Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

2013-01-01

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

Crystal structures of penicillin-binding protein 3 (PBP3) from methicillin-resistant Staphylococcus aureus in the apo and cefotaxime-bound forms.

PubMed

Yoshida, Hisashi; Kawai, Fumihiro; Obayashi, Eiji; Akashi, Satoko; Roper, David I; Tame, Jeremy R H; Park, Sam-Yong

2012-10-26

Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution. Copyright © 2012 Elsevier Ltd. All rights reserved.

A multimode vibronic treatment of absorption, resonance Raman, and hyper-Rayleigh scattering of excitonically coupled molecular dimers

NASA Astrophysics Data System (ADS)

Myers Kelley, Anne

2003-08-01

The linear absorption spectra, resonance Raman excitation profiles and depolarization dispersion curves, and hyper-Rayleigh scattering profiles are calculated for excitonically coupled homodimers of a model electron donor-acceptor "push-pull" conjugated chromophore as a function of dimer geometry. The vibronic eigenstates of the dimer are calculated by diagonalizing the matrix of transition dipole couplings among the vibronic transitions of the constituent monomers. The absorption spectra show the usual red- or blueshifted transitions for J-type or H-type dimers, respectively. When the electronic coupling is large compared with the vibronic width of the monomer spectrum, the dimer absorption spectra exhibit simple Franck-Condon progressions having reduced vibronic intensities compared with the monomer, and the resonance Raman excitation profiles are shifted but otherwise only weakly perturbed. When the coupling is comparable to the vibronic width, the H-dimer absorption spectra exhibit irregular vibronic frequency spacings and intensity patterns and the effects on the Raman excitation profiles are larger. There is strong dispersion in the Raman depolarization ratios for dimer geometries in which both transitions carry oscillator strength. The first hyperpolarizabilities are somewhat enhanced in J-dimers and considerably reduced in H-dimers. These effects on the molecular β will amplify the effects of dimerization on the ground-state dipole moment in electro-optic materials formed from chromophore-doped polymers that must be electric field poled to obtain the net alignment needed for a macroscopic χ(2).

Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

PubMed

Johnston, Jennifer M; Aburi, Mahalaxmi; Provasi, Davide; Bortolato, Andrea; Urizar, Eneko; Lambert, Nevin A; Javitch, Jonathan A; Filizola, Marta

2011-03-15

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

Antiplasmodial dimeric chalcone derivatives from the roots of Uvaria siamensis.

PubMed

Salae, Abdul-Wahab; Chairerk, Orapan; Sukkoet, Piyanut; Chairat, Therdsak; Prawat, Uma; Tuntiwachwuttikul, Pittaya; Chalermglin, Piya; Ruchirawat, Somsak

2017-03-01

Four dimeric chalcone derivatives, 8″,9″-dihydrowelwitschin H, uvarins A-C, a naphthalene derivative, 2-hydroxy-3-methoxy-6-(4'- hydroxyphenyl)naphthalene, and the known dimeric chalcones, dependensin and welwitschin E, flavonoids, a cyclohexane oxide derivative, an aromatic aldehyde were isolated from the roots of Uvaria siamensis (Annonaceae). The structures of the compounds were elucidated by spectroscopic analysis, as well as by comparison with literature data. The isolated compounds with a sufficient amount for biological assays were evaluated for their antimalarial, antimycobacterial, and cytotoxic activities. The dimeric chalcones 8″,9″-dihydrowelwitschin H, uvarins B and C, dependensin and welwitschin E showed strong antiplasmodial activity with IC 50 values of 3.10, 3.02, 3.09, 4.21 and 3.99 μg/mL, respectively. A possible biosynthesis pathway of the dimeric chalcones is discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Elevation of serum CA 125 and D-dimer levels associated with rupture of ovarian endometrioma.

PubMed

Uharcek, P; Mlyncek, M; Ravinger, J

2007-01-01

Patients with endometriosis rarely have a serum CA 125 concentration >100 IU/mL. A raised plasma level of D-dimer indicates active fibrinolysis, either secondary to clot formation or primarily activated. This condition is seldom diagnosed in patients with endometriosis. A 53-year-old woman was referred to our institution for acute abdominal pain. Laparoscopic surgery revealed a large ovarian cyst with rupture on the left side. Preoperative laboratory tests detected high serum CA 125 and D-dimer levels. Adnexectomy was performed, resulting in a sharp decrease in serum CA 125 and D-dimer concentration. We describe the clinical course of the patient. Rupture of a large ovarian endometrioma can lead to a high serum concentration of CA 125, a condition which, in addition to the detected pelvic mass, may mimic a malignant process. The increased D-dimer plasma level indicated that a ruptured endometriotic cyst can induce coagulation reactions.

Theoretical Insights into a CO Dimerization Mechanism in CO2 Electroreduction.

PubMed

Montoya, Joseph H; Shi, Chuan; Chan, Karen; Nørskov, Jens K

2015-06-04

In this work, we present DFT simulations that demonstrate the ability of Cu to catalyze CO dimerization in CO2 and CO electroreduction. We describe a previously unreported CO dimer configuration that is uniquely stabilized by a charged water layer on both Cu(111) and Cu(100). Without this charged water layer at the metal surface, the formation of the CO dimer is prohibitively endergonic. Our calculations also demonstrate that dimerization should have a lower activation barrier on Cu(100) than Cu(111), which, along with a more exergonic adsorption energy and a corresponding higher coverage of *CO, is consistent with experimental observations that Cu(100) has a high activity for C-C coupling at low overpotentials. We also demonstrate that this effect is present with cations other than H(+), a finding that is consistent with the experimentally observed pH independence of C2 formation on Cu.

Restoration of glycoprotein Erns dimerization via pseudoreversion partially restores virulence of classical swine fever virus.

PubMed

Tucakov, Anna Katharina; Yavuz, Sabine; Schürmann, Eva-Maria; Mischler, Manjula; Klingebeil, Anne; Meyers, Gregor

2018-01-01

The classical swine fever virus (CSFV) represents one of the most important pathogens of swine. The CSFV glycoprotein E rns is an essential structural protein and an important virulence factor. The latter is dependent on the RNase activity of this envelope protein and, most likely, its secretion from the infected cell. A further important feature with regard to its function as a virulence factor is the formation of disulfide-linked E rns homodimers that are found in virus-infected cells and virions. Mutant CSFV lacking cysteine (Cys) 171, the residue responsible for intermolecular disulfide bond formation, were found to be attenuated in pigs (Tews BA, Schürmann EM, Meyers G. J Virol 2009;83:4823-4834). In the course of an animal experiment with such a dimerization-negative CSFV mutant, viruses were reisolated from pigs that contained a mutation of serine (Ser) 209 to Cys. This mutation restored the ability to form disulphide-linked E rns homodimers. In transient expression studies E rns mutants carrying the S209C change were found to form homodimers with about wt efficiency. Also the secretion level of the mutated proteins was equivalent to that of wt E rns . Virus mutants containing the Cys171Ser/Ser209Cys configuration exhibited wt growth rates and increased virulence when compared with the Cys171Ser mutant. These results provide further support for the connection between CSFV virulence and E rns dimerization.

Cu,Zn superoxide dismutase structure from a microbial pathogen establishes a class with a conserved dimer interface.

PubMed

Forest, K T; Langford, P R; Kroll, J S; Getzoff, E D

2000-02-11

Macrophages and neutrophils protect animals from microbial infection in part by issuing a burst of toxic superoxide radicals when challenged. To counteract this onslaught, many Gram-negative bacterial pathogens possess periplasmic Cu,Zn superoxide dismutases (SODs), which act on superoxide to yield molecular oxygen and hydrogen peroxide. We have solved the X-ray crystal structure of the Cu,Zn SOD from Actinobacillus pleuropneumoniae, a major porcine pathogen, by molecular replacement at 1.9 A resolution. The structure reveals that the dimeric bacterial enzymes form a structurally homologous class defined by a water-mediated dimer interface, and share with all Cu,Zn SODs the Greek-key beta-barrel subunit fold with copper and zinc ions located at the base of a deep loop-enclosed active-site channel. Our structure-based sequence alignment of the bacterial enzymes explains the monomeric nature of at least two of these, and suggests that there may be at least one additional structural class for the bacterial SODs. Two metal-mediated crystal contacts yielded our C222(1) crystals, and the geometry of these sites could be engineered into proteins recalcitrant to crystallization in their native form. This work highlights structural differences between eukaryotic and prokaryotic Cu,Zn SODs, as well as similarities and differences among prokaryotic SODs, and lays the groundwork for development of antimicrobial drugs that specifically target periplasmic Cu,Zn SODs of bacterial pathogens. Copyright 12000 Academic Press.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds

PubMed Central

Ballet, Steven; Marczak, Ewa D.; Feytens, Debby; Salvadori, Severo; Sasaki, Yusuke; Abell, Andrew D.; Lazarus, Lawrence H.; Balboni, Gianfranco; Tourwé, Dirk

2010-01-01

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced δ/μ antagonists, as determined by the functional [S35]GTPγS binding assay, the dimerization of potent Aia-based ‘parent’ ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds. PMID:20137938

A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

PubMed Central

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Tamada, Taro; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

2012-01-01

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P21 and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances. PMID:22275000

The Pseudomonas aeruginosa AmrZ C-terminal domain mediates tetramerization and is required for its activator and repressor functions

PubMed Central

Xu, Binjie; Ju, Yue; Soukup, Randal J.; Ramsey, Deborah M.; Fishel, Richard; Wysocki, Vicki H.; Wozniak, Daniel J.

2015-01-01

Summary Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher-order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets. PMID:26549743

EDEM1 targets misfolded HLA-B27 dimers for endoplasmic reticulum associated degradation

PubMed Central

Guiliano, David B.; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J.; Campbell, Elaine C.; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J.; Kellam, Paul; Hebert, Daniel N.; Gould, Keith; Powis, Simon J.; Antoniou, Antony N.

2015-01-01

Objective HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We wanted to define the role of the UPR induced ER associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. Methods HeLa cell lines expressing only two copies of a carboxy terminally Sv5 tagged HLA-B27 were generated. The ER stress induced EDEM1 protein was over expressed by transfection and dimer levels monitored by immunoblotting. EDEM1, the UPR associated transcription factor XBP-1, the E3 ubiquitin ligase HRD1, the degradation associated derlin 1 and 2 proteins were inhibited by either short hairpin RNA or dominant negative mutants. The UPR associated ERAD of HLA-B27 was confirmed using ER stress inducing pharamacological agents in kinetic and pulse chase assays. Results We demonstrate that UPR induced machinery can target HLA-B27 dimers, and that dimer formation can be controlled by alterations to expression levels of components of the UPR induced ERAD pathway. HLA-B27 dimers and misfolded MHC class I monomeric molecules were detected bound to EDEM1, with overexpression of EDEM1 inhibiting HLA-B27 dimer formation. EDEM1 inhibition resulted in upregulation of HLA-B27 dimers, whilst UPR induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1 and derlin1/2. Conclusion The UPR ERAD pathway as described here can dispose of HLA-B27 dimers and presents a potential novel therapeutic target for the modulation of HLA-B27 associated inflammatory disease. PMID:25132672

Quantitative Experimental Determination of Primer-Dimer Formation Risk by Free-Solution Conjugate Electrophoresis

PubMed Central

Desmarais, Samantha M.; Leitner, Thomas; Barron, Annelise E.

2012-01-01

DNA barcodes are short, unique ssDNA primers that “mark” individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis (FSCE) approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer-barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 basepairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive basepairs formed, yet non-consecutive basepairs did not create stable dimers even when 20 out of 30 possible basepairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation. PMID:22331820

Molecular dynamics studies of the protein-protein interactions in inhibitor of κB kinase-β.

PubMed

Jones, Michael R; Liu, Cong; Wilson, Angela K

2014-02-24

Activation of the inhibitor of κB kinase subunit β (IKKβ) oligomer initiates a cascade that results in the translocation of transcription factors involved in mediating immune responses. Dimerization of IKKβ is required for its activation. Coarse-grained and atomistic molecular dynamics simulations were used to investigate the conformation-activity and structure-activity relationships within the oligomer assembly of IKKβ that are impacted upon activation, mutation, and binding of ATP. Intermolecular interactions, free energies, and conformational changes were compared among several conformations, including a monomer, two different dimers, and the tetramer. Modifications to the activation segment induce conformational changes that disrupt dimerization and suggest that the multimeric assembly mediates a global stability for the enzyme that influences the activity of IKKβ.

Oligomerization of deoxynucleoside-biphosphate dimers - Template and linkage specificity

NASA Technical Reports Server (NTRS)

Visscher, J.; Van Der Woerd, R.; Bakker, C. G.; Schwartz, Alan W.

1989-01-01

The oligomerization of the activated 3-prime-5-prime pyrophosphate-linked dimer, pdAppdAp, is presently noted to be selectively favored by a poly(U) template over the 3-prime-3-prime and 5-prime-5-prime linked dimers. Both overall yields and the production of the longest oligomers were markedly stimulated by poly(U)'s presence; in its absence, the 5-prime-5-prime linked dimer became the most reactive, yielding chains of the order of 60 monomer-unit lengths. Remarkable self-organization properties are noted for the 5-prime-5-prime dimer of pdAp.

Probing actin polymerization by intermolecular cross-linking.

PubMed

Millonig, R; Salvo, H; Aebi, U

1988-03-01

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.