WorldWideScience

Sample records for surface binding sites

  1. Accurate prediction of peptide binding sites on protein surfaces.

    Directory of Open Access Journals (Sweden)

    Evangelia Petsalaki

    2009-03-01

    Full Text Available Many important protein-protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled but where no structure of the protein-peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein-peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.

  2. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a cat......Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  3. Binding of oxygen on vacuum fractured pyrite surfaces: Reactivity of iron and sulfur surface sites

    Science.gov (United States)

    Berlich, A. G.; Nesbitt, H. W.; Bancroft, G. M.; Szargan, R.

    2013-05-01

    Synchrotron radiation excited photoelectron spectroscopy (SXPS) has been used to study the interaction of oxygen with vacuum fractured pyrite surfaces. Especially valence band spectra obtained with 30 eV photon energy were analyzed to provide a mechanism of the incipient steps of pyrite oxidation. These spectra are far more sensitive to the oxidation than sulfur or iron core level spectra. It is shown that oxygen is adsorbed on Fe(II) surface sites restoring the octahedral coordination of the Fe(II) sites. This process leads to the removal of two surface states in the valence band which are located at the low and high binding energy sides of the outer valence band, respectively. The existence of these surface states which have been proposed by calculations is experimentally proven. Furthermore, it is shown, that the sulfur sites are more reactive than expected. Sulfite like species are already formed after the lowest oxygen exposure of 10 L. This oxidation occurs at sulfur sites neighboring the Fe(II) surface sites. Oxidation of the S2 - surface sites which were considered as the most reactive species in former studies is second. No iron(III) oxides are formed during oxygen exposure, supporting the assumption that water plays an important role in the oxidation mechanism of pyrite surfaces.

  4. Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display.

    Science.gov (United States)

    Najar, Tariq Ahmad; Khare, Shruti; Pandey, Rajesh; Gupta, Satish K; Varadarajan, Raghavan

    2017-03-07

    We describe a facile method for mapping protein:ligand binding sites and conformational epitopes. The method uses a combination of Cys scanning mutagenesis, chemical labeling, and yeast surface display. While Ala scanning is widely used for similar purposes, often mutation to Ala (or other amino acids) has little effect on binding, except at hotspot residues. Many residues in physical contact with a binding partner are insensitive to substitution with Ala. In contrast, we show that labeling of Cys residues in a binding site consistently abrogates binding. We couple this methodology to yeast surface display and deep sequencing to map conformational epitopes targeted by both monoclonal antibodies and polyclonal sera as well as a protein:ligand binding site. The method does not require purified protein, can distinguish buried and exposed residues, and can be extended to other display formats, including mammalian cells and viruses, emphasizing its wide applicability. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Probing and mapping the binding sites on streptavidin imprinted polymer surface

    International Nuclear Information System (INIS)

    Duman, Memed

    2014-01-01

    Molecular imprinting is an effective technique for preparing recognition sites which act as synthetic receptors on polymeric surfaces. Herein, we synthesized MIP surfaces with specific binding sites for streptavidin and characterized them at nanoscale by using two different atomic force microscopy (AFM) techniques. While the single molecule force spectroscopy (SMFS) reveals the unbinding kinetics between streptavidin molecule and binding sites, simultaneous topography and recognition imaging (TREC) was employed, for the first time, to directly map the binding sites on streptavidin imprinted polymers. Streptavidin modified AFM cantilever showed specific unbinding events with an unbinding force around 300 pN and the binding probability was calculated as 35.2% at a given loading rate. In order to prove the specificity of the interaction, free streptavidin molecules were added to AFM liquid cell and the binding probability was significantly decreased to 7.6%. Moreover, the recognition maps show that the smallest recognition site with a diameter of around ∼ 21 nm which corresponds to a single streptavidin molecule binding site. We believe that the potential of combining SMFS and TREC opens new possibilities for the characterization of MIP surfaces with single molecule resolution under physiological conditions. - Graphical abstract: Simultaneous Topography and RECognition (TREC) imaging is a novel characterization technique to reveal binding sites on molecularly imprinted polymer surfaces with single molecule resolution under physiological conditions. - Highlights: • Highly specific streptavidin printed polymer surfaces were synthesized. • Unbinding kinetic rate of single streptavidin molecule was studied by SMFS. • The distribution of binding pockets was revealed for the first time by TREC imaging. • TREC showed that the binding pockets formed nano-domains on MIP surface. • SMFS and TREC are powerful AFM techniques for characterization of MIP surfaces

  6. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was ...

  7. Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

    International Nuclear Information System (INIS)

    Pollack, A.E.; Wooten, G.F.

    1987-01-01

    The binding of the opiate antagonist 3 H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3 H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

  8. Predicting Ligand Binding Sites on Protein Surfaces by 3-Dimensional Probability Density Distributions of Interacting Atoms

    Science.gov (United States)

    Jian, Jhih-Wei; Elumalai, Pavadai; Pitti, Thejkiran; Wu, Chih Yuan; Tsai, Keng-Chang; Chang, Jeng-Yih; Peng, Hung-Pin; Yang, An-Suei

    2016-01-01

    Predicting ligand binding sites (LBSs) on protein structures, which are obtained either from experimental or computational methods, is a useful first step in functional annotation or structure-based drug design for the protein structures. In this work, the structure-based machine learning algorithm ISMBLab-LIG was developed to predict LBSs on protein surfaces with input attributes derived from the three-dimensional probability density maps of interacting atoms, which were reconstructed on the query protein surfaces and were relatively insensitive to local conformational variations of the tentative ligand binding sites. The prediction accuracy of the ISMBLab-LIG predictors is comparable to that of the best LBS predictors benchmarked on several well-established testing datasets. More importantly, the ISMBLab-LIG algorithm has substantial tolerance to the prediction uncertainties of computationally derived protein structure models. As such, the method is particularly useful for predicting LBSs not only on experimental protein structures without known LBS templates in the database but also on computationally predicted model protein structures with structural uncertainties in the tentative ligand binding sites. PMID:27513851

  9. Surface binding sites (SBSs), mechanism and regulation of enzymes degrading amylopectin and α-limit dextrins

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Cockburn, Darrell; Nielsen, Jonas W.

    2013-01-01

    Certain enzymes interact with polysaccharides at surface binding sites (SBSs) situated outside of their active sites. SBSs are not easily identified and their function has been discerned in relatively few cases. Starch degradation is a concerted action involving GH13 hydrolases. New insight...... into barley seed α-amylase 1 (AMY1) and limit dextrinase (LD) includes i. kinetics of bi-exponential amylopectin hydrolysis by AMY1, one reaction having low Km (8 μg/mL) and high kcat (57 s-1) and the other high Km (97 μg/mL) and low kcat (23 s-1). β-Cyclodextrin (β-CD) inhibits the first reaction by binding...

  10. LIGSITEcsc: predicting ligand binding sites using the Connolly surface and degree of conservation

    Directory of Open Access Journals (Sweden)

    Schroeder Michael

    2006-09-01

    Full Text Available Abstract Background Identifying pockets on protein surfaces is of great importance for many structure-based drug design applications and protein-ligand docking algorithms. Over the last ten years, many geometric methods for the prediction of ligand-binding sites have been developed. Results We present LIGSITEcsc, an extension and implementation of the LIGSITE algorithm. LIGSITEcsc is based on the notion of surface-solvent-surface events and the degree of conservation of the involved surface residues. We compare our algorithm to four other approaches, LIGSITE, CAST, PASS, and SURFNET, and evaluate all on a dataset of 48 unbound/bound structures and 210 bound-structures. LIGSITEcsc performs slightly better than the other tools and achieves a success rate of 71% and 75%, respectively. Conclusion The use of the Connolly surface leads to slight improvements, the prediction re-ranking by conservation to significant improvements of the binding site predictions. A web server for LIGSITEcsc and its source code is available at scoppi.biotec.tu-dresden.de/pocket.

  11. Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian

    2014-01-01

    Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half ...

  12. Impacts of Surface Site Coordination on Arsenate Adsorption: Macroscopic Uptake and Binding Mechanisms on Aluminum Hydroxide Surfaces.

    Science.gov (United States)

    Xu, Tingying; Catalano, Jeffrey G

    2016-12-13

    Aluminum hydroxides play important roles in regulating the fate and transport of contaminants and nutrients in soils and aquatic systems. Like many metal oxides, these minerals display surface functional groups in a series of coordination states, each of which may differ in its affinity for adsorbates. The distribution of functional group types varies among distinct surfaces of aluminum hydroxides, and we thus hypothesize that the adsorption behavior and mechanisms will show a dependence on particle morphology. To test this hypothesis, we investigate arsenate adsorption on two aluminum hydroxide polymorphs with distinct particle morphologies, gibbsite [γ-Al(OH) 3 ] and bayerite [α-Al(OH) 3 ], at pH 4 and 7. Synthetic gibbsite platelets expose large (001) basal surfaces predicted to be terminated by doubly coordinated functional groups (>Al 2 OH). In contrast, synthetic bayerite microrods display mainly edge surfaces (parallel to the c axis) containing abundant singly coordinated functional groups (>AlOH 2 ). Macroscopic adsorption studies show that gibbsite adsorbs less arsenate per unit surface area than bayerite at both pH values and suggest that two surface complexes form on each material. Similar electrokinetic behavior is displayed at the same relative coverages of arsenate, suggesting that similar reactive surface groups (>AlOH 2 ) control the surface charging on both particles. EXAFS spectroscopy shows that there is no variation in arsenate surface speciation on a given mineral with surface coverage or pH. Whereas bidentate binuclear inner-sphere species are the dominant complexes present, the EXAFS result suggest that outer-sphere species also occur on both minerals, with a greater abundance on gibbsite. This binding mode likely involves adsorption to >Al 2 OH sites, which have a slow ligand exchange rate that inhibits inner-sphere binding. These results demonstrate that adsorption mechanisms and capacity, even when normalized for specific surface area

  13. Prediction of Carbohydrate Binding Sites on Protein Surfaces with 3-Dimensional Probability Density Distributions of Interacting Atoms

    Science.gov (United States)

    Tsai, Keng-Chang; Jian, Jhih-Wei; Yang, Ei-Wen; Hsu, Po-Chiang; Peng, Hung-Pin; Chen, Ching-Tai; Chen, Jun-Bo; Chang, Jeng-Yih; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall

  14. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  15. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    International Nuclear Information System (INIS)

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  16. Characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein.

    Science.gov (United States)

    Galloux, Marie; Tarus, Bogdan; Blazevic, Ilfad; Fix, Jenna; Duquerroy, Stéphane; Eléouët, Jean-François

    2012-08-01

    The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P(CTD)) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N(NTD)) as a P binding domain. A biochemical characterization of the P(CTD) and molecular modeling of the N(NTD) allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P(CTD) interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

  17. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    Energy Technology Data Exchange (ETDEWEB)

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon (UC); (UIC)

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  18. Discovery and characterization of surface binding sites in polysaccharide converting enzymes

    DEFF Research Database (Denmark)

    Wilkens, Casper

    a generalization and may mask the significance of these sites in catalysis. GH62 α-L-arabinofuranosidase from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) does not contain a CBM, however, AnAbf62A-m2,3 interacts strongly with wheat arabinoxylan, birchwood xylan and oatspelt xylan in affinity gel electrophoresis...... no detectable affinity for maltotriose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable KD for only β-CD (0.94 ± 0.07 mM) and M7 (1.99 ± 0.10 mM). The plant phosphoglucan phosphatases Starch Excess 4 (SEX4) and Like Sex Four 2 (LSF2......) have different affinity for amylopectin, KD being 0.030 ± 0.002 and 1.59 ± 0.08 mg ml-1, respectively. Although corresponding KD values for β-CD of 1.69 ± 0.17 and 0.72 ± 0.06 mM are similar, SEX4 and LSF2 are suggested to have different binding modes and roles in starch dephosphorylation. While SEX4...

  19. Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum; Kramhøft, Birte; Bozonnet, Sophie

    2012-01-01

    Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380...... is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing...... kinetic properties of the wild type AMY1 and the Y380A mutant enzyme in hydrolysis of amylopectin, amylose and β-limit dextrin, and the inhibition by β-cyclodextrin. Progress curves of the release of reducing ends revealed a bi-exponential hydrolysis of amylopectin and β-limit dextrin, whereas hydrolysis...

  20. Two Secondary Carbohydrate Binding Sites on the Surface of Barley alpha-Amylase 1 Have Distinct Functions and Display Synergy in Hydrolysis of Starch Granules

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Bozonnet, Sophie; Seo, Eun-Seong

    2009-01-01

    Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)8-barrel and the noncatalytic C-terminal domain, respective...

  1. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Nielsen, Morten M.; Christiansen, Camilla

    2015-01-01

    degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley alpha-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser...

  2. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    identified in enzymes from a wide variety of families, though almost half are found in the α-amylase family GH13. The roles attributed to SBSs are not limited to targeting the enzyme to the substrate, but also include a variety of others such as guiding the substrate into the active site, altering enzyme...... specificity and acting as an allosteric site. Although SBSs share many roles with CBMs they may not simply be an alternative to CBMs, but rather complementary as SBSs and CBMs frequently co-occur in enzymes. Despite a relatively long history, it is only in recent years that SBSs have been studied in great...

  3. Analysis of Surface Binding Sites (SBS) within GH62, GH13, and GH77

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cockburn, Darrell; Andersen, Susan

    2015-01-01

    to form an SBS situated about 30 Å from the catalytic site. Compared to wild-type, W23A/Y44A AnAbf62A-m2,3 retained 45% activity on WAX and was less retarded in AGE by WAX as well as by barley β-glucan and birchwood xylan, which are neither hydrolysed nor inhibiting activity towards WAX. The presence...... of a functional SBS agrees with W23A/Y44A AnAbf62A-m2,3 retaining only 3-25% activity for arabinoxylo-oligosaccharides (AXOS) of DP 3-5 possibly reflecting allosteric activation of wild-type through SBS occupation by AXOS....

  4. Structural characterization of S100A15 reveals a novel zinc coordination site among S100 proteins and altered surface chemistry with functional implications for receptor binding

    Directory of Open Access Journals (Sweden)

    Murray Jill I

    2012-07-01

    Full Text Available Abstract Background S100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers. While the majority of human S100 proteins share 25-65% sequence similarity, S100A7 and its recently identified paralog, S100A15, display 93% sequence identity. Intriguingly, however, S100A7 and S100A15 serve distinct roles in inflammatory skin disease; S100A7 signals through the receptor for advanced glycation products (RAGE in a zinc-dependent manner, while S100A15 signals through a yet unidentified G-protein coupled receptor in a zinc-independent manner. Of the seven divergent residues that differentiate S100A7 and S100A15, four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface. Results To investigate the structural and functional consequences of these divergent clusters, we report the X-ray crystal structures of S100A15 and S100A7D24G, a hybrid variant where the zinc ligand Asp24 of S100A7 has been substituted with the glycine of S100A15, to 1.7 Å and 1.6 Å resolution, respectively. Remarkably, despite replacement of the Asp ligand, zinc binding is retained at the S100A15 dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. Zinc binding was confirmed using anomalous difference maps and solution binding studies that revealed similar affinities of zinc for S100A15 and S100A7. Additionally, the predicted receptor-binding surface on S100A7 is substantially more basic in S100A15 without incurring structural rearrangement. Conclusions Here we demonstrate that S100A15 retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among S100 proteins. The altered surface chemistry between S100A7 and S100A15 that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct

  5. Two secondary carbohydrate binding sites on the surface of barley alpha-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules.

    Science.gov (United States)

    Nielsen, Morten M; Bozonnet, Sophie; Seo, Eun-Seong; Mótyán, János A; Andersen, Joakim M; Dilokpimol, Adiphol; Abou Hachem, Maher; Gyémánt, Gyöngyi; Naested, Henrik; Kandra, Lili; Sigurskjold, Bent W; Svensson, Birte

    2009-08-18

    Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.

  6. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    International Nuclear Information System (INIS)

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  7. Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

    DEFF Research Database (Denmark)

    Nielsen, Morten Munch; Seo, E.S.; Dilokpimol, Adiphol

    2008-01-01

    Germinating barley seeds contain multiple forms of alpha-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The alpha-amylases are endo-acting and possess a long substrate binding cleft with a charact...

  8. Binding matrix: a novel approach for binding site recognition.

    Science.gov (United States)

    Kim, Jan T; Gewehr, Jan E; Martinetz, Thomas

    2004-06-01

    Recognition of protein-DNA binding sites in genomic sequences is a crucial step for discovering biological functions of genomic sequences. Explosive growth in availability of sequence information has resulted in a demand for binding site detection methods with high specificity. The motivation of the work presented here is to address this demand by a systematic approach based on Maximum Likelihood Estimation. A general framework is developed in which a large class of binding site detection methods can be described in a uniform and consistent way. Protein-DNA binding is determined by binding energy, which is an approximately linear function within the space of sequence words. All matrix based binding word detectors can be regarded as different linear classifiers which attempt to estimate the linear separation implied by the binding energy function. The standard approaches of consensus sequences and profile matrices are described using this framework. A maximum likelihood approach for determining this linear separation leads to a novel matrix type, called the binding matrix. The binding matrix is the most specific matrix based classifier which is consistent with the input set of known binding words. It achieves significant improvements in specificity compared to other matrices. This is demonstrated using 95 sets of experimentally determined binding words provided by the TRANSFAC database.

  9. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  10. LIBRA: LIgand Binding site Recognition Application.

    Science.gov (United States)

    Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

    2015-12-15

    In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Ethylene binding site affinity in ripening apples

    Energy Technology Data Exchange (ETDEWEB)

    Blankenship, S.M. (North Carolina State Univ., Raleigh, NC (United States). Dept. of Horticultural Science); Sisler, E.C. (North Carolina State Univ., Raleigh, NC (United States))

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  12. Definition and display of steric, hydrophobic, and hydrogen-bonding properties of ligand binding sites in proteins using Lee and Richards accessible surface: validation of a high-resolution graphical tool for drug design.

    Science.gov (United States)

    Bohacek, R S; McMartin, C

    1992-05-15

    The accessible surface, described by Lee and Richards (the L&R surface: J. Mol. Biol. 1971, 55, 379), has remarkably useful properties for displaying ligand-protein interactions. The surface is placed one van der Waals radius plus one probe radius away from the protein atoms. The ligands are displayed in skeletal form. With a suitable probe radius, those parts of the ligand in good van der Waals contact with the protein binding site are found superimposed on the L&R surface. Display of the surface using parallel contours therefore provides a very powerful guide for interactive drug design because only ligand atoms lying on or close to the surface are in low-energy contact. The ability of the surface to accurately display steric complementarity between ligands and proteins was optimized using data from small molecule crystal structures. The possibility of displaying the chemical specificity of the binding site was also investigated. The surface can be colored to give precise information about chemical specificity. Electrostatic potential, electrostatic gradient, and distance to hydrogen-bonding groups were tested as methods of displaying chemical specificity. The ability of these methods to describe the complementarity actually observed in the interior of proteins was compared. High-resolution crystal data for ribonuclease and trypsin was used. The environment surrounding extended peptide chains in the protein was treated as a virtual binding site. The peptide chain served as a virtual ligand. This large sample of experimental data was used to measure the correlation between type of ligand atom and the calculated property of the nearest binding site surface. The best correlation was obtained using hydrogen-bonding properties of the binding site. Using this parameter the surface could be divided into three separate zones representing the hydrophobic, hydrogen-bond-acceptor, and hydrogen-bond-donor properties of the binding site. The percentage of hydrophobic ligand

  13. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  14. Tissue specificity of endothelin binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Bolger, G.T.; Liard, F.; Krogsrud, R.; Thibeault, D.; Jaramillo, J. (BioMega, Inc., Laval, Quebec (Canada))

    1990-09-01

    A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was approximately 0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea greater than lung parenchyma = vas deferens much greater than aorta = portal vein = atrium greater than bladder greater than ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45-60 min at 25 degrees C. The calculated microassociation constant was 9.67 x 10(5) s-1 M-1. Only 15-20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent.

  15. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  16. Binding sites for gonadotropins in human postmenopausal ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, R.; Shima, K.; Yamoto, M.; Kobayashi, M.; Nishimori, K.; Hiraoka, J.

    1989-02-01

    The binding of human LH and human FSH to postmenopausal ovarian tissue from 21 patients with cervical carcinoma was analyzed. The binding sites for FSH and LH were demonstrated in postmenopausal ovarian tissue. The surface-binding sites for gonadotropins were localized in the cells of cortical stroma of the postmenopausal ovary. In addition, diffuse cytoplasmic staining of endogenous estrogen and 3 beta-hydroxysteroid dehydrogenase activity were detected immunohistochemically and histochemically in the cells of the cortical stroma. Electron microscopic study also suggested steroidogenic function in the cells of the cortical stroma. The results of the present study suggest that postmenopausal ovaries contain specific binding sites for pituitary gonadotropins and play a role in ovarian steroidogenesis.

  17. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Ribosome binding site recognition using neural networks

    Directory of Open Access Journals (Sweden)

    Márcio Ferreira da Silva Oliveira

    2004-01-01

    Full Text Available Pattern recognition is an important process for gene localization in genomes. The ribosome binding sites are signals that can help in the identification of a gene. It is difficult to find these signals in the genome through conventional methods because they are highly degenerated. Artificial Neural Networks is the approach used in this work to address this problem.

  19. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  20. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  1. Binding sites analyser (BiSA: software for genomic binding sites archiving and overlap analysis.

    Directory of Open Access Journals (Sweden)

    Matloob Khushi

    Full Text Available Genome-wide mapping of transcription factor binding and histone modification reveals complex patterns of interactions. Identifying overlaps in binding patterns by different factors is a major objective of genomic studies, but existing methods to archive large numbers of datasets in a personalised database lack sophistication and utility. Therefore we have developed transcription factor DNA binding site analyser software (BiSA, for archiving of binding regions and easy identification of overlap with or proximity to other regions of interest. Analysis results can be restricted by chromosome or base pair overlap between regions or maximum distance between binding peaks. BiSA is capable of reporting overlapping regions that share common base pairs; regions that are nearby; regions that are not overlapping; and average region sizes. BiSA can identify genes located near binding regions of interest, genomic features near a gene or locus of interest and statistical significance of overlapping regions can also be reported. Overlapping results can be visualized as Venn diagrams. A major strength of BiSA is that it is supported by a comprehensive database of publicly available transcription factor binding sites and histone modifications, which can be directly compared to user data. The documentation and source code are available on http://bisa.sourceforge.net.

  2. Binding site graphs: a new graph theoretical framework for prediction of transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Timothy E Reddy

    2007-05-01

    Full Text Available Computational prediction of nucleotide binding specificity for transcription factors remains a fundamental and largely unsolved problem. Determination of binding positions is a prerequisite for research in gene regulation, a major mechanism controlling phenotypic diversity. Furthermore, an accurate determination of binding specificities from high-throughput data sources is necessary to realize the full potential of systems biology. Unfortunately, recently performed independent evaluation showed that more than half the predictions from most widely used algorithms are false. We introduce a graph-theoretical framework to describe local sequence similarity as the pair-wise distances between nucleotides in promoter sequences, and hypothesize that densely connected subgraphs are indicative of transcription factor binding sites. Using a well-established sampling algorithm coupled with simple clustering and scoring schemes, we identify sets of closely related nucleotides and test those for known TF binding activity. Using an independent benchmark, we find our algorithm predicts yeast binding motifs considerably better than currently available techniques and without manual curation. Importantly, we reduce the number of false positive predictions in yeast to less than 30%. We also develop a framework to evaluate the statistical significance of our motif predictions. We show that our approach is robust to the choice of input promoters, and thus can be used in the context of predicting binding positions from noisy experimental data. We apply our method to identify binding sites using data from genome scale ChIP-chip experiments. Results from these experiments are publicly available at http://cagt10.bu.edu/BSG. The graphical framework developed here may be useful when combining predictions from numerous computational and experimental measures. Finally, we discuss how our algorithm can be used to improve the sensitivity of computational predictions of

  3. Bifunctional avidin with covalently modifiable ligand binding site.

    Directory of Open Access Journals (Sweden)

    Jenni Leppiniemi

    Full Text Available The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (streptavidin to improve the existing applications. Even so, (streptavidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

  4. Autoradiographic localization of benzomorphan binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Crain, B.J.; Kwenjen Chang; McNamara, J.O.; Valdes, F.

    1985-07-17

    The benzomorphan subpopulation of opiate binding sites was labeled by (TH)diprenorphine in the presence of unlabeled ligands selected to quench and delta opiate binding sites. The distribution of benzomorphan binding sites was then localized autoradiographically. The distribution differs from the distributions of , delta and kappa opiate binding and is quite similar to the distribution of US -endorphin immunoreactivity. These observations support the hypothesis, based on biochemical studies in brain membranes, that benzomorphan binding sites may represent the ligand recognition sites of putative epsilon receptors. (Auth.). 34 refs.; 3 figs.

  5. Binding Sites for Amyloid-β Oligomers and Synaptic Toxicity

    Science.gov (United States)

    Smith, Levi M.; Strittmatter, Stephen M.

    2017-01-01

    In Alzheimer’s disease (AD), insoluble and fibrillary amyloid-β (Aβ) peptide accumulates in plaques. However, soluble Aβ oligomers are most potent in creating synaptic dysfunction and loss. Therefore, receptors for Aβ oligomers are hypothesized to be the first step in a neuronal cascade leading to dementia. A number of cell-surface proteins have been described as Aβ binding proteins, and one or more are likely to mediate Aβ oligomer toxicity in AD. Cellular prion protein (PrPC) is a high-affinity Aβ oligomer binding site, and a range of data delineates a signaling pathway leading from Aβ complexation with PrPC to neuronal impairment. Further study of Aβ binding proteins will define the molecular basis of this crucial step in AD pathogenesis. PMID:27940601

  6. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  7. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    Science.gov (United States)

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

  8. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  9. Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis.

    Science.gov (United States)

    Ueki, Tatsuya; Kawakami, Norifumi; Toshishige, Masaaki; Matsuo, Koichi; Gekko, Kunihiko; Michibata, Hitoshi

    2009-10-01

    Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+. These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+.

  10. Incorporating evolution of transcription factor binding sites into ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Incorporating evolution of transcription factor binding sites into annotated alignments. 841. J. Biosci. 32(5), August 2007. 1. Introduction. A majority of computational approaches that aim to predict transcription factor binding sites employ cross- species comparison to focus on conserved locations. Such a comparison helps in ...

  11. Druggability of methyl-lysine binding sites

    Science.gov (United States)

    Santiago, C.; Nguyen, K.; Schapira, M.

    2011-12-01

    Structural modules that specifically recognize—or read—methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions.

  12. Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis.

    Science.gov (United States)

    Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Kuroda, Shun'ichi; Niimi, Tomoaki

    2015-12-21

    Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  14. The impact of the competitive adsorption of ions at surface sites on surface free energies and surface forces

    Science.gov (United States)

    Parsons, Drew F.; Salis, Andrea

    2015-04-01

    The relationship between surface charge and surface potential at the solid-liquid interface is often determined by a charge regulation process, the chemisorption of a potential determining ion such as H+. A subtle ion-specific effect can be observed when other ions compete with the primary potential determining ion to bind to a surface site. Site competition may involve alternative ions competing for a first binding site, e.g., metals ions competing with H+ to bind to a negatively charged oxide or carboxyl site. Second-binding sites with site competition may also be found, including amphoteric OH2+ sites, or anion binding to amine groups. In this work, a general theoretical model is developed to describe the competitive adsorption of ions at surface sites. Applied to the calculation of forces, the theory predicts a 20% increase in repulsion between titania surfaces in 1 mM NaCl, and a 25% reduction in repulsion between silica surfaces in 0.1M NaCl compared to calculations neglecting ion site competition.

  15. Hanford Site surface environmental surveillance

    International Nuclear Information System (INIS)

    Dirkes, R.L.

    1998-01-01

    Environmental surveillance of the Hanford Site and the surrounding region is conducted to demonstrate compliance with environmental regulations, confirm adherence to US Department of Energy (DOE) environmental protection policies, support DOE environmental management decisions, and provide information to the public. The Surface Environmental Surveillance Project (SESP) is a multimedia environmental monitoring program conducted to measure the concentrations of radionuclides and chemical contaminants in the environment and assess the integrated effects of these contaminants on the environment and the public. The monitoring program includes sampling air, surface water, sediments, soil, natural vegetation, agricultural products, fish, and wildlife. Functional elements inherent in the operation of the SESP include project management, quality assurance/control, training, records management, environmental sampling network design and implementation, sample collection, sample analysis, data management, data review and evaluation, exposure assessment, and reporting. The SESP focuses on those contaminant/media combinations calculated to have the highest potential for contributing to off-site exposure. Results of the SESP indicate that contaminant concentrations in the Hanford environs are very low, generally below environmental standards, at or below analytical detection levels, and indicative of environmental levels. However, areas of elevated contaminant concentrations have been identified at Hanford. The extent of these areas is generally limited to past operating areas and waste disposal sites

  16. Characterization of nicotine binding to the rat brain P2 preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

    International Nuclear Information System (INIS)

    Sloan, J.W.

    1984-01-01

    These studies show that nicotine binds to the rat brain P 2 preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) have been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-[ 3 H]nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-[ 3 H]nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine

  17. Salt-mediated two-site ligand binding by the cocaine-binding aptamer.

    Science.gov (United States)

    Neves, Miguel A D; Slavkovic, Sladjana; Churcher, Zachary R; Johnson, Philip E

    2017-02-17

    Multisite ligand binding by proteins is commonly utilized in the regulation of biological systems and exploited in a range of biochemical technologies. Aptamers, although widely utilized in many rationally designed biochemical systems, are rarely capable of multisite ligand binding. The cocaine-binding aptamer is often used for studying and developing sensor and aptamer-based technologies. Here, we use isothermal titration calorimetry (ITC) and NMR spectroscopy to demonstrate that the cocaine-binding aptamer switches from one-site to two-site ligand binding, dependent on NaCl concentration. The high-affinity site functions at all buffer conditions studied, the low-affinity site only at low NaCl concentrations. ITC experiments show the two ligand-binding sites operate independently of one another with different affinities and enthalpies. NMR spectroscopy shows the second binding site is located in stem 2 near the three-way junction. This ability to control ligand binding at the second site by adjusting the concentration of NaCl is rare among aptamers and may prove a useful in biotechnology applications. This work also demonstrates that in vitro selected biomolecules can have functions as complex as those found in nature. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  20. Exploring the composition of protein-ligand binding sites on a large scale.

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

    Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

  1. Binding affinity of a small molecule to an amorphous polymer in a solvent. Part 1: free energy of binding to a binding site.

    Science.gov (United States)

    Chunsrivirot, Surasak; Diao, Ying; Trout, Bernhardt L

    2011-10-18

    Crystallization is commonly used in a separation and purification process in the production of a wide range of materials in various industries. In industry, crystallization usually starts with heterogeneous nucleation on a foreign surface. The complicated mechanism of heterogeneous nucleation is not well understood; however, we hypothesize that there might be a possible correlation between binding affinity to a surface and enhancement of nucleation. Recent studies show that amorphous polymers can be used to control crystallization, selectively produce pharmaceutical polymorphs, and discover novel pharmaceutical polymorphs. To investigate the possible correlation between the binding affinity of one molecule to key binding sites (local binding) and heterogeneous nucleation activity as well as the possibility of using this binding affinity to help guide the selection of polymers that promote heterogeneous nucleation, we computed the free energy of binding of aspirin to four nonporous cross-linked polymers in an ethanol-water 38 v% mixture. These cross-linked polymers are poly(4-acryloylmorpholine) (PAM), poly(2-carboxyethyl acrylate) (PCEA), poly(4-hydroxylbutyl acrylate) (PHBA), and polystyrene (PS); all of them were cross-linked with divinylbenzene (DVB). These systems were used because their heterogeneous nucleation activities are available in literature, and the ranking is PAM > PCEA > PHBA ≈ PS. We generated three independent surfaces for each polymer and computed the free energy of binding of aspirin to the best binding site that we found on each surface. The average free energies of binding to the best sites of PAM, PCEA, PHBA, and PS are -20.4 ± 1.0, -16.7 ± 1.0, -14.4 ± 1.1, and -13.6 ± 1.1 kcal/mol, respectively. We found that the trend of the magnitudes of the average free energies of binding to the best sites is PAM > PCEA > PHBA ≈ PS. This trend is very similar to that of heterogeneous nucleation activity. Our results suggest the importance of the

  2. Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

    Science.gov (United States)

    Haupt, V Joachim; Daminelli, Simone; Schroeder, Michael

    2013-01-01

    Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor

  3. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-04-01

    Autoradiography using /sup 125/I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat.

  4. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  5. Identification of substrate binding sites in enzymes by computational solvent mapping.

    Science.gov (United States)

    Silberstein, Michael; Dennis, Sheldon; Brown, Lawrence; Kortvelyesi, Tamas; Clodfelter, Karl; Vajda, Sandor

    2003-10-03

    Enzyme structures determined in organic solvents show that most organic molecules cluster in the active site, delineating the binding pocket. We have developed algorithms to perform solvent mapping computationally, rather than experimentally, by placing molecular probes (small molecules or functional groups) on a protein surface, and finding the regions with the most favorable binding free energy. The method then finds the consensus site that binds the highest number of different probes. The probe-protein interactions at this site are compared to the intermolecular interactions seen in the known complexes of the enzyme with various ligands (substrate analogs, products, and inhibitors). We have mapped thermolysin, for which experimental mapping results are also available, and six further enzymes that have no experimental mapping data, but whose binding sites are well characterized. With the exception of haloalkane dehalogenase, which binds very small substrates in a narrow channel, the consensus site found by the mapping is always a major subsite of the substrate-binding site. Furthermore, the probes at this location form hydrogen bonds and non-bonded interactions with the same residues that interact with the specific ligands of the enzyme. Thus, once the structure of an enzyme is known, computational solvent mapping can provide detailed and reliable information on its substrate-binding site. Calculations on ligand-bound and apo structures of enzymes show that the mapping results are not very sensitive to moderate variations in the protein coordinates.

  6. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  7. (-)PPAP: a new and selective ligand for sigma binding sites.

    Science.gov (United States)

    Glennon, R A; Battaglia, G; Smith, J D

    1990-11-01

    Most agents employed for the investigation of sigma (sigma) binding sites display relatively low affinity for these sites, bind both at sigma sites and at either phencyclidine (PCP) sites or dopamine receptors with similar affinity, and/or produce some dopaminergic activity in vivo. We describe a new agent, (-)PPAP or R(-)-N-(3-phenyl-n-propyl)-1-phenyl-2-aminopropane hydrochloride, that binds with high affinity and selectivity at sigma (IC50 = 24 nM) versus either PCP sites (IC50 greater than 75,000 nM) or D1 and D2 dopamine receptors (IC50 greater than 5,000 nM). The sigma affinity of this agent is comparable to that of the standard ligands (+)-3-PPP and DTG. Furthermore, although (-)PPAP is structurally related to amphetamine, it neither produces nor antagonizes amphetamine-like stimulus effect in rats trained to discriminate 1 mg/kg of S(+)amphetamine from saline.

  8. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  9. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  10. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs...... have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion...

  11. Biophysical fitness landscapes for transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Allan Haldane

    2014-07-01

    Full Text Available Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions.

  12. Biophysical fitness landscapes for transcription factor binding sites.

    Science.gov (United States)

    Haldane, Allan; Manhart, Michael; Morozov, Alexandre V

    2014-07-01

    Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs) and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions.

  13. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Human chorionic ganodotropin binding sites in the human endometrium

    International Nuclear Information System (INIS)

    Bhattacharya, S.; Banerjee, J.; Sen, S.; Manna, P.R.

    1993-01-01

    The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. The authors have identified the hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5x10 -10 mol/l and in anovulatory women to be 3.1x10 -10 mol/l. The maximum binding capacity varied considerably between ovulatory and anovulatory endometrium. Among the divalent metal ions tested Zn 2+ effected a remarkable increase in [ 125 I]hCG binding to the endometrium, whereas Mn 2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [ 125 I]hCG binding to endometrium. 14 refs., 3 figs

  15. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  16. Transcription factor binding sites prediction based on modified nucleosomes.

    Directory of Open Access Journals (Sweden)

    Mohammad Talebzadeh

    Full Text Available In computational methods, position weight matrices (PWMs are commonly applied for transcription factor binding site (TFBS prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, "modified nucleosomes neighboring" and "modified nucleosomes occupancy", to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method

  17. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  18. Insulin binding sites in various segments of the rabbit nephron

    International Nuclear Information System (INIS)

    Nakamura, R.; Emmanouel, D.S.; Katz, A.I.

    1983-01-01

    Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of 125 I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125 I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the ''diluting segment'' (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone

  19. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.G.

    1987-01-01

    The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics

  20. Computational mapping identifies the binding sites of organic solvents on proteins

    Science.gov (United States)

    Dennis, Sheldon; Kortvelyesi, Tamas; Vajda, Sandor

    2002-01-01

    Computational mapping places molecular probes—small molecules or functional groups—on a protein surface to identify the most favorable binding positions. Although x-ray crystallography and NMR show that organic solvents bind to a limited number of sites on a protein, current mapping methods result in hundreds of energy minima and do not reveal why some sites bind molecules with different sizes and polarities. We describe a mapping algorithm that explains the origin of this phenomenon. The algorithm has been applied to hen egg-white lysozyme and to thermolysin, interacting with eight and four different ligands, respectively. In both cases the search finds the consensus site to which all molecules bind, whereas other positions that bind only certain ligands are not necessarily found. The consensus sites are pockets of the active site, lined with partially exposed hydrophobic residues and with a number of polar residues toward the edge. These sites can accommodate each ligand in a number of rotational states, some with a hydrogen bond to one of the nearby donor/acceptor groups. Specific substrates and/or inhibitors of hen egg-white lysozyme and thermolysin interact with the same side chains identified by the mapping, but form several hydrogen bonds and bind in unique orientations. PMID:11904374

  1. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S. (Oregon HSU)

    2012-05-24

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  2. Multiple [3H]-nemonapride binding sites in calf brain.

    Science.gov (United States)

    Helmeste, D M; Tang, S W; Li, M; Fang, H

    1997-07-01

    [3H]-Nemonapride has been the ligand of choice to label D4 dopamine receptors. Its specificity was questioned when it was discovered that sigma (sigma) sites were also labeled by [3H]-nemonapride. To further characterize the binding of [3H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [3H]-nemonapride labeled multiple sites. Dopaminergic and sigma sites were the most prominent. The sigma binding profile was sigma-1 like with a Ki binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and sigma sites, respectively, revealed additional, not previously characterized binding sites for [3H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [3H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met.

  3. Binding-site assessment by virtual fragment screening.

    Directory of Open Access Journals (Sweden)

    Niu Huang

    2010-04-01

    Full Text Available The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

  4. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  5. Eel calcitonin binding site distribution and antinociceptive activity in rats

    International Nuclear Information System (INIS)

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-01-01

    The distribution of binding site for [ 125 I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [ 125 I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain

  6. Oligomycin frames a common drug-binding site in the ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

    2015-12-01

    We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

  7. Predicting the binding modes and sites of metabolism of xenobiotics.

    Science.gov (United States)

    Mukherjee, Goutam; Lal Gupta, Pancham; Jayaram, B

    2015-07-01

    Metabolism studies are an essential integral part of ADMET profiling of drug candidates to evaluate their safety and efficacy. Cytochrome P-450 (CYP) metabolizes a wide variety of xenobiotics/drugs. The binding modes of these compounds with CYP and their intrinsic reactivities decide the metabolic products. We report here a novel computational protocol, which comprises docking of ligands to heme-containing CYPs and prediction of binding energies through a newly developed scoring function, followed by analyses of the docked structures and molecular orbitals of the ligand molecules, for predicting the sites of metabolism (SOM) of ligands. The calculated binding free energies of 121 heme-containing protein-ligand docked complexes yielded a correlation coefficient of 0.84 against experiment. Molecular orbital analyses of the resultant top three unique poses of the docked complexes provided a success rate of 87% in identifying the experimentally known sites of metabolism of the xenobiotics. The SOM prediction methodology is freely accessible at .

  8. Incorporating evolution of transcription factor binding sites into ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Most current methods in this field adopt a multi-step ap proach that segregates the two aspects. Again, it is widely accepted that the ... In a simulated setting, we provide a proof of concept that the approach works given the ... We study how alignments and binding site predictions interplay at varying evolutionary distances ...

  9. Diversity and evolutionary relationship of nucleotide binding site ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by ...

  10. Dangerous connections : on binding site models of infectious disease dynamics

    NARCIS (Netherlands)

    Leung, Ka Yin; Diekmann, Odo

    2017-01-01

    We formulate models for the spread of infection on networks that are amenable to analysis in the large population limit. We distinguish three different levels: (1) binding sites, (2) individuals, and (3) the population. In the tradition of physiologically structured population models, the

  11. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  12. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  13. Full Length Amelogenin Binds to Cell Surface LAMP-1 on Tooth Root/Periodontium Associated Cells

    Science.gov (United States)

    Zhang, Hai; Tompkins, Kevin; Garrigues, Jacques; Snead, Malcolm L.; Gibson, Carolyn W.; Somerman, Martha J.

    2010-01-01

    Objectives Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells. Design Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0 to 100 μg/ml) and time course (0 to 120 minutes) assays were performed to determine the optimal conditions for live cell surface binding using immuno-fluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern. Results Maximum surface binding was achieved with 50 μg/ml of rp(H)M180 for 120 minutes. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1. Conclusions The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts. PMID:20382373

  14. Phosphorus Binding Sites in Proteins: Structural Preorganization and Coordination

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Greisen, Per Junior; Junker, Märta Caroline

    2014-01-01

    to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced...... by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell...

  15. Opioid binding site in EL-4 thymoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  16. Binding of dinitrogen to an iron-sulfur-carbon site

    Science.gov (United States)

    Čorić, Ilija; Mercado, Brandon Q.; Bill, Eckhard; Vinyard, David J.; Holland, Patrick L.

    2015-10-01

    Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

  17. The Pattern of microRNA Binding Site Distribution

    Directory of Open Access Journals (Sweden)

    Fangyuan Zhang

    2017-10-01

    Full Text Available Micro-RNA (miRNA or miR regulates at least 60% of the genes in the human genome through their target sites at mRNA 3’-untranslated regions (UTR, and defects in miRNA expression regulation and target sites are frequently observed in cancers. We report here a systematic analysis of the distribution of miRNA target sites. Using the evolutionarily conserved miRNA binding sites in the TargetScan database (release 7.1, we constructed a miRNA co-regulation network by connecting genes sharing common miRNA target sites. The network possesses characteristics of the ubiquitous small-world network. Non-hub genes in the network—those sharing miRNA target sites with small numbers of genes—tend to form small cliques with their neighboring genes, while hub genes exhibit high levels of promiscuousness in their neighboring genes. Additionally, miRNA target site distribution is extremely uneven. Among the miRNAs, the distribution concentrates on a small number of miRNAs, in that their target sites occur in an extraordinarily large number of genes, that is, they have large numbers of target genes. The distribution across the genes follows a similar pattern; the mRNAs of a small proportion of the genes contain extraordinarily large numbers of miRNA binding sites. Quantitatively, the patterns fit into the P(K ∝ K−α relationship (P(K: the number of miRNAs with K target genes or genes with K miRNA sites; α: a positive constant, the mathematical description of connection distribution among the nodes and a defining characteristic of the so-called scale-free networks—a subset of small-world networks. Notably, well-known tumor-suppressive miRNAs (Let-7, miR-15/16, 26, 29, 31, 34, 145, 200, 203–205, 223, and 375 collectively have more than expected target genes, and well-known cancer genes contain more than expected miRNA binding sites. In summary, miRNA target site distribution exhibits characteristics of the small-world network. The potential to use this

  18. Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

    Science.gov (United States)

    Gao, Jinxu; Mfuh, Adelphe; Amako, Yuka; Woo, Christina M

    2018-03-15

    Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

  19. Visualization of specific binding sites of benzodiazepine in human brain

    International Nuclear Information System (INIS)

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-01-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

  20. Scoring functions for transcription factor binding site prediction

    Directory of Open Access Journals (Sweden)

    Friberg Markus

    2005-04-01

    Full Text Available Abstract Background Transcription factor binding site (TFBS prediction is a difficult problem, which requires a good scoring function to discriminate between real binding sites and background noise. Many scoring functions have been proposed in the literature, but it is difficult to assess their relative performance, because they are implemented in different software tools using different search methods and different TFBS representations. Results Here we compare how several scoring functions perform on both real and semi-simulated data sets in a common test environment. We have also developed two new scoring functions and included them in the comparison. The data sets are from the yeast (S. cerevisiae genome. Our new scoring function LLBG (least likely under the background model performs best in this study. It achieves the best average rank for the correct motifs. Scoring functions based on positional bias performed quite poorly in this study. Conclusion LLBG may provide an interesting alternative to current scoring functions for TFBS prediction.

  1. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype was effici...... peptide-MHC complexes may have broad significance in the biology of T cell responses, including generation of the T cell repertoire, the specificity of mixed lymphocyte responses, and the immune surveillance of self and nonself antigens in peripheral lymphoid tissues.......Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  2. The next generation of transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Anthony Mathelier

    Full Text Available Finding where transcription factors (TFs bind to the DNA is of key importance to decipher gene regulation at a transcriptional level. Classically, computational prediction of TF binding sites (TFBSs is based on basic position weight matrices (PWMs which quantitatively score binding motifs based on the observed nucleotide patterns in a set of TFBSs for the corresponding TF. Such models make the strong assumption that each nucleotide participates independently in the corresponding DNA-protein interaction and do not account for flexible length motifs. We introduce transcription factor flexible models (TFFMs to represent TF binding properties. Based on hidden Markov models, TFFMs are flexible, and can model both position interdependence within TFBSs and variable length motifs within a single dedicated framework. The availability of thousands of experimentally validated DNA-TF interaction sequences from ChIP-seq allows for the generation of models that perform as well as PWMs for stereotypical TFs and can improve performance for TFs with flexible binding characteristics. We present a new graphical representation of the motifs that convey properties of position interdependence. TFFMs have been assessed on ChIP-seq data sets coming from the ENCODE project, revealing that they can perform better than both PWMs and the dinucleotide weight matrix extension in discriminating ChIP-seq from background sequences. Under the assumption that ChIP-seq signal values are correlated with the affinity of the TF-DNA binding, we find that TFFM scores correlate with ChIP-seq peak signals. Moreover, using available TF-DNA affinity measurements for the Max TF, we demonstrate that TFFMs constructed from ChIP-seq data correlate with published experimentally measured DNA-binding affinities. Finally, TFFMs allow for the straightforward computation of an integrated TF occupancy score across a sequence. These results demonstrate the capacity of TFFMs to accurately model DNA

  3. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2010-03-01

    Full Text Available The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates.We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data.Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct-interaction detection and TFBS-discovery accuracy. We estimated the accuracy

  4. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

    Directory of Open Access Journals (Sweden)

    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  5. Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.

    1985-01-01

    Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [ 125 I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis

  6. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  7. A molecular dynamics study of human serum albumin binding sites.

    Science.gov (United States)

    Artali, Roberto; Bombieri, Gabriella; Calabi, Luisella; Del Pra, Antonio

    2005-01-01

    A 2.0 ns unrestrained Molecular Dynamics was used to elucidate the geometric and dynamic properties of the HSA binding sites. The structure is not stress affected and the rmsds calculated from the published crystallographic data are almost constant for all the simulation time, with an averaged value of 2.4A. The major variability is in the C-terminus region. The trajectory analysis of the IIA binding site put in evidence fast oscillations for the Cgamma@Leu203...Cgamma@Leu275 and Cgamma@Leu219...Cgamma@Leu260 distances, with fluctuations around 250 ps, 1000 ps and over for the first, while the second is smoothly increasing with the simulation time from 7 to 10A. These variations are consistent with a volume increase up to 20% confirmed by the inter-domain contacts analysis, in particular for the pair O@Pro148...Ogamma@Ser283, representing the change of distance between IB-h9 and IIA-h6, O@Glu149...Ogamma@Ser189 for sub-domains IB-h9/IIA-h1 and N@Val339...Odelta2@Asp447 sub-domains IIB-h9/IIIA-h1. These inter-domain motions confirm the flexibility of the unfatted HSA with possible binding site pre-formation.

  8. Balance between hydration enthalpy and entropy is important for ice binding surfaces in Antifreeze Proteins.

    Science.gov (United States)

    Schauperl, Michael; Podewitz, Maren; Ortner, Teresa S; Waibl, Franz; Thoeny, Alexander; Loerting, Thomas; Liedl, Klaus R

    2017-09-19

    Antifreeze Proteins (AFPs) inhibit the growth of an ice crystal by binding to it. The detailed binding mechanism is, however, still not fully understood. We investigated three AFPs using Molecular Dynamics simulations in combination with Grid Inhomogeneous Solvation Theory, exploring their hydration thermodynamics. The observed enthalpic and entropic differences between the ice-binding sites and the inactive surface reveal key properties essential for proteins in order to bind ice: While entropic contributions are similar for all sites, the enthalpic gain for all ice-binding sites is lower than for the rest of the protein surface. In contrast to most of the recently published studies, our analyses show that enthalpic interactions are as important as an ice-like pre-ordering. Based on these observations, we propose a new, thermodynamically more refined mechanism of the ice recognition process showing that the appropriate balance between entropy and enthalpy facilitates ice-binding of proteins. Especially, high enthalpic interactions between the protein surface and water can hinder the ice-binding activity.

  9. Chromatin immunoprecipitation to analyze DNA binding sites of HMGA2.

    Directory of Open Access Journals (Sweden)

    Nina Winter

    Full Text Available BACKGROUND: HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment procedure two consensus sequences for HMGA2 binding have been identified. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. CONCLUSION: After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

  10. Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN)

    Science.gov (United States)

    Wei, Yang; Stec, Boguslaw; Redfield, Alfred G.; Weerapana, Eranthie; Roberts, Mary F.

    2015-01-01

    The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide 31P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling 31P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis. PMID:25429968

  11. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    L Vedula; G Brannigan; N Economou; J Xi; M Hall; R Liu; M Rossi; W Dailey; K Grasty; et. al.

    2011-12-31

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  12. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  13. Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

    Directory of Open Access Journals (Sweden)

    Previato Jose O

    2008-05-01

    Full Text Available Abstract Background The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs. In the present study, nuclear magnetic resonance (NMR was used to map the binding site(s of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. Results The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. Conclusion Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

  14. VASP: a volumetric analysis of surface properties yields insights into protein-ligand binding specificity.

    Directory of Open Access Journals (Sweden)

    Brian Y Chen

    2010-08-01

    Full Text Available Many algorithms that compare protein structures can reveal similarities that suggest related biological functions, even at great evolutionary distances. Proteins with related function often exhibit differences in binding specificity, but few algorithms identify structural variations that effect specificity. To address this problem, we describe the Volumetric Analysis of Surface Properties (VASP, a novel volumetric analysis tool for the comparison of binding sites in aligned protein structures. VASP uses solid volumes to represent protein shape and the shape of surface cavities, clefts and tunnels that are defined with other methods. Our approach, inspired by techniques from constructive solid geometry, enables the isolation of volumetrically conserved and variable regions within three dimensionally superposed volumes. We applied VASP to compute a comparative volumetric analysis of the ligand binding sites formed by members of the steroidogenic acute regulatory protein (StAR-related lipid transfer (START domains and the serine proteases. Within both families, VASP isolated individual amino acids that create structural differences between ligand binding cavities that are known to influence differences in binding specificity. Also, VASP isolated cavity subregions that differ between ligand binding cavities which are essential for differences in binding specificity. As such, VASP should prove a valuable tool in the study of protein-ligand binding specificity.

  15. Synthesis and crystal structure of copper (II) uracil ternary polymeric complex with 1,10-phenanthroline along with the Hirshfeld surface analysis of the metal binding sites for the uracil ligand

    Science.gov (United States)

    Patil, Yogesh Prakash; Nethaji, Munirathinam

    2015-02-01

    The study of models for "metal-enzyme-substrate" interaction has been a proactive area of research owing to its biological and pharmacological importance. In this regard the ternary copper uracil complex with 1,10-phenanthroline represents metal-enzyme-substrate system for DNA binding enzymes. The synthesis of the complex, followed by slow evaporation of the reaction mixture forms two concomitant solvatomorph crystals viz., {[Cu(phen)(μ-ura)(H2O)]n·H2O (1a)} and {[Cu(phen)(μ-ura)(H2O)]n·CH3OH (1b)}. Both complexes are structurally characterized, while elemental analysis, IR and EPR spectra were recorded for 1b (major product). In both complexes, uracil coordinates uniquely via N1 and N3 nitrogen atom acting as a bidentate bridging ligand forming a 1-D polymer. The two solvatomorphs were quantitatively analyzed for the differences with the aid of Hirshfeld surface analysis.

  16. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  17. RH421 binds into the ATP-binding site on the Na+/K+-ATPase.

    Science.gov (United States)

    Huličiak, Miroslav; Bazgier, Václav; Berka, Karel; Kubala, Martin

    2017-10-01

    The Na + /K + -ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na + /K + -ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na + /K + -ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Relationships between cell surface insulin binding and endocytosis in adipocytes

    International Nuclear Information System (INIS)

    Jochen, A.L.

    1988-01-01

    Chymotrypsin substrate analogues, such as N-acetyl-Tyr ethyl ester, have recently been demonstrated to inhibit the endocytic uptake of insulin in isolated rat adipocytes. In this study, the effect of N-acetyl-Tyr ethyl ester on cell surface insulin binding and dissociation were examined. Surface-bound 125 I-insulin was distinguished from intracellular 125 I-insulin by the sensitivity of the former to rapid dissociation with an acidic buffer. Plateau levels of surface-bound insulin at 37 degree C were increased 70% by inhibiting the internalization pathway. This increase was temperature and insulin concentration dependent. Thus differences in surface binding were small at 12 degree C and also at high insulin concentrations. Inhibition of internalization with N-acetyl-Tyr ethyl ester markedly slowed the loss of surface-bound insulin observed during dissociation the loss of surface-bound insulin observed during dissociation studies. After 20-30 min of dissociation, the remaining levels of surface-bound insulin were three- to fourfold higher in treated adipocytes compared with control adipocytes. Added unlabeled insulin retained its ability to accelerate the dissociation of insulin in N-acetyl-Tyr ethyl ester-treated cells. These observations indicate that the internalization pathway is a quantitatively important factor in determining levels of surface binding at 37 degree C and in determining the rat of deactivation of insulin binding

  19. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  20. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    International Nuclear Information System (INIS)

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-01-01

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme

  1. Nicotinic Cholinergic Receptor Binding Sites in the Brain: Regulation in vivo

    Science.gov (United States)

    Schwartz, Rochelle D.; Kellar, Kenneth J.

    1983-04-01

    Tritiated acetylcholine was used to measure binding sites with characteristics of nicotinic cholinergic receptors in rat brain. Regulation of the binding sites in vivo was examined by administering two drugs that stimulate nicotinic receptors directly or indirectly. After 10 days of exposure to the cholinesterase inhibitor diisopropyl fluorophosphate, binding of tritiated acetylcholine in the cerebral cortex was decreased. However, after repeated administration of nicotine for 10 days, binding of tritiated acetylcholine in the cortex was increased. Saturation analysis of tritiated acetylcholine binding in the cortices of rats treated with diisopropyl fluorophosphate or nicotine indicated that the number of binding sites decreased and increased, respectively, while the affinity of the sites was unaltered.

  2. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  3. The structure of the catechin-binding site of human sulfotransferase 1A1.

    Science.gov (United States)

    Cook, Ian; Wang, Ting; Girvin, Mark; Leyh, Thomas S

    2016-12-13

    We are just beginning to understand the allosteric regulation of the human cytosolic sulfotransferase (SULTs) family-13 disease-relevant enzymes that regulate the activities of hundreds, if not thousands, of signaling small molecules. SULT1A1, the predominant isoform in adult liver, harbors two noninteracting allosteric sites, each of which binds a different molecular family: the catechins (naturally occurring flavonols) and nonsteroidal antiinflammatory drugs (NSAIDs). Here, we present the structure of an SULT allosteric binding site-the catechin-binding site of SULT1A1 bound to epigallocatechin gallate (EGCG). The allosteric pocket resides in a dynamic region of the protein that enables EGCG to control opening and closure of the enzyme's active-site cap. Furthermore, the structure offers a molecular explanation for the isozyme specificity of EGCG, which is corroborated experimentally. The binding-site structure was obtained without X-ray crystallography or multidimensional NMR. Instead, a SULT1A1 apoprotein structure was used to guide positioning of a small number of spin-labeled single-Cys mutants that coat the entire enzyme surface with a paramagnetic field of sufficient strength to determine its contribution to the bound ligand's transverse (T 2 ) relaxation from its 1D solution spectrum. EGCG protons were mapped to the protein surface by triangulation using the T 2 values to calculate their distances to a trio of spin-labeled Cys mutants. The final structure was obtained using distance-constrained molecular dynamics docking. This approach, which is readily extensible to other systems, is applicable over a wide range of ligand affinities, requires little protein, avoids the need for isotopically labeled protein, and has no protein molecular weight limitations.

  4. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    International Nuclear Information System (INIS)

    Singh, S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 (angstrom) above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational

  5. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  6. Binding affinity of surface functionalized gold nanoparticles to hydroxyapatite.

    Science.gov (United States)

    Ross, Ryan D; Roeder, Ryan K

    2011-10-01

    Gold nanoparticles (Au NPs) have been investigated for a number of biomedical applications, including drug and gene delivery vehicles, thermal ablation therapy, diagnostic sensors, and imaging contrast agents. Surface functionalization with molecular groups exhibiting calcium affinity can enable targeted delivery of Au NPs to calcified tissue, including damaged bone tissue. Therefore, the objective of this study was to investigate the binding affinity of functionalized Au NPs for targeted delivery to bone mineral, using hydroxyapatite (HA) crystals as a synthetic analog in vitro. Au NPs were synthesized to a mean particle size of 10-15 nm and surface functionalized with either L-glutamic acid, 2-aminoethylphosphonic acid, or alendronate, which exhibit a primary amine for binding gold opposite carboxylate, phosphonate, or bisphosphonate groups, respectively, for targeting calcium. Bisphosphonate functionalized Au NPs exhibited the most rapid binding kinetics and greatest binding affinity to HA, followed by glutamic acid and phosphonic acid. All functional groups reached complete binding after 24 h. Equilibrium binding constants in de-ionized water, determined by nonlinear regression of Langmuir isotherms, were 3.40, 0.69, and 0.25 mg/L for bisphosphonate, carboxylate, and phosphonate functionalized Au NPs, respectively. Functionalized Au NPs exhibited lower overall binding in fetal bovine serum compared to de-ionized water, but relative differences between functional groups were similar. Copyright © 2011 Wiley Periodicals, Inc.

  7. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    International Nuclear Information System (INIS)

    Gates, T.S.; Zimmerman, R.P.; Mantyh, C.R.; Vigna, S.R.; Maggio, J.E.; Welton, M.L.; Passaro, E.P. Jr.; Mantyh, P.W.

    1988-01-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for 125 I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells

  8. Reversible and Irreversible Binding of Nanoparticles to Polymeric Surfaces

    Directory of Open Access Journals (Sweden)

    Wolfgang H. Binder

    2009-01-01

    Full Text Available Reversible and irreversible binding of CdSe-nanoparticles and nanorods to polymeric surfaces via a strong, multiple hydrogen bond (= Hamilton-receptor/barbituric acid is described. Based on ROMP-copolymers, the supramolecular interaction on a thin polymer film is controlled by living polymerization methods, attaching the Hamilton-receptor in various architectures, and concentrations. Strong binding is observed with CdSe-nanoparticles and CdSe-nanorods, whose surfaces are equipped with matching barbituric acid-moieties. Addition of polar solvents, able to break the hydrogen bonds leads to the detachment of the nanoparticles from the polymeric film. Irreversible binding is observed if an azide/alkine-“click”-reaction is conducted after supramolecular recognition of the nanoparticles on the polymeric surface. Thus reversible or irreversible attachment of the nanosized objects can be achieved.

  9. Demonstration of specific binding sites for /sup 3/H-RRR-alpha-tocopherol on human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for /sup 3/H-RRR-alpha-tocopherol (/sup 3/H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for /sup 3/H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for /sup 3/H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane.

  10. Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

  11. Functional impact of HIV coreceptor-binding site mutations

    International Nuclear Information System (INIS)

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W.; Reeves, Jacqueline D.

    2006-01-01

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner

  12. L-(TH)glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Monaghan, D.T.; Yao, D.; Cotman, C.W.

    1985-08-12

    The anatomical distribution of L-(TH)glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-(TH)glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of CaS , Cl and Na ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-(TH)2-amino-5-phosphonopentanoate (D-(TH)AP5), (TH)kainate ((TH)KA) and (TH) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ((TH)AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively. (Auth.). 29 refs.; 1 figure; 1 table.

  13. L-[3H]glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

    International Nuclear Information System (INIS)

    Monaghan, D.T.; Yao, Deborah; Cotman, C.W.

    1985-01-01

    The anatomical distribution of L-[ 3 H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-[ 3 H]glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of Ca 2+ , Cl - and Na + ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-[ 3 H]2-amino-5-phosphonopentanoate (D-[ 3 H]AP5), [ 3 H]kainate ([ 3 H]KA) and [ 3 H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([ 3 H]AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively. (Auth.)

  14. The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

    Science.gov (United States)

    Dayan, Avraham; Babin, Gilad; Ganoth, Assaf; Kayouf, Nivin Samir; Nitoker Eliaz, Neta; Mukkala, Srijana; Tsfadia, Yossi; Fleminger, Gideon

    2017-08-01

    Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO 2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO 2 -binding capabilities with TiBP. Intrigued by the unique TiO 2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO 2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO 2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO 2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO 2 through similar regions located far from the active site and the dimerization sites. The putative TiO 2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Genome-wide prediction, display and refinement of binding sites with information theory-based models

    Directory of Open Access Journals (Sweden)

    Leeder J Steven

    2003-09-01

    Full Text Available Abstract Background We present Delila-genome, a software system for identification, visualization and analysis of protein binding sites in complete genome sequences. Binding sites are predicted by scanning genomic sequences with information theory-based (or user-defined weight matrices. Matrices are refined by adding experimentally-defined binding sites to published binding sites. Delila-Genome was used to examine the accuracy of individual information contents of binding sites detected with refined matrices as a measure of the strengths of the corresponding protein-nucleic acid interactions. The software can then be used to predict novel sites by rescanning the genome with the refined matrices. Results Parameters for genome scans are entered using a Java-based GUI interface and backend scripts in Perl. Multi-processor CPU load-sharing minimized the average response time for scans of different chromosomes. Scans of human genome assemblies required 4–6 hours for transcription factor binding sites and 10–19 hours for splice sites, respectively, on 24- and 3-node Mosix and Beowulf clusters. Individual binding sites are displayed either as high-resolution sequence walkers or in low-resolution custom tracks in the UCSC genome browser. For large datasets, we applied a data reduction strategy that limited displays of binding sites exceeding a threshold information content to specific chromosomal regions within or adjacent to genes. An HTML document is produced listing binding sites ranked by binding site strength or chromosomal location hyperlinked to the UCSC custom track, other annotation databases and binding site sequences. Post-genome scan tools parse binding site annotations of selected chromosome intervals and compare the results of genome scans using different weight matrices. Comparisons of multiple genome scans can display binding sites that are unique to each scan and identify sites with significantly altered binding strengths

  16. Protein kinase Calpha contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities.

    Science.gov (United States)

    Slater, S J; Ho, C; Kelly, M B; Larkin, J D; Taddeo, F J; Yeager, M D; Stubbs, C D

    1996-03-01

    Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC alpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.

  17. Muscarinic acetylcholine receptors: location of the ligand binding site

    International Nuclear Information System (INIS)

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-01-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

  18. A sialic acid binding site in a human picornavirus.

    Directory of Open Access Journals (Sweden)

    Georg Zocher

    2014-10-01

    Full Text Available The picornaviruses coxsackievirus A24 variant (CVA24v and enterovirus 70 (EV70 cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC, a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

  19. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  20. Enhancer transcripts mark active estrogen receptor binding sites.

    Science.gov (United States)

    Hah, Nasun; Murakami, Shino; Nagari, Anusha; Danko, Charles G; Kraus, W Lee

    2013-08-01

    We have integrated and analyzed a large number of data sets from a variety of genomic assays using a novel computational pipeline to provide a global view of estrogen receptor 1 (ESR1; a.k.a. ERα) enhancers in MCF-7 human breast cancer cells. Using this approach, we have defined a class of primary transcripts (eRNAs) that are transcribed uni- or bidirectionally from estrogen receptor binding sites (ERBSs) with an average transcription unit length of ∼3-5 kb. The majority are up-regulated by short treatments with estradiol (i.e., 10, 25, or 40 min) with kinetics that precede or match the induction of the target genes. The production of eRNAs at ERBSs is strongly correlated with the enrichment of a number of genomic features that are associated with enhancers (e.g., H3K4me1, H3K27ac, EP300/CREBBP, RNA polymerase II, open chromatin architecture), as well as enhancer looping to target gene promoters. In the absence of eRNA production, strong enrichment of these features is not observed, even though ESR1 binding is evident. We find that flavopiridol, a CDK9 inhibitor that blocks transcription elongation, inhibits eRNA production but does not affect other molecular indicators of enhancer activity, suggesting that eRNA production occurs after the assembly of active enhancers. Finally, we show that an enhancer transcription "signature" based on GRO-seq data can be used for de novo enhancer prediction across cell types. Together, our studies shed new light on the activity of ESR1 at its enhancer sites and provide new insights about enhancer function.

  1. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    Directory of Open Access Journals (Sweden)

    Gowda Veerabasappa T

    2007-12-01

    Full Text Available Abstract Background The snake venom group IIA secreted phospholipases A2 (SVPLA2, present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL at the membrane/water interface and by highly specific direct binding to: (i presynaptic membrane-bound or intracellular receptors; (ii natural PLA2-inhibitors from snake serum; and (iii coagulation factors present in human blood. Results Using surface plasmon resonance (SPR protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS. The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

  2. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

    DEFF Research Database (Denmark)

    Long, Katherine S; Porse, Bo T

    2003-01-01

    , of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site...... on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome....

  3. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    International Nuclear Information System (INIS)

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  4. Osteopontin: A uranium phosphorylated binding-site characterization

    International Nuclear Information System (INIS)

    Safi, Samir; Jeanson, Aurelie; Roques, Jerome; Simoni, Eric; Creff, Gaelle; Qi, Lei; Basset, Christian; Vidaud, Claude; Solari, Pier Lorenzo; Den Auwer, Christophe

    2013-01-01

    Herein, we describe the structural investigation of one possible uranyl binding site inside a non structured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phospho-peptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U L(III)-edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO 2 2+ /peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein. (authors)

  5. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  6. Use of Functional Polymorphisms To Elucidate the Peptide Binding Site of TAP Complexes.

    Science.gov (United States)

    Geng, Jie; Pogozheva, Irina D; Mosberg, Henry I; Raghavan, Malini

    2015-10-01

    TAP1/TAP2 complexes translocate peptides from the cytosol to the endoplasmic reticulum lumen to enable immune surveillance by CD8(+) T cells. Peptide transport is preceded by peptide binding to a cytosol-accessible surface of TAP1/TAP2 complexes, but the location of the TAP peptide-binding pocket remains unknown. Guided by the known contributions of polymorphic TAP variants to peptide selection, we combined homology modeling of TAP with experimental measurements to identify several TAP residues that interact with peptides. Models for peptide-TAP complexes were generated, which indicate bent conformation for peptides. The peptide binding site of TAP is located at the hydrophobic boundary of the cytosolic membrane leaflet, with striking parallels to the glutathione binding site of NaAtm1, a transporter that functions in bacterial heavy metal detoxification. These studies illustrate the conservation of the ligand recognition modes of bacterial and mammalians transporters involved in peptide-guided cellular surveillance. Copyright © 2015 by The American Association of Immunologists, Inc.

  7. Repository surface design site layout analysis

    International Nuclear Information System (INIS)

    Montalvo, H.R.

    1998-01-01

    The purpose of this analysis is to establish the arrangement of the Yucca Mountain Repository surface facilities and features near the North Portal. The analysis updates and expands the North Portal area site layout concept presented in the ACD, including changes to reflect the resizing of the Waste Handling Building (WHB), Waste Treatment Building (WTB), Carrier Preparation Building (CPB), and site parking areas; the addition of the Carrier Washdown Buildings (CWBs); the elimination of the Cask Maintenance Facility (CMF); and the development of a concept for site grading and flood control. The analysis also establishes the layout of the surface features (e.g., roads and utilities) that connect all the repository surface areas (North Portal Operations Area, South Portal Development Operations Area, Emplacement Shaft Surface Operations Area, and Development Shaft Surface Operations Area) and locates an area for a potential lag storage facility. Details of South Portal and shaft layouts will be covered in separate design analyses. The objective of this analysis is to provide a suitable level of design for the Viability Assessment (VA). The analysis was revised to incorporate additional material developed since the issuance of Revision 01. This material includes safeguards and security input, utility system input (size and location of fire water tanks and pump houses, potable water and sanitary sewage rates, size of wastewater evaporation pond, size and location of the utility building, size of the bulk fuel storage tank, and size and location of other exterior process equipment), main electrical substation information, redundancy of water supply and storage for the fire support system, and additional information on the storm water retention pond

  8. Repository Surface Design Site Layout Analysis

    International Nuclear Information System (INIS)

    Montalvo, H.R.

    1998-01-01

    The purpose of this analysis is to establish the arrangement of the Repository surface facilities and features near the North Portal. The analysis updates and expands the North Portal area site layout concept presented in the ACD (Reference 5.5), including changes to reflect the resizing of the Waste Handling Building (WHB), Waste Treatment Building (WTB), Carrier Preparation Building (CPB), and site parking areas; the addition of the Carrier Washdown Buildings (CWBs); the elimination of the Cask Maintenance Facility (CMF); and the development of a concept for site grading and flood control. The analysis also establishes the layout of the surface features (e.g., roads and utilities) that connect all the repository surface areas (North Portal Operations Area, South Portal Development Operations Area, Emplacement Shaft Surface Operations Area, and Development Shaft Surface Operations Area) and locates an area for a potential lag storage facility. Details of South Portal and shaft layouts will be covered in separate design analyses. The objective of this analysis is to provide a suitable level of design for the Viability Assessment (VA). The analysis was revised to incorporate additional material developed since the issuance of Revision 01. This material includes safeguards and security input, utility system input (size and location of fire water tanks and pump houses, potable water and sanitary sewage rates, size of wastewater evaporation pond, size and location of the utility building, size of the bulk fuel storage tank, and size and location of other exterior process equipment), main electrical substation information, redundancy of water supply and storage for the fire support system, and additional information on the storm water retention pond

  9. The relationship between alterations in spermatozoal deoxyribonucleic acid, heparin binding sites, and semen quality.

    Science.gov (United States)

    Peluso, J J; Luciano, A A; Nulsen, J C

    1992-03-01

    To assess the relationship between spermatozoal deoxyribonucleic acid (DNA) and fertilizing potential. Semen samples were examined from nine fertile donors and six donors without a confirmed pregnancy. All samples were in the normal range for count, morphology, and motility. Spermatozoa from these specimens were stained with acridine orange or Feulgen's reagent. The presence of heparin binding sites was determined by counting the number of spermatozoa that bound to heparin-coated agarose beads. Acridine orange staining demonstrated that in the fertile group 42% +/- 2% of the spermatozoa fluoresced green indicating that the DNA was intact, whereas only 25% +/- 3% of the spermatozoa fluoresced green in the nonfertile group (P less than 0.05). Feulgen's staining revealed that more spermatozoa from infertile donors showed a heterogeneous DNA distribution (P less than 0.05). The DNA content of spermatozoa with heterogeneous distribution of DNA was reduced by 10% compared with those with homogeneous DNA (P less than 0.05). Normal spermatozoa as well as those with DNA anomalies possessed heparin binding sites. These data demonstrated that in donor specimens with normal counts, morphology, and motility, a higher percentage of spermatozoa possess less and/or denatured DNA in the infertile group compared with the fertile donors. In contrast, the surface membranes of spermatozoa with altered DNA have heparin binding sites as do spermatozoa with intact DNA.

  10. Relationships between the catechol substrate binding site and amphetamine, cocaine, and mazindol binding sites in a kinetic model of the striatal transporter of dopamine in vitro.

    Science.gov (United States)

    Wayment, H; Meiergerd, S M; Schenk, J O

    1998-05-01

    Experiments were conducted to determine how (-)-cocaine and S(+)-amphetamine binding sites relate to each other and to the catechol substrate site on the striatal dopamine transporter (sDAT). In controls, m-tyramine and S(+)-amphetamine caused release of dopamine from intracellular stores at concentrations > or = 12-fold those observed to inhibit inwardly directed sDAT activity for dopamine. In preparations from animals pretreated with reserpine, m-tyramine and S(+)-amphetamine caused release of preloaded dopamine at concentrations similar to those that inhibit inwardly directed sDAT activity. S(+)-Amphetamine and m-tyramine inhibited sDAT activity for dopamine by competing for a common binding site with dopamine and each other, suggesting that phenethylamines are substrate analogues at the plasmalemmal sDAT. (-)-Cocaine inhibited sDAT at a site separate from that for substrate analogues. This site is mutually interactive with the substrate site (K(int) = 583 nM). Mazindol competitively inhibited sDAT at the substrate analogue binding site. The results with (-)-cocaine suggest that the (-)-cocaine binding site on sDAT is distinct from that of hydroxyphenethylamine substrates, reinforcing the notion that an antagonist for (-)-cocaine binding may be developed to block (-)-cocaine binding with minimal effects on dopamine transporter activity. However, a strategy of how to antagonize drugs of abuse acting as substrate analogues is still elusive.

  11. Mercury Binding Sites in Thiol-Functionalized Mesostructured Silica

    International Nuclear Information System (INIS)

    Billinge, Simon J.L.; McKimmey, Emily J.; Shatnawi, Mouath; Kim, HyunJeong; Petkov, Valeri; Wermeille, Didier; Pinnavaia, Thomas J.

    2005-01-01

    Thiol-functionalized mesostructured silica with anhydrous compositions of (SiO 2 ) 1-x (LSiO 1.5 ) x , where L is a mercaptopropyl group and x is the fraction of functionalized framework silicon centers, are effective trapping agents for the removal of mercuric(II) ions from water. In the present work, we investigate the mercury-binding mechanism for representative thiol-functionalized mesostructures by atomic pair distribution function (PDF) analysis of synchrotron X-ray powder diffraction data and by Raman spectroscopy. The mesostructures with wormhole framework structures and compositions corresponding to x = 0.30 and 0.50 were prepared by direct assembly methods in the presence of a structure-directing amine porogen. PDF analyses of five mercury-loaded compositions with Hg/S ratios of 0.50-1.30 provided evidence for the bridging of thiolate sulfur atoms to two metal ion centers and the formation of chain structures on the pore surfaces. We find no evidence for Hg-O bonds and can rule out oxygen coordination of the mercury at greater than the 10% level. The relative intensities of the PDF peaks corresponding to Hg-S and Hg-Hg atomic pairs indicate that the mercury centers cluster on the functionalized surfaces by virtue of thiolate bridging, regardless of the overall mercury loading. However, the Raman results indicate that the complexation of mercury centers by thiolate depends on the mercury loading. At low mercury loadings (Hg/S (le) 0.5), the dominant species is an electrically neutral complex in which mercury most likely is tetrahedrally coordinated to bridging thiolate ligands, as in Hg(SBu t ) 2 . At higher loadings (Hg/S 1.0-1.3), mercury complex cations predominate, as evidenced by the presence of charge-balancing anions (nitrate) on the surface. This cationic form of bound mercury is assigned a linear coordination to two bridging thiolate ligands.

  12. Surface barrier research at the Hanford Site

    International Nuclear Information System (INIS)

    Gee, G.W.; Ward, A.L.; Fayer, M.J.

    1997-01-01

    At the DOE Hanford Site, a field-scale prototype surface barrier was constructed in 1994 over an existing waste site as a part of a CERCLA treatability test. The above-grade barrier consists of a fine-soil layer overlying coarse layers of sands, gravels, basalt rock (riprap), and a low permeability asphalt layer. Two sideslope configurations, clean-fill gravel on a 10:1 slope and basalt riprap on a 2:1 slope, were built and are being tested. Design considerations included: constructability; drainage and water balance monitoring, wind and water erosion control and monitoring; surface revegetation and biotic intrusion; subsidence and sideslope stability, and durability of the asphalt layer. The barrier is currently in the final year of a three-year test designed to answer specific questions related to stability and long-term performance. One half of the barrier is irrigated such that the total water applied, including precipitation, is 480 mm/yr (three times the long-term annual average). Each year for the past two years, an extreme precipitation event (71 mm in 8 hr) representing a 1,000-yr return storm was applied in late March, when soil water storage was at a maximum. While the protective sideslopes have drained significant amounts of water, the soil cover (2-m of silt-loam soil overlying coarse sand and rock) has never drained. During the past year there was no measurable surface runoff or wind erosion. This is attributed to extensive revegetation of the surface. In addition, the barrier elevation has shown a small increase of 2 to 3 cm that is attributed to a combination of root proliferation and freeze/thaw activity. Testing will continue through September 1997. Performance data from the prototype barrier will be used by DOE in site-closure decisions at Hanford

  13. Surface system Forsmark. Site descriptive modelling SDM-Site Forsmark

    International Nuclear Information System (INIS)

    Lindborg, Tobias

    2008-12-01

    SKB has undertaken site characterization of two different areas, Forsmark and Laxemar-Simpevarp, in order to find a suitable location for a geological repository for spent nuclear fuel. This report focuses on the site descriptive modelling of the surface system at Forsmark. The characterization of the surface system at the site was primarily made by identifying and describing important properties in different parts of the surface system, properties concerning e.g. hydrology and climate, Quaternary deposits and soils, hydrochemistry, vegetation, ecosystem functions, but also current and historical land use. The report presents available input data, methodology for data evaluation and modelling, and resulting models for each of the different disciplines. Results from the modelling of the surface system are also integrated with results from modelling of the deep bedrock system. The Forsmark site is located within the municipality of Oesthammar, about 120 km north of Stockholm. The investigated area is located along the shoreline of Oeregrundsgrepen, a funnel-shaped bay of the Baltic Sea. The area is characterized by small-scale topographic variations and is almost entirely located at altitudes lower than 20 metres above sea level. The Quaternary deposits in the area are dominated by till, characterized by a rich content of calcite which was transported by the glacier ice to the area from the sedimentary bedrock of Gaevlebukten about 100 km north of Forsmark. As a result, the surface waters and shallow groundwater at Forsmark are characterized by high pH values and high concentrations of certain major constituents, especially calcium and bicarbonate. The annual precipitation and runoff are 560 and 150 mm, respectively. The lakes are small and shallow, with mean and maximum depths ranging from approximately 0.1 to 1 m and 0.4 to 2 m. Sea water flows into the most low-lying lakes during events giving rise to very high sea levels. Wetlands are frequent and cover 25 to 35

  14. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Lovmand, J

    1993-01-01

    Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching t......RNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus...... can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site....

  15. Direct instrumental identification of catalytically active surface sites

    Science.gov (United States)

    Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

    2017-09-01

    The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

  16. Cooperative Binding and Activation of Fibronectin by a Bacterial Surface Protein*

    Science.gov (United States)

    Marjenberg, Zoe R.; Ellis, Ian R.; Hagan, Robert M.; Prabhakaran, Sabitha; Höök, Magnus; Talay, Susanne R.; Potts, Jennifer R.; Staunton, David; Schwarz-Linek, Ulrich

    2011-01-01

    Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens. PMID:21059652

  17. Modeling Complex Equilibria in ITC Experiments: Thermodynamic Parameters Estimation for a Three Binding Site Model

    Science.gov (United States)

    Le, Vu H.; Buscaglia, Robert; Chaires, Jonathan B.; Lewis, Edwin A.

    2013-01-01

    Isothermal Titration Calorimetry, ITC, is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g. Keq (or ΔG), ΔH, ΔS, and n) for a ligand binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combination of equilibrium constant, mass balance, and charge balance equations for the system under study. Commercial ITC instruments are supplied with software that includes a number of simple interaction models, for example one binding site, two binding sites, sequential sites, and n-independent binding sites. More complex models for example, three or more binding sites, one site with multiple binding mechanisms, linked equilibria, or equilibria involving macromolecular conformational selection through ligand binding need to be developed on a case by case basis by the ITC user. In this paper we provide an algorithm (and a link to our MATLAB program) for the non-linear regression analysis of a multiple binding site model with up to four overlapping binding equilibria. Error analysis demonstrates that fitting ITC data for multiple parameters (e.g. up to nine parameters in the three binding site model) yields thermodynamic parameters with acceptable accuracy. PMID:23262283

  18. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  19. Siting of near surface disposal facilities

    International Nuclear Information System (INIS)

    1994-01-01

    Radioactive waste is generated from the production of nuclear energy and from the use of radioactive materials in industrial applications, research and medicine. The importance of safe management of radioactive waste for the protection of human health and the environment has long been recognized and considerable experience has been gained in this field. The Radioactive Waste Safety Standards (RADWASS) programme is the IAEA's contribution to establishing and promoting, in a coherent and comprehensive manner, the basic safety philosophy for radioactive waste management and the steps necessary to ensure its implementation. The Safety Standards are supplemented by a number of Safety Guides and Safety Practices. This Safety Guide defines the site selection process and criteria for identifying suitable near surface disposal facilities for low and intermediate level solid wastes. Management of the siting process and data needed to apply the criteria are also specified. 4 refs

  20. Form, symmetry and packing of biomacromolecules. III. Antigenic, receptor and contact binding sites in picornaviruses.

    Science.gov (United States)

    Janner, A

    2011-03-01

    The relation between serotype differentiation and crystallographic symmetry, revealed by the contact fingerprint diagrams investigated in Part II [Janner (2010). Acta Cryst. A66, 312-326] for the human rhinovirus, is extended to the Picornaviridae family. The approach, outlined in Part I [Janner (2010). Acta Cryst. A66, 301-311] and Part II for biomacromolecules packed in a crystal and based on concepts such as packing lattice, kissing points and crystal-packing parameters, can directly be applied to the picornaviruses. In particular, the contact fingerprint diagrams of 20 different virus strains have been derived. In these cases, as for the rhinovirus, these diagrams are serotype/strain specific, justifying the name fingerprint. The molecular basis for the serotype variability, and the associated conservation requirements, is usually analysed by considering antigenic sites, where capsid residues bind with antibodies, and receptor sites, where other residues bind with molecular receptors of the host cell membrane. Both the antigenic variation and the receptor conservation allow repeated infection of the host cells of the given animals. The graphical description of these sites is usually done by footprints and roadmap diagrams, mapping properties of the capsid surface and using the icosahedral symmetry of the capsid. The alternative fingerprint diagrammatic description, based on the crystal symmetry, adopted in Part II for the contact sites, where a capsid is bound to the next one in the crystal packing, is extended to the antigenic and receptor binding sites. Again, the antigenic/receptor fingerprints are specific, at least for the nine picornaviruses investigated so far, despite the more than a factor of ten larger coarse graining with respect to the corresponding footprint and roadmap diagrams. The latter are based on a grid spacing of about 2 Å, whereas the spacing implied by the packing-lattice approximation adopted in fingerprints varies typically from 20 to

  1. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    Energy Technology Data Exchange (ETDEWEB)

    Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

    2013-03-15

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

  2. Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD.

    Science.gov (United States)

    Hürlimann, Lea M; Hohl, Michael; Seeger, Markus A

    2017-06-01

    Many heterodimeric ATP-binding cassette (ABC) exporters evolved asymmetric ATP-binding sites containing a degenerate site incapable of ATP hydrolysis due to noncanonical substitutions in conserved sequence motifs. Recent studies revealed that nucleotide binding to the degenerate site stabilizes contacts between the nucleotide-binding domains (NBDs) of the inward-facing transporter and regulates ATP hydrolysis at the consensus site via allosteric coupling mediated by the D-loops. However, it is unclear whether nucleotide binding to the degenerate site is strictly required for substrate transport. In this study, we examined the functional consequences of a systematic set of mutations introduced at the degenerate and consensus site of the multidrug efflux pump EfrCD of Enterococcus faecalis. Mutating motifs which differ among the two ATP-binding sites (Walker B, switch loop, and ABC signature) or which are involved in interdomain communication (D-loop and Q-loop) led to asymmetric results in the functional assays and were better tolerated at the degenerate site. This highlights the importance of the degenerate site to allosterically regulate the events at the consensus site. Mutating invariant motifs involved in ATP binding and NBD closure (A-loop and Walker A) resulted in equally reduced transport activities, regardless at which ATP-binding site they were introduced. In contrast to previously investigated heterodimeric ABC exporters, mutation of the degenerate site Walker A lysine completely inactivated ATPase activity and substrate transport, indicating that ATP binding to the degenerate site is essential for EfrCD. This study provides novel insights into the split tasks of asymmetric ATP-binding sites of heterodimeric ABC exporters. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  3. Spectroscopic study of binding of chlorogenic acid with the surface of ZnO nanoparticles

    Science.gov (United States)

    Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

    2017-09-01

    Understanding the interaction properties of biological materials with ZnO NPs is fundamental interest in the field of biotechnological applications as well as in the formation of optoelectronic devices. In this research, the binding of ZnO NPs and chlorogenic acid (CGA) were investigated using fluorescence quenching, UV-Vis absorption spectroscopy, Fourier transform infrared (FTIR), Raman spectroscopy, scanning electron microscopy (TEM), and dynamic light scattering (DLS) techniques. The study results indicated the fluorescence quenching between ZnO NPs and CGA rationalized in terms of static quenching mechanism or the formation of nonfluorescent CGA-ZnO. From fluorescence quenching spectral analysis the binding constant ( K a ), number of binding sites ( n), and thermodynamic properties, were determined. The quenching constants ( K sv) and binding constant ( K a ), decrease with increasing the temperature and their binding sites n are 2. The thermodynamic parameters determined using Van't Hoff equation indicated binding occurs spontaneously involving the hydrogen bond and van der Walls forces played the major role in the reaction of ZnO NPs with CGA. The Raman, SEM, DLS, and Zeta potential measurements were also indicated the differences in the structure, morphology and sizes of CGA, ZnO NPs, and their corresponding CGA-ZnO due to adsorption of CGA on the surface of ZnO NPs

  4. Surface system Forsmark. Site descriptive modelling SDM-Site Forsmark

    Energy Technology Data Exchange (ETDEWEB)

    Lindborg, Tobias (ed.)

    2008-12-15

    SKB has undertaken site characterization of two different areas, Forsmark and Laxemar-Simpevarp, in order to find a suitable location for a geological repository for spent nuclear fuel. This report focuses on the site descriptive modelling of the surface system at Forsmark. The characterization of the surface system at the site was primarily made by identifying and describing important properties in different parts of the surface system, properties concerning e.g. hydrology and climate, Quaternary deposits and soils, hydrochemistry, vegetation, ecosystem functions, but also current and historical land use. The report presents available input data, methodology for data evaluation and modelling, and resulting models for each of the different disciplines. Results from the modelling of the surface system are also integrated with results from modelling of the deep bedrock system. The Forsmark site is located within the municipality of Oesthammar, about 120 km north of Stockholm. The investigated area is located along the shoreline of Oeregrundsgrepen, a funnel-shaped bay of the Baltic Sea. The area is characterized by small-scale topographic variations and is almost entirely located at altitudes lower than 20 metres above sea level. The Quaternary deposits in the area are dominated by till, characterized by a rich content of calcite which was transported by the glacier ice to the area from the sedimentary bedrock of Gaevlebukten about 100 km north of Forsmark. As a result, the surface waters and shallow groundwater at Forsmark are characterized by high pH values and high concentrations of certain major constituents, especially calcium and bicarbonate. The annual precipitation and runoff are 560 and 150 mm, respectively. The lakes are small and shallow, with mean and maximum depths ranging from approximately 0.1 to 1 m and 0.4 to 2 m. Sea water flows into the most low-lying lakes during events giving rise to very high sea levels. Wetlands are frequent and cover 25 to 35

  5. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  6. Structure of Dioclea virgata lectin: relations between carbohydrate binding site and nitric oxide production

    Energy Technology Data Exchange (ETDEWEB)

    Delatorre, P.; Gadelha, C.A.A.; Santi-Gadelha, T. [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil); Nobrega, R.B.; Rocha, B.A.M.; Nascimento, K.S.; Naganao, C.S.; Sampaio, A.H.; Cavada, B.S. [Universidade Federal do Ceara (UFC), Fortaleza, CE (Brazil); Pires, A.F.; Assreuy, A.M.S. [Universidade Estadual do Ceara (UECE), Fortaleza, CE (Brazil)

    2012-07-01

    Full text: Lectins are proteins/glycoproteins with at least one noncatalytic domain binding reversibly to specific monosaccharides or oligosaccharides. By binding to carbohydrate moieties on the cell surface, lectins participate in a range of cellular processes without changing the properties of the carbohydrates involved. The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological proper- ties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. The DvirL diffraction analysis revealed that both the native crystal and the X-Man-complexed form are orthorhombic and belong to space group I222. The cell parameters were: a=65.4 , b=86.6 and c=90.2 (native structure), and a=61.89 , b=87.67 and c=88.78 (X-Man-complexed structure). An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration. (author)

  7. Structure of Dioclea virgata lectin: relations between carbohydrate binding site and nitric oxide production

    International Nuclear Information System (INIS)

    Delatorre, P.; Gadelha, C.A.A.; Santi-Gadelha, T.; Nobrega, R.B.; Rocha, B.A.M.; Nascimento, K.S.; Naganao, C.S.; Sampaio, A.H.; Cavada, B.S.; Pires, A.F.; Assreuy, A.M.S.

    2012-01-01

    Full text: Lectins are proteins/glycoproteins with at least one noncatalytic domain binding reversibly to specific monosaccharides or oligosaccharides. By binding to carbohydrate moieties on the cell surface, lectins participate in a range of cellular processes without changing the properties of the carbohydrates involved. The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological proper- ties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. The DvirL diffraction analysis revealed that both the native crystal and the X-Man-complexed form are orthorhombic and belong to space group I222. The cell parameters were: a=65.4 , b=86.6 and c=90.2 (native structure), and a=61.89 , b=87.67 and c=88.78 (X-Man-complexed structure). An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration. (author)

  8. EasyMIFS and SiteHound: a toolkit for the identification of ligand-binding sites in protein structures.

    Science.gov (United States)

    Ghersi, Dario; Sanchez, Roberto

    2009-12-01

    SiteHound uses Molecular Interaction Fields (MIFs) produced by EasyMIFs to identify protein structure regions that show a high propensity for interaction with ligands. The type of binding site identified depends on the probe atom used in the MIF calculation. The input to EasyMIFs is a PDB file of a protein structure; the output MIF serves as input to SiteHound, which in turn produces a list of putative binding sites. Extensive testing of SiteHound for the detection of binding sites for drug-like molecules and phosphorylated ligands has been carried out. EasyMIFs and SiteHound executables for Linux, Mac OS X, and MS Windows operating systems are freely available for download from http://sitehound.sanchezlab.org/download.html. Supplementary data are available at Bioinformatics online.

  9. Understanding the Lewis Acidity of Co(II) Sites on a Silica Surface.

    Science.gov (United States)

    Estes, Deven P; Cook, Amanda K; Lam, Erwin; Wong, Louise; Copéret, Christophe

    2017-07-17

    Heterogeneous catalysts consisting of isolated transition-metal sites dispersed on the surface of metal oxide supports are commonly used in the chemical industry. Often their reactivity relies on the Lewis acidity of the active sites on the surface of the catalyst. A recent report from our group showed that silica-supported Co(II) sites, prepared via surface organometallic chemistry, are active in both alkene hydrogenation and alkane dehydrogenation, possibly linked to the Lewis acidity of the Co(II) sites. Here we use molecular probes and analogues to both qualitatively and quantitatively model the Lewis acidity of the surface sites. Some sites do not bind probe molecules like carbon monoxide, tetrahydrofuran, and olefins, while others exhibit a continuum of Lewis acidities. This is consistent with variations in the coordination environment of Co. These results suggest that only the most Lewis acidic sites are involved in dehydrogenation and hydrogenation, consistent with catalyst poisoning studies.

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

    Science.gov (United States)

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    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 used 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. PMID:24129575

  11. Exhaustive comparison and classification of ligand-binding surfaces in proteins

    OpenAIRE

    Murakami, Yoichi; Kinoshita, Kengo; Kinjo, Akira R; Nakamura, Haruki

    2013-01-01

    Many proteins function by interacting with other small molecules (ligands). Identification of ligand-binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To e...

  12. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    International Nuclear Information System (INIS)

    James, I.F.; Goldstein, A.

    1984-01-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  13. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  14. Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

    International Nuclear Information System (INIS)

    Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.; Goberna, R.; Guerrero, J.M.

    1991-01-01

    The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using [ 125 I]melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37 degree C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of [ 125 I]melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of [ 125 I]melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the [ 125 I]melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland

  15. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

    2017-01-01

    γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherentl...

  16. Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors.

    Science.gov (United States)

    Yamashita, Tetsuo; Inaoka, Daniel Ken; Shiba, Tomoo; Oohashi, Takumi; Iwata, So; Yagi, Takao; Kosaka, Hiroaki; Miyoshi, Hideto; Harada, Shigeharu; Kita, Kiyoshi; Hirano, Katsuya

    2018-02-05

    Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

  17. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    Science.gov (United States)

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. MotifMap-RNA: a genome-wide map of RBP binding sites.

    Science.gov (United States)

    Liu, Yu; Sun, Sha; Bredy, Timothy; Wood, Marcelo; Spitale, Robert C; Baldi, Pierre

    2017-07-01

    RNA plays a critical role in gene expression and its regulation. RNA binding proteins (RBPs), in turn, are important regulators of RNA. Thanks to the availability of large scale data for RBP binding motifs and in vivo binding sites results in the form of eCLIP experiments, it is now possible to computationally predict RBP binding sites across the whole genome. We describe MotifMap-RNA, an extension of MotifMap which predicts binding sites for RBP motifs across human and mouse genomes and allows large scale querying of predicted binding sites. The data and corresponding web server are available from: http://motifmap-rna.ics.uci.edu/ as part of the MotifMap web portal. rspitale@uci.edu or pfbaldi@uci.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  19. Decomposition of the factors that govern binding site preference in a multiple rotaxane.

    Science.gov (United States)

    Angelo, Joseph P; Sohlberg, Karl

    2009-06-18

    A particularly interesting class of multiple rotaxanes consists of complexes where one long shaft threads two rings. If the shaft contains three or more potential binding sites for the rings, multiple co-conformations are possible. Such a complex is a molecular topological analogue to an abacus. Here we address the question, how does strength of ring binding to the shaft vary with respect to position on the shaft? Previous studies have found that a shaft with three binding sites exhibits strongest ring binding at the center site. Here a five-binding-site shaft is studied. We employ a novel method to partition the total energy of the system into contributions from intercomponent binding and intracomponent distortion. The method uses the output of quantum mechanical electronic structure calculations to determine fitting parameters in a set of coupled equations. The solution of the equations yields the energy partitioning and reveals the influence of long-range intercomponent interactions.

  20. Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle

    DEFF Research Database (Denmark)

    Kjeldsen, K; Richter, Erik; Galbo, H

    1986-01-01

    ]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0...

  1. Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites

    International Nuclear Information System (INIS)

    Bento, Isabel; Peixoto, Cristina; Zaitsev, Vjacheslav N.; Lindley, Peter F.

    2007-01-01

    The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 Å, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca 2+ -binding and Na + -binding sites. The Ca 2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na + sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis

  2. Localization of tachykinin binding sites (NK1, NK2, NK3 ligands) in the rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Saffroy, M.; Beaujouan, J.C.; Torrens, Y.; Besseyre, J.; Bergstroem, L.G.; Glowinski, J.

    1988-03-01

    A comparative autoradiographic analysis of the distribution of tachykinin binding sites was made on brain serial sections using several ligands. (1) /sup 3/H-SP, /sup 125/I-BHSP and /sup 3/H-physalaemin labeled identical binding sites (NK1 type). (2) /sup 3/H-NKB, /sup 125/I-BHE and /sup 3/H-eledoisin also labeled identical sites (NK3 type). (3) /sup 125/I-BHNKA preferentially labeled NK3 binding sites, the distribution of /sup 125/I-BHNKA binding sites being identical to that of /sup 3/H-NKB or /sup 125/I-BHE binding sites. (4) The distributions of /sup 3/H-SP and /sup 3/H-NKB binding sites were markedly different. (5) A very low density of labeling was found with /sup 3/H-NKA or /sup 125/I-NKA, and these binding sites were distributed only in areas rich in either /sup 3/H-SP or /sup 3/H-NKB binding sites. (6) Particular efforts were made to look for the presence of tachykinin binding sites in the substantia nigra, since this structure is particularly rich in SP and NKA and contains functional tachykinin receptors of the NK1 and NK2 types as suggested by physiological studies. Confirming previous reports, low or very low labeling was observed in the substantia nigra with /sup 3/H-SP or /sup 125/I-BHSP and /sup 3/H-NKB or /sup 125/I-BHE. Similar results were found with /sup 3/H-NKA, /sup 125/I-NKA or /sup 125/I-BHNKA. In conclusion, our data do not provide evidence yet for the existence of NK2 binding sites in the rat brain.

  3. New type of tachykinin binding site in the rat brain characterized by specific binding of a labeled eledoisin derivative

    Energy Technology Data Exchange (ETDEWEB)

    Beaujouan, J.C.; Torrens, Y.; Viger, A.; Glowinski, J.

    1984-09-01

    A new ligand for investigating tachykinin-binding site subtypes was synthesized by coupling the /sup 125/I-Bolton and Hunter reagent to eledoisin (/sup 125/I-BHE). Using a synaptosomal preparation (P2 fraction) of rat cerebral cortex, /sup 125/I-BHE was shown to bind with apparent high affinity (apparent Kd . 15.3 nM). When concentrations of up to 30 nM /sup 125/I-BHE were used, /sup 125/I-BHE binding was specific, saturable, reversible, and temperature-dependent. In contrast to (/sup 3/H)dopamine, /sup 125/I-BHE was not taken up within synaptosomes by an ouabain-sensitive process. Eledoisin, kassinin, and substance P were examined for their ability to inhibit specific /sup 125/I-BHE binding to cortical synaptosomes. Eledoisin and kassinin were considerably more potent than substance P, in contrast to the order of potency observed for specific /sup 125/I-Bolton-Hunter substance P (/sup 125/I-BHSP) binding. Specific /sup 125/I-BHE binding was highest in the cerebral cortex and hypothalamus; intermediate in the hippocampus, striatum, and thalamus; low in the mesencephalon, septum, and substantia nigra; and absent in the cerebellum. Comparison of these data with those previously obtained for /sup 125/I-BHSP binding to synaptosomes indicated that /sup 125/I-BHE-labeled binding sites differ markedly from those of /sup 125/I-BHSP-labeled binding sites. Therefore, tachykinin receptors other than substance P receptors seem to be present in the central nervous system.

  4. A surface structural model for ferrihydrite I: Sites related to primary charge, molar mass, and mass density

    NARCIS (Netherlands)

    Hiemstra, T.; Riemsdijk, van W.H.

    2009-01-01

    A multisite surface complexation (MUSIC) model for ferrihydrite (Fh) has been developed. The surface structure and composition of Fh nanoparticles are described in relation to ion binding and surface charge development. The site densities of the various reactive surface groups, the molar mass, the

  5. Blood coagulation factor XIa binds specifically to a site on activated human platelets distinct from that for factor XI

    International Nuclear Information System (INIS)

    Sinha, D.; Seaman, F.S.; Koshy, A.; Knight, L.C.; Walsh, P.N.

    1984-01-01

    Binding of 125 I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 37 degrees C. Factor XIa binding was specific: 50- to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversible and was saturable at concentrations of added Factor XIa of 0.2-0.4 microgram/ml (1.25-2.5 microM). The number of Factor XIa binding sites on activated platelets was estimated to be 225 per platelet (range, 110-450). We conclude that specific, high affinity, saturable binding sites for Factor XIa are present on activated platelets, are distinct from those previously demonstrated for Factor XI, and require the presence of high molecular weight kininogen

  6. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    Science.gov (United States)

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  7. A screening system to identify transcription factors that induce binding site-directed DNA demethylation.

    Science.gov (United States)

    Suzuki, Takahiro; Maeda, Shiori; Furuhata, Erina; Shimizu, Yuri; Nishimura, Hajime; Kishima, Mami; Suzuki, Harukazu

    2017-12-08

    DNA methylation is a fundamental epigenetic modification that is involved in many biological systems such as differentiation and disease. We and others recently showed that some transcription factors (TFs) are involved in the site-specific determination of DNA demethylation in a binding site-directed manner, although the reports of such TFs are limited. Here, we develop a screening system to identify TFs that induce binding site-directed DNA methylation changes. The system involves the ectopic expression of target TFs in model cells followed by DNA methylome analysis and overrepresentation analysis of the corresponding TF binding motif at differentially methylated regions. It successfully identified binding site-directed demethylation of SPI1, which is known to promote DNA demethylation in a binding site-directed manner. We extended our screening system to 15 master TFs involved in cellular differentiation and identified eight novel binding site-directed DNA demethylation-inducing TFs (RUNX3, GATA2, CEBPB, MAFB, NR4A2, MYOD1, CEBPA, and TBX5). Gene ontology and tissue enrichment analysis revealed that these TFs demethylate genomic regions associated with corresponding biological roles. We also describe the characteristics of binding site-directed DNA demethylation induced by these TFs, including the targeting of highly methylated CpGs, local DNA demethylation, and the overlap of demethylated regions between TFs of the same family. Our results show the usefulness of the developed screening system for the identification of TFs that induce DNA demethylation in a site-directed manner.

  8. Crystallographic study of novel transthyretin ligands exhibiting negative-cooperativity between two thyroxine binding sites.

    Directory of Open Access Journals (Sweden)

    Divya Tomar

    Full Text Available BACKGROUND: Transthyretin (TTR is a homotetrameric serum and cerebrospinal fluid protein that transports thyroxine (T4 and retinol by binding to retinol binding protein. Rate-limiting tetramer dissociation and rapid monomer misfolding and disassembly of TTR lead to amyloid fibril formation in different tissues causing various amyloid diseases. Based on the current understanding of the pathogenesis of TTR amyloidosis, it is considered that the inhibition of amyloid fibril formation by stabilization of TTR in native tetrameric form is a viable approach for the treatment of TTR amyloidosis. METHODOLOGY AND PRINCIPAL FINDINGS: We have examined interactions of the wtTTR with a series of compounds containing various substitutions at biphenyl ether skeleton and a novel compound, previously evaluated for binding and inhibiting tetramer dissociation, by x-ray crystallographic approach. High resolution crystal structures of five ligands in complex with wtTTR provided snapshots of negatively cooperative binding of ligands in two T4 binding sites besides characterizing their binding orientations, conformations, and interactions with binding site residues. In all complexes, the ligand has better fit and more potent interactions in first T4 site i.e. (AC site than the second T4 site (BD site. Together, these results suggest that AC site is a preferred ligand binding site and retention of ordered water molecules between the dimer interfaces further stabilizes the tetramer by bridging a hydrogen bond interaction between Ser117 and its symmetric copy. CONCLUSION: Novel biphenyl ether based compounds exhibit negative-cooperativity while binding to two T4 sites which suggests that binding of only single ligand molecule is sufficient to inhibit the TTR tetramer dissociation.

  9. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  10. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

    Science.gov (United States)

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug

  11. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Directory of Open Access Journals (Sweden)

    Kyle C Wilcox

    Full Text Available Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs. AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs. This method gives a soluble membrane protein library (SMPL--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can

  12. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Science.gov (United States)

    Wilcox, Kyle C; Marunde, Matthew R; Das, Aditi; Velasco, Pauline T; Kuhns, Benjamin D; Marty, Michael T; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G; Klein, William L

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery

  13. On the specificity of heparin/heparan sulfate binding to proteins. Anion-binding sites on antithrombin and thrombin are fundamentally different.

    Directory of Open Access Journals (Sweden)

    Philip D Mosier

    Full Text Available The antithrombin-heparin/heparan sulfate (H/HS and thrombin-H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG-protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics.Analyses of solvent accessibility and exposed surface areas, gyrational mobility, symmetry, cavity shape/size, conserved water molecules and crystallographic parameters were performed for 12 X-ray structures, which include 12 thrombin and 16 antithrombin chains. Novel calculations are described for gyrational mobility and prediction of water loci and conservation.The solvent accessibilities and gyrational mobilities of arginines and lysines in the binding sites of the two proteins reveal sharp contrasts. The distribution of positive charges shows considerable asymmetry in antithrombin, but substantial symmetry for thrombin. Cavity analyses suggest the presence of a reasonably sized bifurcated cavity in antithrombin that facilitates a firm 'hand-shake' with H/HS, but with thrombin, a weaker 'high-five'. Tightly bound water molecules were predicted to be localized in the pentasaccharide binding pocket of antithrombin, but absent in thrombin. Together, these differences in the binding sites explain the major H/HS recognition characteristics of the two prototypic proteins, thus affording an explanation of the specificity of binding. This provides a foundation for understanding specificity of interaction at an atomic level, which will greatly aid the design of natural or synthetic H/HS sequences that target proteins in a specific manner.

  14. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

    Energy Technology Data Exchange (ETDEWEB)

    Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

    2016-06-13

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

  15. Characterization and autoradiographic localization of multiple tachykinin binding sites in gastrointestinal tract and bladder

    Energy Technology Data Exchange (ETDEWEB)

    Burcher, E.; Buck, S.H.; Lovenberg, W.; O' Donohue, T.L.

    1986-03-01

    Binding sites for the (125I)Bolton-Hunter-labeled tachykinins substance K (BHSK), eledoisin (BHE) and substance P (BHSP) were investigated using crude membrane suspensions and autoradiography. In smooth muscle membranes from guinea-pig small intestine and rat duodenum, specific binding of BHSK was saturable and reversible, showing a single class of sites with a KD of 1 to 3 nM and maximum number of specific binding sites of 1 to 2 fmol/mg of wet weight tissue. Pharmacological characterization of this binding revealed a novel receptor site (K) with affinity for substance K greater than kassinin greater than or equal to eledoisin greater than neuromedin K greater than substance P greater than physalaemin. Inhibition of the binding of BHSK in membranes from mouse urinary bladder exhibited a similar K-type pattern. In rat duodenum and mouse bladder membranes, the binding of BHE was inhibited by substance K greater than kassinin greater than eledoisin greater than neuromedin K greater than substance P greater than physalaemin indicating the same receptor site as for BHSK. In rat cerebral cortex membranes BHE binding was inhibited by neuromedin K = kassinin = eledoisin greater than physalaemin greater than substance K greater than substance P indicating a definitive tachykinin E receptor site. The same displacement pattern of BHE binding was also detected in longitudinal muscle membranes from the guinea-pig small intestine. In mouse bladder membranes and in rat and guinea-pig intestine, the binding of BHSP was inhibited by substance P greater than physalaemin greater than substance K greater than or equal to eledoisin = kassinin greater than neuromedin K indicating a definitive tachykinin P receptor site. Autoradiographic binding sites for both BHSK and BHSP were seen in circular muscle of the rat stomach, small intestine and colon and in circular and longitudinal muscle of the guinea-pig small intestine and colon.

  16. Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

    Science.gov (United States)

    Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2013-07-01

    Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

  17. An overview of the prediction of protein DNA-binding sites.

    Science.gov (United States)

    Si, Jingna; Zhao, Rui; Wu, Rongling

    2015-03-06

    Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  18. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  19. Location and nature of calcium-binding sites in salivary acidic proline-rich phosphoproteins

    International Nuclear Information System (INIS)

    Bennick, A.; McLaughlin, A.C.; Grey, A.A.; Madapallimattam, G.

    1981-01-01

    The location of the calcium-binding sites in the human acidic proline-rich proteins, salivary proteins A and C, was determined by equilibrium dialysis of the tryptic peptides with buffers containing 45 Ca. All the calcium-binding sites are located in the NH 2 -terminal tryptic peptide (TX peptide). The nature of the calcium binding sites in the TX peptide and native salivary proteins A and C, as well as dephosphorylated proteins was compared. Two types of sites can be distinguished in peptide TX. Type I sites have an apparent dissociation constant (K) of 38 μM and are responsible for the binding of 2.6 mol of Ca/mol of peptide. The corresponding figures for Type II sites are 780 μM and 5.3 mol of Ca/mol of peptide. In the native proteins, the amount of calcium bound at the type II sites decreases to 3.9 mol of Ca/mol of proteins A and C and K increases to 1100 μM. The amount of calcium bound at type I sites decreases to 1.5 mol/mol of protein A and 0.6 mol/mol of protein C, but there is no change in K. Dephosphorylation affects the calcium binding at both types of sites. The experiments indicate that the COOH-terminal parts of the native proteins affect the number and the nature of the protein calcium-binding sites. Proton and phosphorous NMR data demonstrate that β-COOH in aspartic acid, as well as phosphoserine, are part of the calcium-binding sites. The difference in calcium binding to salivary proteins A and C may be due at least partially to differences in the environment of one or more aspartic acids

  20. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

    Directory of Open Access Journals (Sweden)

    Oscar Harari

    2010-07-01

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  1. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong; Orr, Galya; Xiong, Yijia; Squier, Thomas; Rorrer, Gregory L.; Roesijadi, Guritno

    2016-01-08

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

  2. Ligand-binding sites in human serum amyloid P component

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.

    1996-01-01

    Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes, AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly...

  3. Use of thallium to identify monovalent cation binding sites in GroEL

    Science.gov (United States)

    Kiser, Philip D.; Lorimer, George H.; Palczewski, Krzysztof

    2009-01-01

    GroEL is a bacterial chaperone protein that assembles into a homotetra­decameric complex exhibiting D 7 symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg2+ and K+, to bind and hydrolyze ATP. A K+-binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K+-binding site may exist within GroEL. Because K+ has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K+ mimetic Tl+ was incorporated into GroEL crystals, a moderately redundant 3.94 Å resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K+ next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl+ for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low. PMID:19851000

  4. [3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites.

    Science.gov (United States)

    Javitch, J A; Blaustein, R O; Snyder, S H

    1984-07-01

    [3H]Mazindol labels neuronal dopamine uptake sites in corpus striatum membranes (KD = 18 nM) and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes (KD = 4 nM). The potencies of various inhibitors of biogenic amine uptake in reducing [3H]mazindol binding in striatal membranes correlate with their potencies for inhibition of neuronal [3H]dopamine accumulation, whereas their potencies in reducing [3H]mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal [3H]norepinephrine accumulation. Similar to the dopamine and norepinephrine uptake systems, [3H]mazindol binding in all three tissues is dependent upon sodium (with potassium, lithium, rubidium, and Tris being ineffective substitutes) and chloride (with sulfate and phosphate being ineffective substitutes). In membranes of the cerebral cortex and salivary gland, half-maximal stimulation is observed at 50-80 mM NaCl, whereas in membranes of the corpus striatum half-maximal stimulation occurs at 240 mM NaCl. In striatal membranes NaCl increases the affinity of [3H]mazindol binding with no effect on the maximal number of sites. The enhancement of affinity is due to a selective slowing of the dissociation of the ligand from its binding site. The association of [3H]mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of [3H]mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, the association of [3H]mazindol binding sites with neuronal norepinephrine uptake sites in cerebral cortex is supported by the reduction of [3H]mazindol binding to cortical membranes following destruction of noradrenergic neurons by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine.

  5. A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant.

    Science.gov (United States)

    Sureau, Camille; Salisse, Jessica

    2013-03-01

    Two determinants of infectivity have been identified in the hepatitis B virus (HBV) envelope proteins: a pre-S1 receptor-binding site and an uncharacterized determinant in the antigenic loop (AGL), which is structurally related to the antigenic a-determinant. Infection would proceed through virus attachment to cell surface heparan sulfate (HS) proteoglycans (HSPGs) before pre-S1 engages a specific receptor for uptake. Using heparin binding and in vitro infection assays with hepatitis D virus as a surrogate for HBV, we established that HS binding is mediated by the AGL. Electrostatic interaction was shown to depend upon AGL residues R122 and K141, because their substitution with alanine modified the virus net-charge and prevented binding to heparin, attachment to hepatocytes, and infection. In addition to R122 and K141, the HS binding determinant was mapped to cysteines and prolines, which also define the conformational a-determinant. The importance of AGL conformation was further demonstrated by the concomitant loss of a-determinant and heparin binding upon treatment of viral particles with membrane-impermeable reducing agent. Furthermore, envelope proteins extracted from the viral membrane with a nonionic detergent were shown to conserve the a-determinant but to lose heparin affinity/avidity. Our findings support a model in which attachment of HBV to HSPGs is mediated by the AGL HS binding site, including only two positively charged residues (R122 and K141) positioned precisely in a three-dimensional AGL structure that is stabilized by disulfide bonds. HBV envelope proteins would individually bind to HS with low affinity, but upon their clustering in the viral membrane, they would reach sufficient avidity for a stable interaction between virus and cell surface HSPGs. Our data provide new insight into the HBV entry pathway, including the opportunity to design antivirals directed to the AGL-HS interaction. Copyright © 2012 American Association for the Study of

  6. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Science.gov (United States)

    Das, Shubhadip; Paul, Sandip

    2018-01-01

    The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS) cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  7. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  8. Quantitative autoradiography of (/sup 125/I) apamin binding sites in the central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Janicki, P.K.; Horvath, E.; Habermann, E. (Giessen Univ. (Germany, F.R.). Rudolf-Buchheim-Institut fuer Pharmakologie); Seibold, G. (Giessen Univ. (Germany, F.R.). Strahlenzentrum)

    1984-12-01

    The binding sites for (/sup 125/I) apamin in the central nervous system of rat, guinea-pig, chicken and frog were assessed by quantitative autoradiography on X-ray film. In rat and guinea-pig brain apamin labels preferentially the limbic-olfactory system, i.e. nucleus olfactorius, nuclei septi, habenula and hippocampus. In the rat spinal cord the peptide binds preferentially to the substantia gelatinosa. Tectum opticum and nuclei isthmi are labelled in chicken brain. In frog brain no preferentially 'apamin-stained' area was found. The role of the cerebral binding sites is still unknown, whereas the spinal sites may be involved in apamin poisoning.

  9. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5′-Phosphosulfate Kinase*

    Science.gov (United States)

    Ravilious, Geoffrey E.; Jez, Joseph M.

    2012-01-01

    Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3′-phosphate 5′-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis. PMID:22810229

  10. Autoradiographic characterization of L-[3H]glutamate binding sites in the central nervous system

    International Nuclear Information System (INIS)

    Greenamyre, J.T.

    1986-01-01

    A quantitative autoradiographic technique was developed to study L-[ 3 H[glutamate binding in sections of central nervous system tissue. This technique circumvented some problems associated with conventional receptor binding methodologies and allowed direct assessment of regional distribution, numbers and affinities of glutamate binding sites. The sensitivity and high degree of anatomical resolution attainable by autoradiography obviated the need for pooled samples of microdissected specimens. Under assay conditions, [ 4 H]glutamate bound rapidly and reversibly to sections of rat brain and was not metabolized appreciably. The distribution of glutamate binding sites corresponded to the projection areas of putative glutamatergic pathways. Thus, there was heavy glutamate binding in regions where there is evidence for glutamatergic innervation and little binding in nuclei which apparently do not receive glutamatergic input. Scatchard and Hill plots suggested that glutamate was interacting with a single population of sites; however, competition studies revealed binding site heterogeneity. Anatomical and pharmacological evidence suggested that the NMDA-, high affinity quisqualate-, and kainate-sensitive glutamate binding sites may correspond to physiologically-defined NMDA, quisqualate and kainate receptors

  11. Calcium and initial surface binding phase of pinocytosis in Amoeba proteus

    Energy Technology Data Exchange (ETDEWEB)

    Prusch, R.D.

    1986-08-01

    The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca/sup 2 +/ in the external medium. Increasing external Ca/sup 2 +/ to approx.10/sup -4/ M increases pinocytotic intensity, while increases in Ca/sup 2 +/ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca/sup +2/ at the surface of A moeba proteus was therefore examined. Alcain blue and Na/sup +/, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sites on the amoeba surface. Low levels of external Ca/sup 2 +/ in the range of 3 x 10/sup -5/ to 4.5 x 10/sup -4/ M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca/sup 2 +/ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity. Surface binding of Na/sup +/, Ca/sup 2 +/ and Cl/sup -/ was determined by adding /sup 22/Na, /sup 45/Ca or /sup 36/Cl.

  12. Investigation of the metal binding site in methionine aminopeptidase by density functional theory

    DEFF Research Database (Denmark)

    Jørgensen, Anne Techau; Norrby, Per-Ola; Liljefors, Tommy

    2002-01-01

    All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions...

  13. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

    Energy Technology Data Exchange (ETDEWEB)

    Makyio, Hisayoshi [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan); Shimabukuro, Junpei; Suzuki, Tatsuya [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Imamura, Akihiro; Ishida, Hideharu [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Kiso, Makoto [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Ando, Hiromune, E-mail: hando@gifu-u.ac.jp [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Kato, Ryuichi, E-mail: ryuichi.kato@kek.jp [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan)

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

  14. Quantitative autoradiographic distribution of L-[3H]glutamate-binding sites in rat central nervous system

    International Nuclear Information System (INIS)

    Greenamyre, J.T.; Young, A.B.; Penney, J.B.

    1984-01-01

    Quantitative autoradiography was used to determine the distribution of L-[3H]glutamate-binding sites in the rat central nervous system. Autoradiography was carried out in the presence of Cl- and Ca2+ ions. Scatchard plots and Hill coefficients of glutamate binding suggested that glutamate was interacting with a single population of sites having a K-D of about 300 nM and a capacity of 14.5 pmol/mg of protein. In displacement studies, ibotenate also appeared to bind to a single class of non-interacting sites with a KI of 28 microM. However, quisqualate displacement of [3H]glutamate binding revealed two well-resolved sites with KIS of 12 nM and 114 microM in striatum. These sites were unevenly distributed, representing different proportions of specific glutamate binding in different brain regions. The distribution of glutamate-binding sites correlated very well with the projection areas of putative glutamatergic pathways. This technique provides an extremely sensitive assay which can be used to gather detailed pharmacological and anatomical information about L-[3H]glutamate binding in the central nervous system

  15. Rat submaxillary gland contains predominantly P-type tachykinin binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Buck, S.H.; Burcher, E.

    1985-11-01

    The specific binding of the /sup 125/I-Bolton-Hunter labeled tachykinins substance K (BHSK), eledoisin (BHE), and substance P (BHSP) was examined in crude membrane suspensions and by autoradiography in rat submaxillary gland. All three ligands at 0.1 nM concentrations exhibited binding that was inhibited by tachykinins in a potency rank order of substance P greater than physalaemin greater than substance K greater than eledoisin greater than kassinin greater than neuromedin K with slope factors essentially equal to unity. All tachykinins were 5 to 10 times more potent in inhibiting BHSK and BHE binding compared to BHSP binding. Autoradiographic visualization of BHSK and BHSP binding sites in the gland revealed extensive labeling of mucous and serous acini. The intensity of labeling was much less for BHSK than for BHSP. The results indicate that the rat submaxillary gland contains predominantly P-type tachykinin binding sites.

  16. Photoaffinity studies of the tubulin-colchicine binding site

    International Nuclear Information System (INIS)

    Hahn, K.M.

    1987-01-01

    A variety of colchicine derivatives were synthesized and coupled with 3,3,3-trifluoro-2-diazapropionyl chloride (TFDP-Cl) to produce colchicine photoaffinity analogs for use in tubulin labelling studies. Photoaffinity analogs of allocolchicine and podophylotoxin were also made using the same photoreactive moiety. Several labels were found to be effective inhibitors of tubulin polymerization. The approximate tubulin binding constants of the labels, calculated from polymerization inhibition data, varied between 2.2 x 10 5 to 2.5 x 10 3 M -1 . The labels chosen for use in tubulin labelling experiments were (N-TFDP) deacetyl-thiocolchicine 1, (O-TFDP)thiocolchifoline 2, and (O-TFDP)-2-demethylthiocolchicine 3. Compound 1 was found to bind tubulin reversibly and to competitively inhibit colchicine binding. Methods for the incorporation of tritium and 14 C in these labels were developed. Conditions were found which caused labels to insert into solvent without photorearrangement of the colchicine skeleton. Catalytic base caused the α-diazo amide of 1 to rearrange to a triazole

  17. Prolactin binding sites on human chorion-decidua tissue

    International Nuclear Information System (INIS)

    McWey, L.A.; Singhas, C.A.; Rogol, A.D.

    1982-01-01

    An effective procedure has been developed and utilized to demonstrate the presence of prolactin receptors on the plasma membranes of human chorion-decidua cells. Particulate fractions from human chorion-decidua sedimenting between 1,500 and 45,000 x g display optimal binding of 125 I-labeled ovine prolactin when incubated at a membrane protein concentration of 200 micrograms per assay tube for 2 hours at 22 degrees C. Specific binding was increased by pretreatment of the membrane particles with 5M magnesium chloride to remove endogenous prolactin. These receptors show binding parameters (affinity, 0.92 x 10(9) L/mode; capacity, approximately 80 fmoles/mg) similar to those of lactogenic receptors in the rabbit mammary gland and, the rabbit and rat liver. The presence of prolactin receptors in human chorion-decidua suggests that may play a role in mediating local action(s) of prolactin such as involvement in the decidualization reaction or in maintaining fetal osmoregulation

  18. Europium ion as a probe for binding sites to carrageenans

    International Nuclear Information System (INIS)

    Ramos, Ana P.; Goncalves, Rogeria R.; Serra, Osvaldo A.; Zaniquelli, Maria Elisabete D.; Wong, Kenneth

    2007-01-01

    Carrageenans, sulfated polysaccharides extracted from red algae, present a coil-helix transition and helix aggregation dependence on the type and concentration of counterions. In this study, we focus attention on a mixed valence counterion system: Eu 3+ /Na + or K + with different gel-forming carrageenans: kappa, iota, and kappa-2. Results of stationary and time-dependent luminescence showed to be a suitable tool to probe ion binding to both the negatively charged sulfate group and the hydroxyl groups present in the biopolymer. For lower europium ion concentrations, a single longer decay emission lifetime was detected, which was attributed to the binding of europium ion to the carrageenan sulfate groups. An additional decay ascribed to europium binding to hydroxyl groups was observed above a threshold concentration, and this decay was dependent on the carrageenan charge density. Symmetry of the europium ion microenvironment was estimated by the ratio between the intensities of its emission bands, which has been shown to depend on the concentration of europium ions and on the specificity of the monovalent counterion bound to the carrageenan

  19. The binding sites for cocaine and dopamine in the dopamine transporter overlap

    DEFF Research Database (Denmark)

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L

    2008-01-01

    Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... mutagenesis and by trapping the radiolabeled cocaine analog [3H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn2+-binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive...

  20. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly...... be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...... approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence...

  1. Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A.

    Science.gov (United States)

    Strotmeier, Jasmin; Mahrhold, Stefan; Krez, Nadja; Janzen, Constantin; Lou, Jianlong; Marks, James D; Binz, Thomas; Rummel, Andreas

    2014-04-02

    Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Spectroscopic study of interaction between osthole and human serum albumin: Identification of possible binding site of the compound

    International Nuclear Information System (INIS)

    Bijari, Nooshin; Shokoohinia, Yalda; Ashrafi-Kooshk, Mohammad Reza; Ranjbar, Samira; Parvaneh, Shahram; Moieni-Arya, Maryam; Khodarahmi, Reza

    2013-01-01

    The studies on the interaction between human serum albumin (HSA) and drugs have been an interesting research field in life science, chemistry and clinical medicine. Osthole possesses a variety of pharmacological activities including anti-tumor, anti-inflammation, anti-seizure, anti-hyperlipidemic and anti-osteoporosis effects. The interaction of osthole with HSA and its binding site in HSA by spectroscopic methods is the subject of this work. By monitoring the intrinsic fluorescence of the single Trp 214 residue and performing site markers displacement measurements, the specific binding of osthole in the vicinity of Sudlow's site I of HSA has been clarified. The changes in the secondary structure of HSA after its complexation with ligand were studied with CD spectroscopy, which indicate that osthole induced only a slight decrease in the helix structural content of the protein. In addition, the mean distance between osthole and HSA fluorophores is estimated to be 4.96 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Osthole can quench the intrinsic fluorescence of HSA by dynamic quenching, and analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play an important role in the stabilizing of the complex. Increase of protein surface hydrophobicity (PSH) was also observed upon the osthole binding. -- Highlights: • Hydrophobic interactions play an important role in osthole–HSA interaction. • Sudlow's I site is possible binding site of osthole. • Osthole inhibits esterase activity of HSA. • Osthole binding induces no gross protein structural changes

  3. Spectroscopic study of interaction between osthole and human serum albumin: Identification of possible binding site of the compound

    Energy Technology Data Exchange (ETDEWEB)

    Bijari, Nooshin [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Shokoohinia, Yalda [Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza; Ranjbar, Samira; Parvaneh, Shahram [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Moieni-Arya, Maryam [Student Research Committee, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

    2013-11-15

    The studies on the interaction between human serum albumin (HSA) and drugs have been an interesting research field in life science, chemistry and clinical medicine. Osthole possesses a variety of pharmacological activities including anti-tumor, anti-inflammation, anti-seizure, anti-hyperlipidemic and anti-osteoporosis effects. The interaction of osthole with HSA and its binding site in HSA by spectroscopic methods is the subject of this work. By monitoring the intrinsic fluorescence of the single Trp{sub 214} residue and performing site markers displacement measurements, the specific binding of osthole in the vicinity of Sudlow's site I of HSA has been clarified. The changes in the secondary structure of HSA after its complexation with ligand were studied with CD spectroscopy, which indicate that osthole induced only a slight decrease in the helix structural content of the protein. In addition, the mean distance between osthole and HSA fluorophores is estimated to be 4.96 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Osthole can quench the intrinsic fluorescence of HSA by dynamic quenching, and analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play an important role in the stabilizing of the complex. Increase of protein surface hydrophobicity (PSH) was also observed upon the osthole binding. -- Highlights: • Hydrophobic interactions play an important role in osthole–HSA interaction. • Sudlow's I site is possible binding site of osthole. • Osthole inhibits esterase activity of HSA. • Osthole binding induces no gross protein structural changes.

  4. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-05-29

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Localization of 125I-insulin binding sites in the rat hypothalamus by quantitative autoradiography

    International Nuclear Information System (INIS)

    Corp, E.S.; Woods, S.C.; Figlewicz, D.P.; Porte, D. Jr.; Baskin, D.G.; Dorsa, D.M.

    1986-01-01

    In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125 I-insulin in the hypothalamus of the rat brain. Highest specific binding was found in the arculate, dorsomedial, suprachiasmatic, paraventricular and periventricular regions. Significantly lower binding was present in the ventromedial nucleus and median eminence. The results are consistent with the hypothesis that insulin modulates the neural regulation of feeding by acting at sites in the hypothalamus. (author)

  6. Evolutionary mirages: selection on binding site composition creates the illusion of conserved grammars in Drosophila enhancers.

    Directory of Open Access Journals (Sweden)

    Richard W Lusk

    2010-01-01

    Full Text Available The clustering of transcription factor binding sites in developmental enhancers and the apparent preferential conservation of clustered sites have been widely interpreted as proof that spatially constrained physical interactions between transcription factors are required for regulatory function. However, we show here that selection on the composition of enhancers alone, and not their internal structure, leads to the accumulation of clustered sites with evolutionary dynamics that suggest they are preferentially conserved. We simulated the evolution of idealized enhancers from Drosophila melanogaster constrained to contain only a minimum number of binding sites for one or more factors. Under this constraint, mutations that destroy an existing binding site are tolerated only if a compensating site has emerged elsewhere in the enhancer. Overlapping sites, such as those frequently observed for the activator Bicoid and repressor Krüppel, had significantly longer evolutionary half-lives than isolated sites for the same factors. This leads to a substantially higher density of overlapping sites than expected by chance and the appearance that such sites are preferentially conserved. Because D. melanogaster (like many other species has a bias for deletions over insertions, sites tended to become closer together over time, leading to an overall clustering of sites in the absence of any selection for clustered sites. Since this effect is strongest for the oldest sites, clustered sites also incorrectly appear to be preferentially conserved. Following speciation, sites tend to be closer together in all descendent species than in their common ancestors, violating the common assumption that shared features of species' genomes reflect their ancestral state. Finally, we show that selection on binding site composition alone recapitulates the observed number of overlapping and closely neighboring sites in real D. melanogaster enhancers. Thus, this study calls into

  7. Characterization of the part of N-terminal PIP2 binding site of the TRPM1 channel

    Czech Academy of Sciences Publication Activity Database

    Jirků, Michaela; Bumba, Ladislav; Bednárová, Lucie; Kubala, M.; Šulc, Miroslav; Franěk, M.; Vyklický ml., Ladislav; Vondrášek, Jiří; Teisinger, Jan; Boušová, Kristýna

    2015-01-01

    Roč. 207, Dec (2015), s. 135-142 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GAP207/11/0717; GA ČR(CZ) GA15-11851S; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 ; RVO:61388971 ; RVO:61388963 Keywords : TRPM1 channel * binding site * PIP2 * surface plasmon resonance * FRET * circular dichroism Subject RIV: CE - Biochemistry Impact factor: 2.363, year: 2015

  8. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  9. Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2018-02-01

    The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

  10. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  11. An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site

    Science.gov (United States)

    Lu, Yan; Chen, Jianjun; Xiao, Min; Li, Wei

    2013-01-01

    Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding site is one of the most important pockets for potential tubulin polymerization destabilizers. Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. A large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers. CBSIs are believed to act by a common mechanism via binding to the colchicine site on tubulin. The present review is a synopsis of compounds that have been reported in the past decade that have provided an increase in our understanding of the actions of CBSIs. PMID:22814904

  12. Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine, mazindol, and GBR 12935.

    Science.gov (United States)

    Reith, M E; Selmeci, G

    1992-03-01

    This study addressed the possibility of a unique binding interaction between cocaine and the dopamine transporter as compared with other blockers of dopamine uptake. Cocaine binding sites in a fresh P2 fraction of mouse striatum were labeled with [3H]CFT, a phenyltropane analog of cocaine also known as WIN 35,428, and compared with sites labeled with [3H]mazindol or [3H]GBR 12935. Under the conditions used, homogeneous binding was observed that was inhibited monophasically by cocaine, CFT, and mazindol; the same potencies were observed with the three radioligands. Saturation analysis in the presence and in the absence of unlabeled inhibitor (CFT, mazindol, cocaine) indicated a change in the Kd but not the Bmax, consonant with a competitive mechanisms. Tris-HCl reduced the affinity of each radioligand and unlabeled inhibitor without changing the Bmax. N-Ethylmaleimide reduced the binding of all radioligands equally and cocaine offered protection. The dissociation rate of [3H]CFT and [3H]mazindol binding was not affected by the presence of mazindol and CFT, respectively. The Bmax of [3H]CFT and [3H]mazindol binding was the same; the relatively higher value for [3H]GBR 12935 binding in analyses involving varying tritiated GBR 12935 only, was due primarily to an underestimation of the specific activity of [3H]GBR 12935. All results are in agreement with a one-site model in which cocaine, CFT, mazindol, and GBR 12935 share a common binding site in mouse striatum.

  13. Radiolabelling of phoneutria nigriventer spider toxin (Tx1): a tool to study its binding site

    International Nuclear Information System (INIS)

    Santos, Raquel Gouvea dos; Diniz, Carlos Roberto; Nascimento, Marta Cordeiro; Lima, Maria Elena de

    1996-01-01

    The neurotoxin Tx1, isolated from the venom of the South American spider Phoneutria nigriventer produces tail elevation and spastic paralysis of posterior limbs after intracerebral ventricular injection in mice. Tx1 also produces ileum contraction in bioassay. We have investigated the binding of radioiodinated-Tx1 ( 125 I-Tx1) on the preparation of myenteric plexus-longitudinal muscle membrane from guinea pig ileum (MPLM) as a tool to characterize the interaction of this neurotoxin with its site. The neurotoxin Tx1 was radioiodinated with Na 125 I by the lactoperoxidase method. 125 I-Tx1 specifically binds to a single class of noninteracting binding sites of high affinity (Kd= 3.5 x 10 -10 M) and low capacity (1.2 pmol/mg protein). The specific binding increased in parallel with the protein concentration. In competition experiments the ligands of ionic channels used (sodium, potassium and calcium) did not affect the binding of 125 I-Tx1 to MPLM neither did the cholinergic ligands (hemicholinium-3, hexamethonium, d-tubocurarine and atropine). Another neurotoxin (Tx2-6, one of the isoforms of Tx2 pool) decreased toxin with MPLM and showed that toxin has a specific and saturable binding site in guinea pig ileum and this binding site appears to be related to the Tx2 site. (author)

  14. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    DEFF Research Database (Denmark)

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.

    2012-01-01

    -target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn2+-binding group. The lead compounds (e.g., 15 and 26) display good...... potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with Ki values of 49, 33, and 37 nM, respectively. Our proof...

  15. Estimating the relative position of risperidone primary binding site in Sera Albumins. Modeling from spectrofluorimetric data

    Science.gov (United States)

    Cortez, Celia Martins; Fragoso, Viviane Muniz S.; Silva, Dilson

    2014-10-01

    In this work, we used a mathematical model to study the interaction of risperidone with human and bovine serum albumins estimating the relative position of the primary binding site, based on the fluorescence quenching theory. Results have shown that the model was able to demonstrate that primary binding site for risperidone in HSA and BSA is very close to the position where is tryptophan 134 of BSA, possibly in domain 1B.

  16. Effect of positional dependence and alignment strategy on modeling transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Quader Saad

    2012-07-01

    Full Text Available Abstract Background Many consensus-based and Position Weight Matrix-based methods for recognizing transcription factor binding sites (TFBS are not well suited to the variability in the lengths of binding sites. Besides, many methods discard known binding sites while building the model. Moreover, the impact of Information Content (IC and the positional dependence of nucleotides within an aligned set of TFBS has not been well researched for modeling variable-length binding sites. In this paper, we propose ML-Consensus (Mixed-Length Consensus: a consensus model for variable-length TFBS which does not exclude any reported binding sites. Methods We consider Pairwise Score (PS as a measure of positional dependence of nucleotides within an alignment of TFBS. We investigate how the prediction accuracy of ML-Consensus is affected by the incorporation of IC and PS with a particular binding site alignment strategy. We perform cross-validations for datasets of six species from the TRANSFAC public database, and analyze the results using ROC curves and the Wilcoxon matched-pair signed-ranks test. Results We observe that the incorporation of IC and PS in ML-Consensus results in statistically significant improvement in the prediction accuracy of the model. Moreover, the existence of a core region among the known binding sites (of any length is witnessed by the pairwise coexistence of nucleotides within the core length. Conclusions These observations suggest the possibility of an efficient multiple sequence alignment algorithm for aligning TFBS, accommodating known binding sites of any length, for optimal (or near-optimal TFBS prediction. However, designing such an algorithm is a matter of further investigation.

  17. Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro

    International Nuclear Information System (INIS)

    Leysen, J.E.

    1981-01-01

    In vitro binding studies to serotoninergic receptors were performed using 3 H-LSD, 3 H-5-HT and 3 H-spiperone. An overwiew is given on findings using these three ligands with respect to the following: localization of specific binding sites, in various animal species, the regional distribution in the brain and periphery, the subcellular and cellular distribution. Properties of the binding sites, influence of the composition of the assay medium, binding kinetic properties, receptor regulation in vivo. Identity of the binding sites, differences between site for various 3 H-ligands, pharmacological specificity of the membranous binding sites, chemical composition of the macromolecular complex constituting the binding site. Function of the receptor. Binding affinities of 44 compounds were measured in binding assays using 3 H-spiperone and 3 H-LSD with rat frontal cortex membrane preparations and using 3 H-5-HT and 3 H-LSD with rat hippocampal membrane preparations

  18. Three complement-like repeats compose the complete alpha2-macroglobulin binding site in the second ligand binding cluster of the low density lipoprotein receptor-related protein.

    Science.gov (United States)

    Dolmer, Klavs; Gettins, Peter G W

    2006-11-10

    Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of alpha2-macroglobulin (alpha2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity alpha2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for alpha2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nM) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.

  19. PocketMatch: a new algorithm to compare binding sites in protein structures.

    Science.gov (United States)

    Yeturu, Kalidas; Chandra, Nagasuma

    2008-12-17

    Recognizing similarities and deriving relationships among protein molecules is a fundamental requirement in present-day biology. Similarities can be present at various levels which can be detected through comparison of protein sequences or their structural folds. In some cases similarities obscure at these levels could be present merely in the substructures at their binding sites. Inferring functional similarities between protein molecules by comparing their binding sites is still largely exploratory and not as yet a routine protocol. One of the main reasons for this is the limitation in the choice of appropriate analytical tools that can compare binding sites with high sensitivity. To benefit from the enormous amount of structural data that is being rapidly accumulated, it is essential to have high throughput tools that enable large scale binding site comparison. Here we present a new algorithm PocketMatch for comparison of binding sites in a frame invariant manner. Each binding site is represented by 90 lists of sorted distances capturing shape and chemical nature of the site. The sorted arrays are then aligned using an incremental alignment method and scored to obtain PMScores for pairs of sites. A comprehensive sensitivity analysis and an extensive validation of the algorithm have been carried out. A comparison with other site matching algorithms is also presented. Perturbation studies where the geometry of a given site was retained but the residue types were changed randomly, indicated that chance similarities were virtually non-existent. Our analysis also demonstrates that shape information alone is insufficient to discriminate between diverse binding sites, unless combined with chemical nature of amino acids. A new algorithm has been developed to compare binding sites in accurate, efficient and high-throughput manner. Though the representation used is conceptually simplistic, we demonstrate that along with the new alignment strategy used, it is sufficient to

  20. Up-regulatory effect of triphasic oral contraceptive on platelet 3H-imipramine binding sites.

    Science.gov (United States)

    Weizman, A; Morgenstern, H; Kaplan, B; Amiri, Z; Tyano, S; Ovadia, Y; Rehavi, M

    1988-01-01

    Triphasic oral contraceptive (Logynon) induced a significant increase (36%) in the maximal binding capacity of platelet membranes for [3H]imipramine. The increase was achieved in the second Logynon cycle as compared to pretreatment and first Logynon cycle binding values. The pill contains a combination of ethinyl estradiol and levonorgestrel, and it is as yet unclear which of the two hormones is responsible for the up-regulatory effect. The increase in the density of platelet imipramine binding sites may reflect a similar alteration in brain. The increase in imipramine binding did not correlate with alteration in mood as assessed by Beck Depression Inventory scores.

  1. Design and creation of a Ca2+ binding site in human lysozyme to enhance structural stability.

    OpenAIRE

    Kuroki, R; Taniyama, Y; Seko, C; Nakamura, H; Kikuchi, M; Ikehara, M

    1989-01-01

    A Ca2+ binding site like an EF-hand motif was designed and created in human lysozyme by replacing both Gln-86 and Ala-92 with aspartic acids by site-directed mutagenesis. The mutant human lysozyme (D86/92-lysozyme) was expressed and secreted by yeast. One Ca2+ was found to bind one molecule of the purified protein with the binding constant 5.0 x 10(6) M-1. The enzymatic activity of holo-D86/92-lysozyme against glycol chitin at 40 degrees C was 2-fold higher than that of the native lysozyme. M...

  2. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

  3. Evaluation of a novel virtual screening strategy using receptor decoy binding sites.

    Science.gov (United States)

    Patel, Hershna; Kukol, Andreas

    2016-08-23

    Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated.

  4. Weakly Hydrated Surfaces and the Binding Interactions of Small Biological Solutes

    Energy Technology Data Exchange (ETDEWEB)

    Brady, J. W.; Tavagnacco, L.; Ehrlich, L.; Chen, M.; Schnupf, U.; Himmel, M. E.; Saboungi, M. L.; Cesaro, A.

    2012-04-01

    Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

  5. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Amy Sebeson

    Full Text Available The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  6. Thyrotropin-releasing hormone receptor binding sites: autoradiographic distribution in the rat and guinea pig brain

    Energy Technology Data Exchange (ETDEWEB)

    Pazos, A.; Cortes, R.; Palacios, J.M.

    1985-11-01

    Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with (TH)methyl TRH and localized autoradiographically using TH-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.

  7. Copper(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization.

    Science.gov (United States)

    Ramis, Rafael; Ortega-Castro, Joaquín; Vilanova, Bartolomé; Adrover, Miquel; Frau, Juan

    2017-08-03

    The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.

  8. 2[125I]Iodomelatonin binding sites in spleens of guinea pigs

    International Nuclear Information System (INIS)

    Poon, A.M.S.; Pang, S.F.

    1992-01-01

    2-[ 125 I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8±4.12 pmol/l and binding site density (Bmax) of 0.69±0.082 fmol/mg protein at mid-light. There was no significant change in the Kd or the Bmax at mid-dark. Kinetic analysis showed a Kd of 23.13±4.81 pmol/l, in agreement to that derived from the saturation studies. The 2-[ 125 I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin much-gt N-acetylserotonin, 6-hydroxymelatonin > 5-methoxytryptamine, 5-methoxytryptophol > serotonin, 5-methoxyindole-3-acetic acid > 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan > tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction, the rest are distributed in the microsomal fraction, mitochondrial fraction and cytosolic fraction. The demonstration of 2-[ 125 I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system

  9. Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

    International Nuclear Information System (INIS)

    Kumar, K.P.; Chatterji, D.

    1990-01-01

    Terbium(III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K d between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31 P and 1 H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K d values of 4 μM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the β-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 angstrom for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the β-subunit of E. coli RNA polymerase was measured to be around 30 angstrom. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate

  10. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Thuesen, Cathrine K; Bøtkjær, Kenneth A

    2015-01-01

    around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro...

  11. Analysis of functional importance of binding sites in the Drosophila gap gene network model.

    Science.gov (United States)

    Kozlov, Konstantin; Gursky, Vitaly V; Kulakovskiy, Ivan V; Dymova, Arina; Samsonova, Maria

    2015-01-01

    The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. We elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

  12. Two glycosylation sites in H5N1 influenza virus hemagglutinin that affect binding preference by computer-based analysis.

    Directory of Open Access Journals (Sweden)

    Wentian Chen

    Full Text Available Increasing numbers of H5N1 influenza viruses (IVs are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA on the viral surface to host sialic acid (SA receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs.

  13. Two glycosylation sites in H5N1 influenza virus hemagglutinin that affect binding preference by computer-based analysis.

    Science.gov (United States)

    Chen, Wentian; Sun, Shisheng; Li, Zheng

    2012-01-01

    Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs.

  14. Tubulin inhibitors targeting the colchicine binding site: a perspective of privileged structures.

    Science.gov (United States)

    Li, Wenlong; Sun, Honghao; Xu, Shengtao; Zhu, Zheying; Xu, Jinyi

    2017-10-01

    The vital roles of microtubule in mitosis and cell division make it an attractive target for antitumor therapy. Colchicine binding site of tubulin is one of the most important pockets that have been focused on to design tubulin-destabilizing agents. Over the past few years, a large number of colchicine binding site inhibitors (CBSIs) have been developed inspired by natural products or synthetic origins, and many moieties frequently used in these CBSIs are structurally in common. In this review, we will classify the CBSIs into classical CBSIs and nonclassical CBSIs according to their spatial conformations and binding modes with tubulin, and highlight the privileged structures from these CBSIs in the development of tubulin inhibitors targeting the colchicine binding site.

  15. Properties of achetakinin binding sites on malpighian tubule membranes from the house cricket, Acheta domesticus.

    Science.gov (United States)

    Chung, J S; Wheeler, C H; Goldsworthy, G J; Coast, G M

    1995-01-01

    A biologically active 125I-labeled analogue of AK-II (3'-hydroxyphenyl propionic-Gly-Gly-Gly-Phe-Ser-Pro-Trp-Gly-NH2) was used to investigate the properties of achetakinin binding sites on plasma membranes from Malpighian tubules of Acheta domesticus. With optimized conditions, binding was rapid, reversible, and specific, and saturation studies revealed a single class of binding sites with Kd 0.55 nM and Bmax 39.9 fmol/mg membrane protein. The affinities of achetakinins for binding sites on tubule membranes ranked AK-V > AK III > AK-II > AK-I > or = AK-IV, in general agreement with their potencies in functional assays. However, IC50 values were several orders of magnitude higher than corresponding values for EC50, which suggests a considerable receptor reserve.

  16. Subtype-Specific Agonists for NMDA Receptor Glycine Binding Sites

    DEFF Research Database (Denmark)

    Maolanon, Alex R.; Risgaard, Rune; Wang, Shuang Yan

    2017-01-01

    A series of analogues based on serine as lead structure were designed, and their agonist activities were evaluated at recombinant NMDA receptor subtypes (GluN1/2A-D) using two-electrode voltage-clamp (TEVC) electrophysiology. Pronounced variation in subunit-selectivity, potency, and agonist...... efficacy was observed in a manner that was dependent on the GluN2 subunit in the NMDA receptor. In particular, compounds 15a and 16a are potent GluN2C-specific superagonists at the GluN1 subunit with agonist efficacies of 398% and 308% compared to glycine. This study demonstrates that subunit......-selectivity among glycine site NMDA receptor agonists can be achieved and suggests that glycine-site agonists can be developed as pharmacological tool compounds to study GluN2C-specific effects in NMDA receptor-mediated neurotransmission....

  17. Residue propensities, discrimination and binding site prediction of adenine and guanine phosphates

    Directory of Open Access Journals (Sweden)

    Ahmad Zulfiqar

    2011-05-01

    Full Text Available Abstract Background Adenine and guanine phosphates are involved in a number of biological processes such as cell signaling, metabolism and enzymatic cofactor functions. Binding sites in proteins for these ligands are often detected by looking for a previously known motif by alignment based search. This is likely to miss those where a similar binding site has not been previously characterized and when the binding sites do not follow the rule described by predefined motif. Also, it is intriguing how proteins select between adenine and guanine derivative with high specificity. Results Residue preferences for AMP, GMP, ADP, GDP, ATP and GTP have been investigated in details with additional comparison with cyclic variants cAMP and cGMP. We also attempt to predict residues interacting with these nucleotides using information derived from local sequence and evolutionary profiles. Results indicate that subtle differences exist between single residue preferences for specific nucleotides and taking neighbor environment and evolutionary context into account, successful models of their binding site prediction can be developed. Conclusion In this work, we explore how single amino acid propensities for these nucleotides play a role in the affinity and specificity of this set of nucleotides. This is expected to be helpful in identifying novel binding sites for adenine and guanine phosphates, especially when a known binding motif is not detectable.

  18. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A

    Directory of Open Access Journals (Sweden)

    Manuela Maurer

    2016-04-01

    Full Text Available The periplasmic oligopeptide binding protein A (OppA represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK, but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

  19. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

  20. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277......-793nM)). Eight amino acids, which correspond to amino acids that are critical for ligand binding to other NMDA receptor subunits, situated within the S1S2 predicted ligand binding domain of hNR3A were mutated, which resulted in complete or near complete loss of [(3)H]-glycine binding to hNR3A. The NMDA...

  1. Kinetics of Cation and Oxyanion Adsorption and Desorption on Ferrihydrite: Roles of Ferrihydrite Binding Sites and a Unified Model.

    Science.gov (United States)

    Tian, Lei; Shi, Zhenqing; Lu, Yang; Dohnalkova, Alice C; Lin, Zhang; Dang, Zhi

    2017-09-19

    Quantitative understanding the kinetics of toxic ion reactions with various heterogeneous ferrihydrite binding sites is crucial for accurately predicting the dynamic behavior of contaminants in environment. In this study, kinetics of As(V), Cr(VI), Cu(II), and Pb(II) adsorption and desorption on ferrihydrite was studied using a stirred-flow method, which showed that metal adsorption/desorption kinetics was highly dependent on the reaction conditions and varied significantly among four metals. High resolution scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed that all four metals were distributed within the ferrihydrite aggregates homogeneously after adsorption reactions. Based on the equilibrium model CD-MUSIC, we developed a novel unified kinetics model applicable for both cation and oxyanion adsorption and desorption on ferrihydrite, which is able to account for the heterogeneity of ferrihydrite binding sites, different binding properties of cations and oxyanions, and variations of solution chemistry. The model described the kinetic results well. We quantitatively elucidated how the equilibrium properties of the cation and oxyanion binding to various ferrihydrite sites and the formation of various surface complexes controlled the adsorption and desorption kinetics at different reaction conditions and time scales. Our study provided a unified modeling method for the kinetics of ion adsorption/desorption on ferrihydrite.

  2. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

    Science.gov (United States)

    Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

    2013-01-01

    In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

  3. Location and effect of obesity on putative anorectic binding sites in the rat brain.

    Science.gov (United States)

    Dunn-Meynell, A A; Levin, B E

    1997-05-01

    Anorectic drugs such as mazindol bind to a class of low-affinity, sodium-sensitive sites in the brain which are affected by ambient glucose concentrations and a predisposition to develop diet-induced obesity (DIO). This study used quantitative autoradiography of 10 nM 3H-mazindol binding to identify the cellular location of these putative anorectic binding sites in the brain and to assess the way in which the development of DIO affected their binding. We previously showed that chow-fed, obesity-prone rats have widespread increases in brain 3H-mazindol binding to these low-affinity sites as compared with diet-resistant (DR) rats. Here, low-affinity 3H-mazindol binding was assessed in the brains of eight rats which developed DIO vs. eight which were DR after three months on a high-energy diet. DIO rats gained 89% more weight and had 117% higher plasma insulin levels but no difference in plasma glucose levels compared with DR rats. Along with these differences, low-affinity 3H-mazindol binding in DIO rats was identical to that in DR rats in all of the 23 brain areas assessed. This suggested that this binding was downregulated by the development of obesity in DIO rats. In other chow-fed rats, stereotaxic injections of 5,7-dihydroxytryptamine and 6-hydroxydopamine (6OHDA) to ablate serotonin and catecholamine nerve terminals in the ventromedial nucleus of the hypothalamus (VMN) had no effect on 3H-mazindol binding. However, ibotenic acid injected into the VMN, substantia nigra, pars reticulata, and pars compacta destroyed intrinsic neurons and/or their local processes and decreased low-affinity 3H-mazindol binding by 13%-22%. Destruction of dopamine neurons in the substantia nigra, pars compacta, and noradrenergic neurons in the locus ceruleus with 6OHDA also reduced 3H-mazindol binding in those areas by 9% and 12%, respectively. This suggested that up to 22% of putative anorectic binding sites may be located on the cell bodies of dopamine, norepinephrine, and other

  4. Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane.

    Science.gov (United States)

    Marek, Patrick; Senecal, Kris; Nida, Dawn; Magnone, Joshua; Senecal, Andre

    2011-10-24

    The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs) 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed. A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black) membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655). Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R(2) = .9973) while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R(2) = .999). A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.

  5. Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane

    Directory of Open Access Journals (Sweden)

    Magnone Joshua

    2011-10-01

    Full Text Available Abstract Background The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed. Results A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655. Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R2 = .9973 while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R2 = .999. Conclusion A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.

  6. Searching for the low affinity ubiquinone binding site in cytochrome bo3from Escherichia coli.

    Science.gov (United States)

    Choi, Sylvia K; Lin, Myat T; Ouyang, Hanlin; Gennis, Robert B

    2017-05-01

    The cytochrome bo 3 ubiquinol oxidase is one of three respiratory oxygen reductases in the aerobic respiratory chain of Escherichia coli. The generally accepted model of catalysis assumes that cyt bo 3 contains two distinct ubiquinol binding sites: (i) a low affinity (Q L ) site which is the traditional substrate binding site; and (ii) a high affinity (Q H ) site where a "permanently" bound quinone acts as a cofactor, taking two electrons from the substrate quinol and passing them one-by-one to the heme b component of the enzyme which, in turn, transfers them to the heme o 3 /Cu B active site. Whereas the residues at the Q H site are well defined, the location of the Q L site remains unknown. The published X-ray structure does not contain quinone, and substantial amounts of the protein are missing as well. A recent bioinformatics study by Bossis et al. [Biochem J. (2014) 461, 305-314] identified a sequence motif G 163 EFX 3 GWX 2 Y 173 as the likely Q L site in the family of related quinol oxidases. In the current work, this was tested by site-directed mutagenesis. The results show that these residues are not important for catalytic function and do not define the Q L substrate binding site. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  8. Phocid seal leptin: tertiary structure and hydrophobic receptor binding site preservation during distinct leptin gene evolution.

    Directory of Open Access Journals (Sweden)

    John A Hammond

    Full Text Available The cytokine hormone leptin is a key signalling molecule in many pathways that control physiological functions. Although leptin demonstrates structural conservation in mammals, there is evidence of positive selection in primates, lagomorphs and chiropterans. We previously reported that the leptin genes of the grey and harbour seals (phocids have significantly diverged from other mammals. Therefore we further investigated the diversification of leptin in phocids, other marine mammals and terrestrial taxa by sequencing the leptin genes of representative species. Phylogenetic reconstruction revealed that leptin diversification was pronounced within the phocid seals with a high dN/dS ratio of 2.8, indicating positive selection. We found significant evidence of positive selection along the branch leading to the phocids, within the phocid clade, but not over the dataset as a whole. Structural predictions indicate that the individual residues under selection are away from the leptin receptor (LEPR binding site. Predictions of the surface electrostatic potential indicate that phocid seal leptin is notably different to other mammalian leptins, including the otariids. Cloning the grey seal leptin binding domain of LEPR confirmed that this was structurally conserved. These data, viewed in toto, support a hypothesis that phocid leptin divergence is unlikely to have arisen by random mutation. Based upon these phylogenetic and structural assessments, and considering the comparative physiology and varying life histories among species, we postulate that the unique phocid diving behaviour has produced this selection pressure. The Phocidae includes some of the deepest diving species, yet have the least modified lung structure to cope with pressure and volume changes experienced at depth. Therefore, greater surfactant production is required to facilitate rapid lung re-inflation upon surfacing, while maintaining patent airways. We suggest that this additional

  9. Specificity of cellular DNA-binding sites of microbial populations in a Florida reservoir

    International Nuclear Information System (INIS)

    Paul, J.H.; Pichard, S.L.

    1989-01-01

    The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of [ 3 H]- or [ 32 P]DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nuclei acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments

  10. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    Energy Technology Data Exchange (ETDEWEB)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  11. Characterization of a second ligand binding site of the insulin receptor

    International Nuclear Information System (INIS)

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan

    2006-01-01

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

  12. Autoradiographic demonstration of oxytocin-binding sites in the macula densa

    International Nuclear Information System (INIS)

    Stoeckel, M.E.; Freund-Mercier, M.J.

    1989-01-01

    Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective 125 I-labeled OT antagonist ( 125 I-OTA). High concentrations of OT binding sites were detected on the juxtaglomerular apparatus with use of the conventional film autoradiographic technique. No labeling occurred on other renal structures. The cellular localization of the OT binding sites within the juxtaglomerular apparatus was studied in light microscope autoradiography, on semithin sections from paraformaldehyde-fixed kidney slices incubated in the presence of 125 I-OTA. These preparations revealed selective labeling of the macula densa, mainly concentrated at the basal pole of the cells. Control experiments showed first that 125 I-OTA binding characteristics were not noticeably altered by prior paraformaldehyde fixation of the kidneys and second that autoradiographic detection of the binding sites was not impaired by histological treatments following binding procedures. In view of the role of the macula densa in the tubuloglomerular feedback, the putative OT receptors of this structure might mediate the stimulatory effect of OT on glomerular filtration

  13. Autoradiographic demonstration of oxytocin-binding sites in the macula densa

    Energy Technology Data Exchange (ETDEWEB)

    Stoeckel, M.E.; Freund-Mercier, M.J. (Centre National de la Recherche Scientifique, Strasbourg (France))

    1989-08-01

    Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective {sup 125}I-labeled OT antagonist ({sup 125}I-OTA). High concentrations of OT binding sites were detected on the juxtaglomerular apparatus with use of the conventional film autoradiographic technique. No labeling occurred on other renal structures. The cellular localization of the OT binding sites within the juxtaglomerular apparatus was studied in light microscope autoradiography, on semithin sections from paraformaldehyde-fixed kidney slices incubated in the presence of {sup 125}I-OTA. These preparations revealed selective labeling of the macula densa, mainly concentrated at the basal pole of the cells. Control experiments showed first that {sup 125}I-OTA binding characteristics were not noticeably altered by prior paraformaldehyde fixation of the kidneys and second that autoradiographic detection of the binding sites was not impaired by histological treatments following binding procedures. In view of the role of the macula densa in the tubuloglomerular feedback, the putative OT receptors of this structure might mediate the stimulatory effect of OT on glomerular filtration.

  14. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta 9 tetrahydrocannabinol (delta 9 THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 3 H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  15. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  16. Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses.

    Science.gov (United States)

    Grünewald, Nora; Jan, Audric; Salvatico, Charlotte; Kress, Vanessa; Renner, Marianne; Triller, Antoine; Specht, Christian G; Schwarz, Guenter

    2018-01-01

    The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 3 10 -helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells.

  17. Molecularly imprinted protein recognition cavities bearing exchangeable binding sites for postimprinting site-directed introduction of reporter molecules for readout of binding events.

    Science.gov (United States)

    Sunayama, Hirobumi; Takeuchi, Toshifumi

    2014-11-26

    Protein-imprinted cavities bearing exchangeable domains to be used for postimprinting fluorophore introduction to transform binding events into fluorescence changes were constructed in molecularly imprinted polymer (MIPs) matrixes prepared on glass substrates. Copolymerization was performed with acrylamide, N,N'-methylenebisaclylamide, and a newly designed functional group-exchangeable monomer, ({[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy)acetic acid (MDTA), in the presence of a model basic protein, lysozyme (Lyso); MDTA can interact with Lyso and assemble close to Lyso in the resulting polymer. After removal of Lyso, followed by a disulfide reduction to cleave the (ethylcarbamoylmethoxy)acetic acid moiety from the MDTA residues, the exposed thiol groups within the imprinted cavities were modified by aminoethylpyridyldisulfide to be transformed into aminoethyl groups that function as active sites for amine-reactive fluorophores. Fluorescein isothiocyanate (FITC) was then coupled with the aminoethyl groups, yielding site specifically FITC-modified signaling imprinted cavities for Lyso binding. Because the in-cavity fluorescent labeling was achieved via a disulfide linkage, it was easy to remove, exchange, and/or replace amine-reactive fluorophores. This facilitated the screening of fluorophores to select the highest readout for binding events, replace fluorophores when photobleaching occurred, and introduce other functions. The proposed molecular imprinting process, combined with postimprinting modifications, is expected to provide an affordable route to develop multifunctional MIPs for specific detection of protein binding events.

  18. Fluorone dyes have binding sites on both cytoplasmic and extracellular domains of Na,K-ATPase.

    Science.gov (United States)

    Havlíková, Marika; Huličiak, Miroslav; Bazgier, Václav; Berka, Karel; Kubala, Martin

    2013-02-01

    Combination of fluorescence techniques and molecular docking was used to monitor interaction of Na,K-ATPase and its large cytoplasmic loop connecting fourth and fifth transmembrane helices (C45) with fluorone dyes (i.e. eosin Y, 5(6)-carboxyeosin, rose bengal, fluorescein, and erythrosine B). Our data suggested that there are at least two binding sites for all used fluorone dyes, except of 5(6)-carboxyeosin. The first binding site is located on C45 loop, and it is sensitive to the presence of nucleotide. The other site is located on the extracellular part of the enzyme, and it is sensitive to the presence of Na(+) or K(+) ions. The molecular docking revealed that in the open conformation of C45 loop (which is obtained in the presence of ATP) all used fluorone dyes occupy position directly inside the ATP-binding pocket, while in the closed conformation (i.e. in the absence of any ligand) they are located only near the ATP-binding site depending on their different sizes. On the extracellular part of the protein, the molecular docking predicts two possible binding sites with similar binding energy near Asp897(α) or Gln69(β). The former was identified as a part of interaction site between α- and β-subunits, the latter is in contact with conserved FXYD sequence of the γ-subunit. Our findings provide structural explanation for numerous older studies, which were performed with fluorone dyes before the high-resolution structures were known. Further, fluorone dyes seem to be good probes for monitoring of intersubunit interactions influenced by Na(+) and K(+) binding. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. A web server for analysis, comparison and prediction of protein ligand binding sites.

    Science.gov (United States)

    Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

    2016-03-25

    One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

  20. Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands

    Science.gov (United States)

    Richter, Lars; de Graaf, Chris; Sieghart, Werner; Varagic, Zdravko; Mörzinger, Martina; de Esch, Iwan J P; Ecker, Gerhard F; Ernst, Margot

    2012-01-01

    Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABAA receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an un biased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design. PMID:22446838

  1. Egernia stokesii (gidgee skink) MHC I positively selected sites lack concordance with HLA peptide binding regions.

    Science.gov (United States)

    Pearson, Sarah K; Bull, C Michael; Gardner, Michael G

    2017-01-01

    Genes of the major histocompatibility complex (MHC) play an important role in vertebrate disease resistance, kin recognition and mate choice. Mammalian MHC is the most widely characterised of all vertebrates, and attention is often given to the peptide binding regions of the MHC because they are presumed to be under stronger selection than non-peptide binding regions. For vertebrates where the MHC is less well understood, researchers commonly use the amino acid positions of the peptide binding regions of the human leukocyte antigen (HLA) to infer the peptide binding regions within the MHC sequences of their taxon of interest. However, positively selected sites within MHC have been reported to lack correspondence with the HLA in fish, frogs, birds and reptiles including squamates. Despite squamate diversity, the MHC has been characterised in few snakes and lizards. The Egernia group of scincid lizards is appropriate for investigating mechanisms generating MHC variation, as their inclusion will add a new lineage (i.e. Scincidae) to studies of selection on the MHC. We aimed to identify positively selected sites within the MHC of Egernia stokesii and then determine if these sites corresponded with the peptide binding regions of the HLA. Six positively selected sites were identified within E. stokesii MHC I, only two were homologous with the HLA. E. stokesii positively selected sites corresponded more closely to non-lizard than other lizard taxa. The characterisation of the MHC of more intermediate taxa within the squamate order is necessary to understand the evolution of the MHC across all vertebrates.

  2. Validating metal binding sites in macromolecule structures using the CheckMyMetal web server

    Science.gov (United States)

    Zheng, Heping; Chordia, Mahendra D.; Cooper, David R.; Chruszcz, Maksymilian; Müller, Peter; Sheldrick, George M.

    2015-01-01

    Metals play vital roles in both the mechanism and architecture of biological macromolecules. Yet structures of metal-containing macromolecules where metals are misidentified and/or suboptimally modeled are abundant in the Protein Data Bank (PDB). This shows the need for a diagnostic tool to identify and correct such modeling problems with metal binding environments. The "CheckMyMetal" (CMM) web server (http://csgid.org/csgid/metal_sites/) is a sophisticated, user-friendly web-based method to evaluate metal binding sites in macromolecular structures in respect to 7350 metal binding sites observed in a benchmark dataset of 2304 high resolution crystal structures. The protocol outlines how the CMM server can be used to detect geometric and other irregularities in the structures of metal binding sites and alert researchers to potential errors in metal assignment. The protocol also gives practical guidelines for correcting problematic sites by modifying the metal binding environment and/or redefining metal identity in the PDB file. Several examples where this has led to meaningful results are described in the anticipated results section. CMM was designed for a broad audience—biomedical researchers studying metal-containing proteins and nucleic acids—but is equally well suited for structural biologists to validate new structures during modeling or refinement. The CMM server takes the coordinates of a metal-containing macromolecule structure in the PDB format as input and responds within a few seconds for a typical protein structure modeled with a few hundred amino acids. PMID:24356774

  3. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  4. The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding

    Directory of Open Access Journals (Sweden)

    E. Gout

    2010-01-01

    Full Text Available Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors.

  5. 3D-QSAR model of flavonoids binding at benzodiazepine site in GABAA receptors.

    Science.gov (United States)

    Huang, X; Liu, T; Gu, J; Luo, X; Ji, R; Cao, Y; Xue, H; Wong, J T; Wong, B L; Pei, G; Jiang, H; Chen, K

    2001-06-07

    With flavone as a structural template, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies and ab initio calculations were performed on a series of flavonoids. A reasonable pharmacophore model was built through CoMFA, CoMSIA, and HQSAR analyses and electrostatic potential calculations. A plausible binding mode for flavonoids with GABA(A) receptors was rationalized. On the basis of the commonly recognized binding site, the specific S1 and S2 subsites relating to substituent positions were proposed. The different binding affinities could be explained according to the frontier orbitals and electrostatic potential (ESP) maps. The ESP could be used as a novel starting point for designing more selective BZ-binding-site ligands.

  6. Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

    Science.gov (United States)

    Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

    2016-11-01

    Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

  7. Modular insulators: genome wide search for composite CTCF/thyroid hormone receptor binding sites.

    Directory of Open Access Journals (Sweden)

    Oliver Weth

    Full Text Available The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, including insulation and enhancer blocking. We had previously identified two composite elements consisting of a CTCF and a TR binding site at the chicken lysozyme and the human c-myc genes. Using these it has been demonstrated that thyroid hormone mediates the relief of enhancer blocking even though CTCF remains bound to its binding site. Here we wished to determine whether CTCF and TR combined sites are representative of a general feature of the genome, and whether such sites are functional in regulating enhancer blocking. Genome wide analysis revealed that about 18% of the CTCF regions harbored at least one of the four different palindromic or repeated sequence arrangements typical for the binding of TR homodimers or TR/RXR heterodimers. Functional analysis of 10 different composite elements of thyroid hormone responsive genes was performed using episomal constructs. The episomal system allowed recapitulating CTCF mediated enhancer blocking function to be dependent on poly (ADP-ribose modification and to mediate histone deacetylation. Furthermore, thyroid hormone sensitive enhancer blocking could be shown for one of these new composite elements. Remarkably, not only did the regulation of enhancer blocking require functional TR binding, but also the basal enhancer blocking activity of CTCF was dependent on the binding of the unliganded TR. Thus, a number of composite CTCF/TR binding sites may represent a subset of other modular CTCF composite sites, such as groups of multiple CTCF sites or of CTCF/Oct4, CTCF/Kaiso or CTCF/Yy1 combinations.

  8. Assessing the model transferability for prediction of transcription factor binding sites based on chromatin accessibility.

    Science.gov (United States)

    Liu, Sheng; Zibetti, Cristina; Wan, Jun; Wang, Guohua; Blackshaw, Seth; Qian, Jiang

    2017-07-27

    Computational prediction of transcription factor (TF) binding sites in different cell types is challenging. Recent technology development allows us to determine the genome-wide chromatin accessibility in various cellular and developmental contexts. The chromatin accessibility profiles provide useful information in prediction of TF binding events in various physiological conditions. Furthermore, ChIP-Seq analysis was used to determine genome-wide binding sites for a range of different TFs in multiple cell types. Integration of these two types of genomic information can improve the prediction of TF binding events. We assessed to what extent a model built upon on other TFs and/or other cell types could be used to predict the binding sites of TFs of interest. A random forest model was built using a set of cell type-independent features such as specific sequences recognized by the TFs and evolutionary conservation, as well as cell type-specific features derived from chromatin accessibility data. Our analysis suggested that the models learned from other TFs and/or cell lines performed almost as well as the model learned from the target TF in the cell type of interest. Interestingly, models based on multiple TFs performed better than single-TF models. Finally, we proposed a universal model, BPAC, which was generated using ChIP-Seq data from multiple TFs in various cell types. Integrating chromatin accessibility information with sequence information improves prediction of TF binding.The prediction of TF binding is transferable across TFs and/or cell lines suggesting there are a set of universal "rules". A computational tool was developed to predict TF binding sites based on the universal "rules".

  9. The binding sites for benztropines and dopamine in the dopamine transporter overlap

    DEFF Research Database (Denmark)

    Jensen, Heidi Bisgaard; Larsen, M Andreas B; Mazier, Sonia

    2011-01-01

    Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational...... with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine....

  10. Identification of Ubiquinol Binding Motifs at the Qo-Site of the Cytochrome bc1 Complex

    DEFF Research Database (Denmark)

    Barragan, Angela M.; Crofts, Antony R.; Schulten, Klaus

    2015-01-01

    all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton......-coupled electron transfer from quinol to iron-sulfur cluster through a bridging hydrogen bond from histidine that stabilizes the reaction complex....

  11. Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2.

    Science.gov (United States)

    Kafurke, Uwe; Erijman, Ariel; Aizner, Yonatan; Shifman, Julia M; Eichler, Jutta

    2015-09-01

    Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  12. Identification of binding sites on protein targeting to glycogen for enzymes of glycogen metabolism.

    Science.gov (United States)

    Fong, N M; Jensen, T C; Shah, A S; Parekh, N N; Saltiel, A R; Brady, M J

    2000-11-10

    The activation of protein phosphastase-1 (PP1) by insulin plays a critical role in the regulation of glycogen metabolism. PTG is a PP1 glycogen-targeting protein, which also binds the PP1 substrates glycogen synthase, glycogen phosphorylase, and phosphorylase kinase (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Through a combination of deletion analysis and site-directed mutagenesis, the regions on PTG responsible for binding PP1 and its substrates have been delineated. Mutagenesis of Val-62 and Phe-64 in the highly conserved (K/R)VXF PP1-binding motif to alanine was sufficient to ablate PP1 binding to PTG. Phosphorylase kinase, glycogen synthase, and phosphorylase binding all mapped to the same C-terminal region of PTG. Mutagenesis of Asp-225 and Glu-228 to alanine completely blocked the interaction between PTG and these three enzymes, without affecting PP1 binding. Disruption of either PP1 or substrate binding to PTG blocked the stimulation of PP1 activity in vitro against phosphorylase, indicating that both binding sites may be important in PTG action. Transient overexpression of wild-type PTG in Chinese hamster ovary cells overexpressing the insulin receptor caused a 50-fold increase in glycogen levels. Expression of PTG mutants that do not bind PP1 had no effect on glycogen accumulation, indicating that PP1 targeting is essential for PTG function. Likewise, expression of the PTG mutants that do not bind PP1 substrates did not increase glycogen levels, indicating that PP1 targeting glycogen is not sufficient for the metabolic effects of PTG. These results cumulatively demonstrate that PTG serves as a molecular scaffold, allowing PP1 to recognize its substrates at the glycogen particle.

  13. Marked reduction in the number of platelet-tritiated imipramine binding sites in geriatric depression

    International Nuclear Information System (INIS)

    Nemeroff, C.B.; Knight, D.L.; Krishnan, R.R.; Slotkin, T.A.; Bissette, G.; Melville, M.L.; Blazer, D.G.

    1988-01-01

    The number (Bmax) and affinity (Kd) of platelet-tritiated imipramine binding sites was determined in young and middle-aged controls 50 years of age and younger (n = 25), elderly normal controls over 60 years of age (n = 18), patients who fulfilled DSM-III criteria for major depression who were under 50 years of age (n = 29), patients who fulfilled DSM-III criteria for major depression who were 60 years of age and older (n = 19), and patients who fulfilled both DSM-III criteria for primary degenerative dementia and National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria for probable Alzheimer's disease (n = 13). Both groups of depressed patients (under 50 and over 60 years of age) exhibited significant reductions (decreases 42%) in the number of platelet-tritiated imipramine binding sites with no change in affinity, when compared with their age-matched controls. There was little overlap in Bmax values between the elderly depressed patients and their controls. The patients with probable Alzheimer's disease showed no alteration in platelet-tritiated imipramine binding. There was no statistically significant relationship between postdexamethasone plasma cortisol concentrations and tritiated imipramine binding. These results indicate that platelet-tritiated imipramine binding may have potential utility as a diagnostic adjunct in geriatric depression, and moreover that the reduction in the number of platelet-tritiated imipramine binding sites is not due to hypercortisolemia

  14. Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle

    Energy Technology Data Exchange (ETDEWEB)

    Shima, K.; Kitayama, S.; Nakano, R.

    1987-05-01

    Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with /sup 125/I-labeled gonadotropins. The binding sites for /sup 125/I-labeled human follicle-stimulating hormone (/sup 125/I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The /sup 125/I-labeled human luteinizing hormone (/sup 125/I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of /sup 125/I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3 beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the /sup 125/I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.

  15. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated to be 230 nM by displacement of two different concentrations of 125 I-azidocalmodulin with increasing concentrations of unmodified calmodulin followed by Dixon plot analysis. This protein is present in red cells at approximately 30,000 copies per cell and contains a very tight binding site(s) on cytoskeletons. The protein can be only partially solubilized from isolated cytoskeletons in buffers containing high salt, but can be totally solubilized from red cell ghost membranes by extraction in low ionic strength buffers. Affinity purified IgG against this calmodulin-binding protein identifies crossreacting polypeptide(s) in brain, kidney, testes and retina. Visualization of the calmodulin-binding protein by negative staining, rotary shadowing and unidirectional shadowing indicate that it is a flattened circular molecule with molecular height of 5.4 nm and a diameter of 12.4 nm. Preliminary cosedimentation studies with purified spectrin and F-actin indicate that the site of interaction of this calmodulin-binding protein with the cytoskeleton resides on spectrin

  16. Asap: a framework for over-representation statistics for transcription factor binding sites

    DEFF Research Database (Denmark)

    Marstrand, Troels T; Frellsen, Jes; Moltke, Ida

    2008-01-01

    -founded choice. METHODOLOGY: We introduce a software package, Asap, for fast searching with position weight matrices that include several standard methods for assessing over-representation. We have compared the ability of these methods to detect over-represented transcription factor binding sites in artificial......BACKGROUND: In studies of gene regulation the efficient computational detection of over-represented transcription factor binding sites is an increasingly important aspect. Several published methods can be used for testing whether a set of hypothesised co-regulated genes share a common regulatory...... regime based on the occurrence of the modelled transcription factor binding sites. However there is little or no information available for guiding the end users choice of method. Furthermore it would be necessary to obtain several different software programs from various sources to make a well...

  17. Radioligands for PET studies of central benzodiazepine receptors and PK (peripheral benzodiazepine) binding sites -current status

    International Nuclear Information System (INIS)

    Pike, V.W.; Osman, S.; Shah, F.; Turton, D.R.; Waters, S.L.; Crouzel, C.; Nutt, D.J.

    1993-01-01

    The status of the radiochemical development and biological evaluation of radioligands for PET studies of central benzodiazepine (BZ) receptors and the so-called peripheral benzodiazepine binding sites, here discriminated and referred to as PK binding sites, is reviewed against current pharmacological knowledge, indicating those agents with present value and those with future potential. Practical recommendations are given for the preparation of two useful radioligands for PET studies, [N-methyl- 11 C]flumazenil for central BZ receptors, and [N-methyl- 11 C]PK 11195 for PK binding sites. Quality assurance and plasma metabolite analysis are also reviewed for these radioligands and practical recommendations are given on methodology for their performance. (Author)

  18. Autoradiographic distribution of /sup 125/I-galanin binding sites in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Skofitsch, G.; Sills, M.A.; Jacobowitz, D.M.

    1986-11-01

    Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of /sup 125/I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. /sup 125/I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H1 and H2, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.

  19. The nucleotide-binding site of the Escherichia coli DnaC protein: molecular topography of DnaC protein-nucleotide cofactor complexes.

    Science.gov (United States)

    Galletto, Roberto; Jezewska, Maria J; Maillard, Rodrigo; Bujalowski, Wlodzimierz

    2005-01-01

    The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3'-O-(N-methylantraniloyl)-5'-triphosphate (MANT-ATP) and 3'-O-(N-methylantraniloyl)-5'-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.

  20. Cortisol decreases 2[125I] iodomelatonin binding sites in the duck thymus

    International Nuclear Information System (INIS)

    Poon, A.M.S.; Liu, Z.M.; Tang, F.; Pang, S.F.

    1994-01-01

    The immunosuppressive effect of chronic glucocorticoid treatment on 2[ 125 I] iodomelatonin binding in the duck thymus was studied. Two-week-old ducks were injected intraperitoneally with either 1 mg of cortisol per day (experimental group) or an equivalent volume of vehicle (control group) in the middle of the light period for seven days. 2[ 125 I] iodomelatonin binding assays were performed on thymic membranes. Cortisol injection reduced the body weight gain, size of the bursa of Fabricius and absolute weights of the primary lymphoid organs but had no effect on the spleen weights. The relative weights of the spleen were increased while those of the primary lymphoid organs were unchanged. The density of the thymus 2[ 125 I] iodomelatonin binding sites was decreased while the affinity was not affected. The modulation of the thymic 2[ 125 I] iodomelatonin binding sites by changes in the immune status of the duck suggests that these binding sites represent physiologically relevant melatonin receptors and that melatonin exerts its action on the lymphoid tissues directly. The authors findings support the hypothesis that the thymus is the target site for the immunomodulatory interactions between the pineal melatonin and the adrenal steroids. A possible inhibitory influence of adrenal steroids on the immuno-enhancing effect of melatonin is also suggested. 34 refs., 3 tabs

  1. Cortisol decreases 2[[sup 125]I] iodomelatonin binding sites in the duck thymus

    Energy Technology Data Exchange (ETDEWEB)

    Poon, A.M.S.; Liu, Z.M.; Tang, F.; Pang, S.F. (Univ. of Hong Kong (China))

    1994-03-01

    The immunosuppressive effect of chronic glucocorticoid treatment on 2[[sup 125]I] iodomelatonin binding in the duck thymus was studied. Two-week-old ducks were injected intraperitoneally with either 1 mg of cortisol per day (experimental group) or an equivalent volume of vehicle (control group) in the middle of the light period for seven days. 2[[sup 125]I] iodomelatonin binding assays were performed on thymic membranes. Cortisol injection reduced the body weight gain, size of the bursa of Fabricius and absolute weights of the primary lymphoid organs but had no effect on the spleen weights. The relative weights of the spleen were increased while those of the primary lymphoid organs were unchanged. The density of the thymus 2[[sup 125]I] iodomelatonin binding sites was decreased while the affinity was not affected. The modulation of the thymic 2[[sup 125]I] iodomelatonin binding sites by changes in the immune status of the duck suggests that these binding sites represent physiologically relevant melatonin receptors and that melatonin exerts its action on the lymphoid tissues directly. The authors findings support the hypothesis that the thymus is the target site for the immunomodulatory interactions between the pineal melatonin and the adrenal steroids. A possible inhibitory influence of adrenal steroids on the immuno-enhancing effect of melatonin is also suggested. 34 refs., 3 tabs.

  2. FunFOLDQA: a quality assessment tool for protein-ligand binding site residue predictions.

    Directory of Open Access Journals (Sweden)

    Daniel B Roche

    Full Text Available The estimation of prediction quality is important because without quality measures, it is difficult to determine the usefulness of a prediction. Currently, methods for ligand binding site residue predictions are assessed in the function prediction category of the biennial Critical Assessment of Techniques for Protein Structure Prediction (CASP experiment, utilizing the Matthews Correlation Coefficient (MCC and Binding-site Distance Test (BDT metrics. However, the assessment of ligand binding site predictions using such metrics requires the availability of solved structures with bound ligands. Thus, we have developed a ligand binding site quality assessment tool, FunFOLDQA, which utilizes protein feature analysis to predict ligand binding site quality prior to the experimental solution of the protein structures and their ligand interactions. The FunFOLDQA feature scores were combined using: simple linear combinations, multiple linear regression and a neural network. The neural network produced significantly better results for correlations to both the MCC and BDT scores, according to Kendall's τ, Spearman's ρ and Pearson's r correlation coefficients, when tested on both the CASP8 and CASP9 datasets. The neural network also produced the largest Area Under the Curve score (AUC when Receiver Operator Characteristic (ROC analysis was undertaken for the CASP8 dataset. Furthermore, the FunFOLDQA algorithm incorporating the neural network, is shown to add value to FunFOLD, when both methods are employed in combination. This results in a statistically significant improvement over all of the best server methods, the FunFOLD method (6.43%, and one of the top manual groups (FN293 tested on the CASP8 dataset. The FunFOLDQA method was also found to be competitive with the top server methods when tested on the CASP9 dataset. To the best of our knowledge, FunFOLDQA is the first attempt to develop a method that can be used to assess ligand binding site

  3. Nonequivalence of alpha-bungarotoxin binding sites in the native nicotinic receptor molecule

    International Nuclear Information System (INIS)

    Conti-Tronconi, B.M.; Tang, F.; Walgrave, S.; Gallagher, W.

    1990-01-01

    In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist alpha-bungarotoxin (alpha-BGT) have different binding properties. One site has high affinity, and the M-AcChR/alpha-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD approximately 59 +/- 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 degrees C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on alpha-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of alpha-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with alpha-BGT, the long alpha-neurotoxin from Naja naja siamensis venom (alpha-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR. We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of alpha-NTX

  4. Fine definition of the CXCR4-binding region on the V3 loop of feline immunodeficiency virus surface glycoprotein.

    Directory of Open Access Journals (Sweden)

    Qiong-Ying Hu

    2010-05-01

    Full Text Available The chemokine receptor CXCR4 is shared by primary and laboratory-adapted strains of feline immunodeficiency virus (FIV for viral entry. Our previous studies implicated a contiguous nine-amino-acid region of the V3 loop of the FIV envelope surface as important in CXCR4 binding and virus entry. The binding is specific for CXCR4 since it can be inhibited by AMD3100, a selective CXCR4 inhibitor. Additional site-directed mutagenesis was used to further reveal the key residues. Binding studies indicated that basic residues R395, K397, R399 as well as N398 are critical for CXCR4 binding. The effect of other amino acid residues on receptor binding depends on the type of amino acid residue substituted. The binding study results were confirmed on human CXCR4-expressing SupT1 cells and correlated with entry efficiency using a virus entry assay. Amino acid residues critical for CXCR4 are not critical for interactions with the primary binding receptor CD134, which has an equivalent role as CD4 for HIV-1 binding. The ELISA results show that W394 and W400 are crucial for the recognition by neutralizing anti-V3 antibodies. Since certain strains of HIV-1 also use CXCR4 as the entry receptor, the findings make the feline model attractive for development of broad-based entry antagonists and for study of the molecular mechanism of receptor/virus interactions.

  5. Identification of a functional hepatocyte nuclear factor 4 binding site in the neutral ceramidase promoter

    DEFF Research Database (Denmark)

    Maltesen, Henrik R; Troelsen, Jesper T; Olsen, Jørgen

    2010-01-01

    in ceramide digestion. It was the purpose of the present work to experimentally verify the functional importance of a HNF-4a binding site predicted by bioinformatic analysis to be present in the Asah2 promoter. Using supershift analysis, HNF-4a overexpression, and HNF-4a knockdown experiments it was confirmed...... that the predicted HNF-4a binding site identified in the Asah2 promoter is functional. The results support the hypothesis that HNF-4a might be important for intestinal glycolipid metabolism....

  6. Adenovirus-Mediated Delivery of Decoy Hyper Binding Sites Targeting Oncogenic HMGA1 Reduces Pancreatic and Liver Cancer Cell Viability.

    Science.gov (United States)

    Hassan, Faizule; Ni, Shuisong; Arnett, Tyler C; McKell, Melanie C; Kennedy, Michael A

    2018-03-30

    High mobility group AT-hook 1 (HMGA1) protein is an oncogenic architectural transcription factor that plays an essential role in early development, but it is also implicated in many human cancers. Elevated levels of HMGA1 in cancer cells cause misregulation of gene expression and are associated with increased cancer cell proliferation and increased chemotherapy resistance. We have devised a strategy of using engineered viruses to deliver decoy hyper binding sites for HMGA1 to the nucleus of cancer cells with the goal of sequestering excess HMGA1 at the decoy hyper binding sites due to binding competition. Sequestration of excess HMGA1 at the decoy binding sites is intended to reduce HMGA1 binding at the naturally occurring genomic HMGA1 binding sites, which should result in normalized gene expression and restored sensitivity to chemotherapy. As proof of principle, we engineered the replication defective adenovirus serotype 5 genome to contain hyper binding sites for HMGA1 composed of six copies of an individual HMGA1 binding site, referred to as HMGA-6. A 70%-80% reduction in cell viability and increased sensitivity to gemcitabine was observed in five different pancreatic and liver cancer cell lines 72 hr after infection with replication defective engineered adenovirus serotype 5 virus containing the HMGA-6 decoy hyper binding sites. The decoy hyper binding site strategy should be general for targeting overexpression of any double-stranded DNA-binding oncogenic transcription factor responsible for cancer cell proliferation.

  7. Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations.

    Science.gov (United States)

    Ge, Yushu; van der Kamp, Marc; Malaisree, Maturos; Liu, Dan; Liu, Yi; Mulholland, Adrian J

    2017-11-01

    Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

  8. Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

    Science.gov (United States)

    Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

    2015-01-01

    SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

  9. [3H]aniracetam binds to specific recognition sites in brain membranes.

    Science.gov (United States)

    Fallarino, F; Genazzani, A A; Silla, S; L'Episcopo, M R; Camici, O; Corazzi, L; Nicoletti, F; Fioretti, M C

    1995-08-01

    [3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  11. A Parzen window-based approach for the detection of locally enriched transcription factor binding sites.

    Science.gov (United States)

    Vandenbon, Alexis; Kumagai, Yutaro; Teraguchi, Shunsuke; Amada, Karlou Mar; Akira, Shizuo; Standley, Daron M

    2013-01-21

    Identification of cis- and trans-acting factors regulating gene expression remains an important problem in biology. Bioinformatics analyses of regulatory regions are hampered by several difficulties. One is that binding sites for regulatory proteins are often not significantly over-represented in the set of DNA sequences of interest, because of high levels of false positive predictions, and because of positional restrictions on functional binding sites with regard to the transcription start site. We have developed a novel method for the detection of regulatory motifs based on their local over-representation in sets of regulatory regions. The method makes use of a Parzen window-based approach for scoring local enrichment, and during evaluation of significance it takes into account GC content of sequences. We show that the accuracy of our method compares favourably to that of other methods, and that our method is capable of detecting not only generally over-represented regulatory motifs, but also locally over-represented motifs that are often missed by standard motif detection approaches. Using a number of examples we illustrate the validity of our approach and suggest applications, such as the analysis of weaker binding sites. Our approach can be used to suggest testable hypotheses for wet-lab experiments. It has potential for future analyses, such as the prediction of weaker binding sites. An online application of our approach, called LocaMo Finder (Local Motif Finder), is available at http://sysimm.ifrec.osaka-u.ac.jp/tfbs/locamo/.

  12. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W. (Toronto); (UBC)

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  13. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  14. Characterization of [3H]mazindol binding sites in cultured monkey amniotic epithelial cells.

    Science.gov (United States)

    Elwan, M A; Sakuragawa, N

    2000-01-21

    Our previous studies showed that monkey amniotic epithelial cells (MAEC) synthesize and release catecholamines and possess D1 and D2 dopamine (DA) receptors (Elwan, M.A., Ishii, T., Ono, F. and Sakuragawa, N., Evidence for the presence of dopamine D1 receptor mRNA and binding sites in monkey amniotic epithelial cells. Neurosci. Lett., 262 (1999) 9-12; Elwan, M.A., Ishii, T. and Sakuragawa, N., Detection of dopamine D2 receptor mRNA and binding sites in monkey amniotic epithelial cells. J. Neurosci. Res., 56 (1999) 316-322; Elwan, M.A., Thangavel, R., Ono, F. and Sakuragawa, N., Synthesis and release of catecholamines by cultured monkey amniotic epithelial cells. J. Neurosci. Res., 53 (1998) 107-113). In the present study we tested the presence of DA transporter (DAT) in MAEC using radioligand binding experiments. Saturation studies showed that [3H]mazindol binds to a high affinity site with K(D) and Bmax values of 7.85 +/- 1.25 nM and 123.22 +/- 18.34 fmol/mg protein, respectively. Competition studies indicated that selective DAT inhibitors are potent displacers of [3H]mazindol binding, compared to inhibitors of other types of transporters. The rank order of potency of the competing drugs is consistent with the pharmacology of DAT. These results provide, for the first time, clear evidence that MAEC natively possess DAT binding sites and suggest that MAEC may provide a potential primate cell model to study DA release and uptake processes and to explore new drugs active at this site.

  15. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  16. The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules*

    Science.gov (United States)

    Atkin, Kate E.; Brentnall, Andrew S.; Harris, Gemma; Bingham, Richard J.; Erat, Michele C.; Millard, Christopher J.; Schwarz-Linek, Ulrich; Staunton, David; Vakonakis, Ioannis; Campbell, Iain D.; Potts, Jennifer R.

    2010-01-01

    Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the 8F19F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to 8F19F1 and that UR binding to 8F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn. PMID:20843804

  17. Functional analysis of a potential regulatory K+-binding site in the Na+, K+-ATPase

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Vilsen, Bente

    The Na+, K+-ATPase functions by actively transporting 3 Na+ ions out of and 2 K+ ions into the cell, thereby creating ion gradients crucial for many physiological processes. Recently, a combined structural and functional study of the closely related Ca2+-ATPase indicated the presence...... of a regulatory K+-binding site in the P-domain of the enzyme, identifying E732 as being of particular importance (Sorensen, Clausen et al. 2004). In addition, P709 is thought to play a significant role in the structural organization of this site. Both E732 and P709 are highly conserved among P-type ATPases (E732...... is present as either glutamic acid or aspartic acid), which supports their importance and additionally raises the question whether this site may play a general role among P-type ATPases. In Na+, K+-ATPase, K+ functions directly as a substrate for membrane binding sites, however, an additional regulatory...

  18. Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis.

    Directory of Open Access Journals (Sweden)

    Simon Oakley

    Full Text Available Barbiturates potentiate GABA actions at the GABA(A receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10-500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

  19. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Doktor, Thomas K; Borch-Jensen, Jonas

    2016-01-01

    for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing...... regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP......-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site...

  20. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  1. Two disparate ligand binding sites in the human P2Y1 receptor

    Science.gov (United States)

    Zhang, Dandan; Gao, Zhan-Guo; Zhang, Kaihua; Kiselev, Evgeny; Crane, Steven; Wang, Jiang; Paoletta, Silvia; Yi, Cuiying; Ma, Limin; Zhang, Wenru; Han, Gye Won; Liu, Hong; Cherezov, Vadim; Katritch, Vsevolod; Jiang, Hualiang; Stevens, Raymond C.; Jacobson, Kenneth A.; Zhao, Qiang; Wu, Beili

    2015-01-01

    In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. PMID:25822790

  2. Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

    Directory of Open Access Journals (Sweden)

    Barna Dey

    2009-05-01

    Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

  3. Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.

    Science.gov (United States)

    Paiz-Candia, Bertin; Islas, Angel A; Sánchez-Solano, Alfredo; Mancilla-Simbro, Claudia; Scior, Thomas; Millan-PerezPeña, Lourdes; Salinas-Stefanon, Eduardo M

    2017-02-05

    Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na + channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na + channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Na v 1.4 currents (IC 50 =60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Na v 1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na + channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Na-K pump site density and ouabain binding affinity in cultured chick heart cells

    International Nuclear Information System (INIS)

    Lobaugh, L.A.; Lieberman, M.

    1987-01-01

    The possible existence of multiple [ 3 H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [ 3 H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s. Maximal specific [ 3 H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [ 3 H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [ 3 H]ouabain and 125 I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [ 3 H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42 K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42 K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells

  5. Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S.R.; Bencze, K.Z.; Russ, K.A.; Wasiukanis, K.; Benore-Parsons, M.; Stemmler, T.L.

    2009-05-26

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 {angstrom}, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O{sub 3}N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  6. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    DEFF Research Database (Denmark)

    Long, Katherine S.; Vester, Birte

    2012-01-01

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations...... to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation...... of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little...

  7. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    International Nuclear Information System (INIS)

    Maruyama, Yukie; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2011-01-01

    Research highlights: → Bacterial alginate-binding Algp7 is similar to component EfeO of Fe 2+ transporter. → We determined the crystal structure of Algp7 with a metal-binding motif. → Algp7 consists of two helical bundles formed through duplication of a single bundle. → A deep cleft involved in alginate binding locates around the metal-binding site. → Algp7 may function as a Fe 2+ -chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  8. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  9. Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

    Directory of Open Access Journals (Sweden)

    A.M. Kettlun

    2000-07-01

    Full Text Available Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10 and Desirée (ATPase/ADPase = 1 isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

  10. Characterisation of the zebrafish serotonin transporter functionally links TM10 to the ligand binding site

    DEFF Research Database (Denmark)

    Severinsen, Kasper; Müller, Heidi Kaastrup; Wiborg, Ove

    2008-01-01

    and [(3)H]-escitalopram binding in transiently transfected human embryonic kidney cells; HEK-293-MSR. Residues responsible for altered affinities inhibitors were pinpointed by generating cross-species chimeras and subsequent point mutations by site directed mutagenesis. drSERT has a higher affinity...

  11. AthaMap: from in silico data to real transcription factor binding sites.

    Science.gov (United States)

    Bülow, Lorenz; Steffens, Nils Ole; Galuschka, Claudia; Schindler, Martin; Hehl, Reinhard

    2006-01-01

    AthaMap generates a map for cis-regulatory sequences for the whole Arabidopsis thaliana genome. AthaMap was initially developed by matrix-based detection of putative transcription factor binding sites (TFBS) mostly determined from random binding site selection experiments. Now, also experimentally verified TFBS have been included for 48 different Arabidopsis thaliana transcription factors (TF). Based on these sequences, 89,416 very similar putative TFBS were determined within the genome of A. thaliana and annotated to AthaMap. Matrix- and single sequence-based binding sites can be included in colocalization analysis for the identification of combinatorial cis-regulatory elements. As an example, putative target genes of the WRKY18 transcription factor that is involved in plant-pathogen interaction were determined. New functions of AthaMap include descriptions for all annotated Arabidopsis thaliana genes and direct links to TAIR, TIGR and MIPS. Transcription factors used in the binding site determination are linked to TAIR and TRANSFAC databases. AthaMap is freely available at http://www.athamap.de.

  12. An Integrated Approach to Sequence-Independent Local Alignment of Protein Binding Sites.

    Science.gov (United States)

    Pang, Bin; Schlessman, David; Kuang, Xingyan; Zhao, Nan; Shyu, Daniel; Korkin, Dmitry; Shyu, Chi-Ren

    2015-01-01

    Accurate alignment of protein-protein binding sites can aid in protein docking studies and constructing templates for predicting structure of protein complexes, along with in-depth understanding of evolutionary and functional relationships. However, over the past three decades, structural alignment algorithms have focused predominantly on global alignments with little effort on the alignment of local interfaces. In this paper, we introduce the PBSalign (Protein-protein Binding Site alignment) method, which integrates techniques in graph theory, 3D localized shape analysis, geometric scoring, and utilization of physicochemical and geometrical properties. Computational results demonstrate that PBSalign is capable of identifying similar homologous and analogous binding sites accurately and performing alignments with better geometric match measures than existing protein-protein interface comparison tools. The proportion of better alignment quality generated by PBSalign is 46, 56, and 70 percent more than iAlign as judged by the average match index (MI), similarity index (SI), and structural alignment score (SAS), respectively. PBSalign provides the life science community an efficient and accurate solution to binding-site alignment while striking the balance between topological details and computational complexity.

  13. Identification of anesthetic binding sites on human serum albumin using a novel etomidate photolabel

    NARCIS (Netherlands)

    Bright, Damian P.; Adham, Sara D.; Lemaire, Lucienne C. J. M.; Benavides, Rodrigo; Gruss, Marco; Taylor, Graham W.; Smith, Edward H.; Franks, Nicholas P.

    2007-01-01

    We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as

  14. Substrate binding in the active site of cytochrome P450cam

    NARCIS (Netherlands)

    Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

    2005-01-01

    We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

  15. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  16. Differential alterations of cortical glutamatergic binding sites in senile dementia of the Alzheimer type

    International Nuclear Information System (INIS)

    Chalmers, D.T.; Dewar, D.; Graham, D.I.; Brooks, D.N.; McCulloch, J.

    1990-01-01

    Involvement of cortical glutamatergic mechanisms in senile dementia of the Alzheimer type (SDAT) has been investigated with quantitative ligand-binding autoradiography. The distribution and density of Na(+)-dependent glutamate uptake sites and glutamate receptor subtypes--kainate, quisqualate, and N-methyl-D-aspartate--were measured in adjacent sections of frontal cortex obtained postmortem from six patients with SDAT and six age-matched controls. The number of senile plaques was determined in the same brain region. Binding of D-[3H]aspartate to Na(+)-dependent uptake sites was reduced by approximately 40% throughout SDAT frontal cortex relative to controls, indicating a general loss of glutamatergic presynaptic terminals. [3H]Kainate receptor binding was significantly increased by approximately 70% in deep layers of SDAT frontal cortex compared with controls, whereas this binding was unaltered in superficial laminae. There was a positive correlation (r = 0.914) between kainate binding and senile plaque number in deep cortical layers. Quisqualate receptors, as assessed by 2-amino-3-hydroxy-5-[3H]methylisoxazole-4-propionic acid binding, were unaltered in SDAT frontal cortex compared with controls. There was a small reduction (25%) in N-methyl-D-aspartate-sensitive [3H]glutamate binding only in superficial cortical layers of SDAT brains relative to control subjects. [3H]Glutamate binding in SDAT subjects was unrelated to senile plaque number in superficial cortical layers (r = 0.104). These results indicate that in the presence of cortical glutamatergic terminal loss in SDAT plastic alterations occur in some glutamate receptor subtypes but not in others

  17. Pt(CN)42- and Au(CN)2-: potential general probes for anion-binding sites of proteins

    International Nuclear Information System (INIS)

    Norne, J.E.; Lilja, H.; Lindman, B.; Einarsson, R.; Zeppezauer, M.

    1975-01-01

    Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35 Cl - and 81 Br - in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, α-chymotrypsin, human carbonic anhydrase, fructose-1.6-biphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35 Cl - and 81 Br - relaxation rate on addition of functional ligands. (orig./HK) [de

  18. Protecting Surface Water Systems on Forest Sites Through Herbicide Use

    Science.gov (United States)

    J.L. Michael; H.L. Gibbs; J.B. Fischer; E.C. Webber

    2000-01-01

    Sediment, nutrients, and pesticides are universally accepted as the greatest threats to surface water quality world-wide. Sedimentation in surface waters is a natural phenomenon, but is magnified by human activities. Intensive forest management practices, particularly road building, harvesting and planting site preparation, result in the greatest increases in erosion...

  19. BSSF: a fingerprint based ultrafast binding site similarity search and function analysis server

    Directory of Open Access Journals (Sweden)

    Jiang Hualiang

    2010-01-01

    Full Text Available Abstract Background Genome sequencing and post-genomics projects such as structural genomics are extending the frontier of the study of sequence-structure-function relationship of genes and their products. Although many sequence/structure-based methods have been devised with the aim of deciphering this delicate relationship, there still remain large gaps in this fundamental problem, which continuously drives researchers to develop novel methods to extract relevant information from sequences and structures and to infer the functions of newly identified genes by genomics technology. Results Here we present an ultrafast method, named BSSF(Binding Site Similarity & Function, which enables researchers to conduct similarity searches in a comprehensive three-dimensional binding site database extracted from PDB structures. This method utilizes a fingerprint representation of the binding site and a validated statistical Z-score function scheme to judge the similarity between the query and database items, even if their similarities are only constrained in a sub-pocket. This fingerprint based similarity measurement was also validated on a known binding site dataset by comparing with geometric hashing, which is a standard 3D similarity method. The comparison clearly demonstrated the utility of this ultrafast method. After conducting the database searching, the hit list is further analyzed to provide basic statistical information about the occurrences of Gene Ontology terms and Enzyme Commission numbers, which may benefit researchers by helping them to design further experiments to study the query proteins. Conclusions This ultrafast web-based system will not only help researchers interested in drug design and structural genomics to identify similar binding sites, but also assist them by providing further analysis of hit list from database searching.

  20. Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography

    International Nuclear Information System (INIS)

    Rosier, A.M.; Vandesande, F.; Orban, G.A.

    1991-01-01

    The distribution of galanin (GAL) binding sites in the visual cortex of cat and monkey was determined by autoradiographic visualization of [ 125 I]-GAL binding to tissue sections. Binding conditions were optimized and, as a result, the binding was saturable and specific. In cat visual cortex, GAL binding sites were concentrated in layers I, IVc, V, and VI. Areas 17, 18, and 19 exhibited a similar distribution pattern. In monkey primary visual cortex, the highest density of GAL binding sites was observed in layers II/III, lower IVc, and upper V. Layers IVA and VI contained moderate numbers of GAL binding sites, while layer I and the remaining parts of layer IV displayed the lowest density. In monkey secondary visual cortex, GAL binding sites were mainly concentrated in layers V-VI. Layer IV exhibited a moderate density, while the supragranular layers contained the lowest proportion of GAL binding sites. In both cat and monkey, we found little difference between regions subserving central and those subserving peripheral vision. Similarities in the distribution of GAL and acetylcholine binding sites are discussed

  1. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  2. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    Directory of Open Access Journals (Sweden)

    Deng W-M

    2009-02-01

    Full Text Available Abstract Background Dystroglycan (Dg is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

  3. Identification of an allosteric binding site for RORγt inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Scheepstra, Marcel; Leysen, Seppe; vanAlmen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc (Merck); (Eindhoven)

    2015-12-07

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

  4. Autoradiographic localization of (125I-Tyr4)bombesin-binding sites in rat brain

    International Nuclear Information System (INIS)

    Zarbin, M.A.; Kuhar, M.J.; O'Donohue, T.L.; Wolf, S.S.; Moody, T.W.

    1985-01-01

    The binding of ( 125 I-Tyr 4 )bombesin to rat brain slices was investigated. Radiolabeled (Tyr 4 )bombesin bound with high affinity (K/sub d/ . 4 nM) to a single class of sites (B/sub max/ . 130 fmol/mg of protein); the ratio of specific to nonspecific binding was 6/1. Also, pharmacology studies indicated that the C-terminal of bombesin was important for the high affinity binding activity. Autoradiographic studies indicated that the ( 125 I-Tyr4)bombesin-binding sites were discretely distributed in certain gray but not white matter regions of rat brain. Highest grain densities were present in the olfactory bulb and tubercle, nucleus accumbens, suprachiasmatic and periventricular nuclei of the hypothalamus, central medial thalamic nucleus, medial amygdaloid nucleus, hippocampus, dentate gyrus, subiculum, nucleus of the solitary tract, and substantia gelatinosa. Moderate grain densities were present in the parietal cortex, deep layers of the neocortex, rhinal cortex, caudate putamen, stria terminalis, locus ceruleus, parabrachial nucleus, and facial nucleus. Low grain densities were present in the globus pallidus, lateral thalamus, and midbrain. Negligible grain densities were present in the cerebellum, corpus callosum, and all regions treated with 1 microM unlabeled bombesin. The discrete regional distribution of binding suggests that endogenous bombesin-like peptides may function as important regulatory agents in certain brain loci

  5. In vivo receptor binding of opioid drugs at the mu site

    Energy Technology Data Exchange (ETDEWEB)

    Rosenbaum, J.S.; Holford, N.H.; Sadee, W.

    1985-06-01

    The in vivo receptor binding of a series of opioid drugs was investigated in intact rats after s.c. administration of (/sup 3/H)etorphine tracer, which selectively binds to mu sites in vivo. Receptor binding was determined by a membrane filtration assay immediately after sacrifice of the animals and brain homogenization. Coadministration of unlabeled opioid drugs together with tracer led to a dose-dependent decrease of in vivo tracer binding. Estimates of the doses required to occupy 50% of the mu sites in vivo established the following potency rank order: diprenorphine, naloxone, buprenorphine, etorphine, levallorphan, cyclazocine, sufentanil, nalorphine, ethylketocyclazocine, ketocyclazocine, pentazocine, morphine. In vivo-in vitro differences among the relative receptor binding potencies were only partially accounted for by differences in their access to the brain and the regulatory effects of Na+ and GTP, which are expected to reduce agonist affinities in vivo. The relationship among mu receptor occupancy in vivo and pharmacological effects of the opioid drugs is described.

  6. Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

    Directory of Open Access Journals (Sweden)

    Jan Bily

    Full Text Available The transient receptor potential (TRP protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864 were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

  7. Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

    Science.gov (United States)

    Bily, Jan; Grycova, Lenka; Holendova, Blanka; Jirku, Michaela; Janouskova, Hana; Bousova, Kristyna; Teisinger, Jan

    2013-01-01

    The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

  8. Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Tsung-Fu Yu

    Full Text Available The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1 encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

  9. Hanford Site surface soil radioactive contamination control plan, March 1993

    International Nuclear Information System (INIS)

    Mix, P.D.; Winship, R.A.

    1993-04-01

    The Decommissioning and Resource Conservation and Recovery Act Closure Program is responsible to the US Department of Energy Richland Field Office, for the safe and cost-effective surveillance, maintenance, and decommissioning of surplus facilities and Resource Conservation and Recovery Act of 1976 closures at the Hanford Site. This program also manages the Radiation Area Remedial Action that includes the surveillance, maintenance, decontamination, and/or interim stabilization of inactive burial grounds, cribs, ponds, trenches, and unplanned release sites. This plan addresses only the Radiation Area Remedial Action activity requirements for managing and controlling the contaminated surface soil areas associated with these inactive sites until they are remediated as part of the Hanford Site environmental restoration process. All officially numbered Radiation Area Remedial Action and non-Radiation Area Remedial Action contaminated surface soil areas are listed in this document so that a complete list of the sites requiring remediation is contained in one document

  10. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    Science.gov (United States)

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  11. Engineering of specific uranyl-coordination sites in the calcium-binding motif of Calmodulin

    International Nuclear Information System (INIS)

    Beccia, M.; Pardoux, R.; Sauge-Merle, S.; Bremond, N.; Lemaire, D.; Berthomieu, C.; Delangle, P.; Guilbaud, P.

    2014-01-01

    Complete text of publication follows: Characterization of heavy metals interactions with proteins is fundamental for understanding the molecular factors and mechanisms governing ions toxicity and speciation in cells. This line of research will also help in developing new molecules able to selectively and efficiently bind toxic metal ions, which could find application for bio-detection or bioremediation purposes. We have used the regulatory calcium-binding protein Calmodulin (CaM) from A. thaliana as a structural model and, starting from it, we have designed various mutants by site-directed mutagenesis. We have analysed thermodynamics of uranyl ion binding to both sites I and II of CaM N-terminal domain and we have identified structural factors governing this interaction. Selectivity for uranyl ion has been tested by studying reactions of the investigated peptides with Ca 2+ , in the same conditions used for UO 2 2+ . Spectro-fluorimetric titrations and FTIR analysis have shown that the affinity for uranyl increases by phosphorylation of a threonine in site I, especially approaching the physiological pH, where the phospho-threonine side chain is deprotonated. Based on structural models obtained by Molecular Dynamics, we tested the effect of a two residues deletion on site I properties. We obtained an almost two orders of magnitude increase in affinity for uranyl, with a sub-nanomolar dissociation constant for the uranyl complex with the non phosphorylated peptide, and an improved uranyl/calcium selectivity. Allosteric effects depending on Ca 2+ and UO 2 2+ binding have been investigated by comparing thermodynamic parameters obtained for mutants having both sites I and II able to chelate metal ions with those of mutants consisting of just one active site

  12. A model-based approach to identify binding sites in CLIP-Seq data.

    Directory of Open Access Journals (Sweden)

    Tao Wang

    Full Text Available Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq has made it possible to identify the targeting sites of RNA-binding proteins in various cell culture systems and tissue types on a genome-wide scale. Here we present a novel model-based approach (MiClip to identify high-confidence protein-RNA binding sites from CLIP-seq datasets. This approach assigns a probability score for each potential binding site to help prioritize subsequent validation experiments. The MiClip algorithm has been tested in both HITS-CLIP and PAR-CLIP datasets. In the HITS-CLIP dataset, the signal/noise ratios of miRNA seed motif enrichment produced by the MiClip approach are between 17% and 301% higher than those by the ad hoc method for the top 10 most enriched miRNAs. In the PAR-CLIP dataset, the MiClip approach can identify ∼50% more validated binding targets than the original ad hoc method and two recently published methods. To facilitate the application of the algorithm, we have released an R package, MiClip (http://cran.r-project.org/web/packages/MiClip/index.html, and a public web-based graphical user interface software (http://galaxy.qbrc.org/tool_runner?tool_id=mi_clip for customized analysis.

  13. Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

    International Nuclear Information System (INIS)

    Pougeois, R.; Lauquin, G.J.

    1985-01-01

    The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (P /sub i/ ), could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca 2+ -ATPase in the presence of Ca 2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase, abolished P /sub i/ binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites

  14. Identification of metal ion binding sites based on amino acid sequences

    Science.gov (United States)

    Cao, Xiaoyong; Zhang, Xiaojin; Gao, Sujuan; Ding, Changjiang; Feng, Yonge; Bao, Weihua

    2017-01-01

    The identification of metal ion binding sites is important for protein function annotation and the design of new drug molecules. This study presents an effective method of analyzing and identifying the binding residues of metal ions based solely on sequence information. Ten metal ions were extracted from the BioLip database: Zn2+, Cu2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Na+, K+ and Co2+. The analysis showed that Zn2+, Cu2+, Fe2+, Fe3+, and Co2+ were sensitive to the conservation of amino acids at binding sites, and promising results can be achieved using the Position Weight Scoring Matrix algorithm, with an accuracy of over 79.9% and a Matthews correlation coefficient of over 0.6. The binding sites of other metals can also be accurately identified using the Support Vector Machine algorithm with multifeature parameters as input. In addition, we found that Ca2+ was insensitive to hydrophobicity and hydrophilicity information and Mn2+ was insensitive to polarization charge information. An online server was constructed based on the framework of the proposed method and is freely available at http://60.31.198.140:8081/metal/HomePage/HomePage.html. PMID:28854211

  15. Involvement of batrachotoxin binding sites in ginsenoside-mediated voltage-gated Na+ channel regulation.

    Science.gov (United States)

    Lee, Jun-Ho; Lee, Byung-Hwan; Choi, Sun-Hye; Yoon, In-Soo; Shin, Tae-Joon; Pyo, Mi Kyung; Lee, Sang-Mok; Kim, Hyoung-Chun; Nah, Seung-Yeol

    2008-04-08

    Recently, we showed that the 20(S)-ginsenoside Rg3 (Rg3), an active ingredient of Panax ginseng, inhibits rat brain NaV1.2 channel peak currents (INa). Batrachotoxin (BTX) is a steroidal alkaloid neurotoxin and activates NaV channels through interacting with transmembrane domain-I-segment 6 (IS6) of channels. Recent report shows that ginsenoside inhibits BTX binding in rat brain membrane fractions. However, it needs to be confirmed whether biochemical mechanism is relevant physiologically and which residues of the BTX binding sites are important for ginsenoside regulations. Here, we demonstrate that mutations of BTX binding sites such as N418K and L421K of rat brain NaV1.2 and L437K of mouse skeletal muscle NaV1.4 channel reduce or abolish Rg3 inhibition of I(Na) and attenuate Rg3-mediated depolarizing shift of the activation voltage and use-dependent inhibition. These results indicate that BTX binding sites play an important role in modifying Rg3-mediated Na+ channel properties.

  16. Spectroscopic Signature of a Ubiquitous Metal Binding Site in the Metallo-beta-lactamase Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    V Campos-Bermudez; J Gonzalez; D Tierney; A Vila

    2011-12-31

    The metallo-{beta}-lactamase (M{beta}L) superfamily is a functionally diverse group of metalloproteins sharing a distinctive {alpha}{beta}/{alpha}{beta} fold and a characteristic metal binding motif. A large number of open reading frames identified in genomic sequencing efforts have been annotated as members of this superfamily through sequence comparisons. However, structural and functional studies performed on purified proteins are normally needed to unequivocally include a newly discovered protein in the M{beta}L superfamily. Here we report the spectroscopic characterization of recombinant YcbL, a gene product annotated as a member of the M{beta}L superfamily whose function in vivo remains unknown. By taking advantage of the structural features characterizing the M{beta}L superfamily metal binding motif, we performed spectroscopic studies on Zn(II)- and Co(II)-substituted YcbL to structurally interrogate the metal binding site. The dinuclear center in Co(II)-YcbL was shown to display characteristic electronic absorption features in the visible region, which were also observed in an engineered M{beta}L aimed at mimicking this metal site. Thus, the spectroscopic features reported herein can be employed as a signature to readily identify and characterize the presence of these ubiquitous metal binding sites.

  17. Validation of binding of SE-75 labeled sucralfate to sites of gastrointestinal ulceration

    International Nuclear Information System (INIS)

    Maurer, A.H.; Knight, L.C.; Kollman, M.; Krevsky, B.; Pleet, D.; D'Ercole, F.; Siegel, J.A.; Fisher, R.S.; Malmud, L.S.

    1985-01-01

    This study was performed to determine if and for how long sucralfate (SU) binds selectively to sites of gastro-intestinal (GI) ulceration. Se-Su was prepared by sulfating sucrose with tracer Se-75 and precipitating it as the basic Al salt. All patients (pts) had endoscopy to confirm the presence of either: esophagitis (n=5), gastritis (GA) (n=5), gastric ulcers (GU) (n=5), duodenal ulcers (DU) (n=5), or no ulceration (NU) (n=5). Following an overnight fast the pts swallowed 1 gm with 100 μCi of Se-SU and were imaged continuously over 24 hours or until no activity remained in the upper GI tract. Pts with GU visually demonstrated focal SU binding at the ulcers for an average of 3.9 +- 1.1 hrs. with a mean GET of 68 +- 25 min. Mean GET for pts with DU was prolonged, 171 +- 63 min, however focal binding at duodenal ulcers was not seen. All pts with GA had diffuse retention of SU in the stomach with a mean GET of 118 +- 34 min. Focal binding of SU at all sites of esophagitis was seen with a T-1/2 of 65 +- 32 min at the ulcerations. In conclusion these data support the theory that the mechanism of ulcer healing with SU is related to its ability to adhere to the ulcer site forming a protective barrier. In addition Se-SU is a potential ulcer imaging agent which can be used to noninvasively assess healing

  18. Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2Receptors.

    Science.gov (United States)

    Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I; Lanciego, José L; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

    2017-01-01

    The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

  19. Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

    Directory of Open Access Journals (Sweden)

    Eva Martínez-Pinilla

    2017-10-01

    Full Text Available The mechanism of action of cannabidiol (CBD, the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs; however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

  20. Adherence of platelets to in situ albumin-binding surfaces under flow conditions: role of surface-adsorbed albumin

    International Nuclear Information System (INIS)

    Guha Thakurta, Sanjukta; Miller, Robert; Subramanian, Anuradha

    2012-01-01

    Surfaces that preferentially bind human serum albumin (HSA) were generated by grafting albumin-binding linear peptide (LP1) onto silicon surfaces. The research aim was to evaluate the adsorption pattern of proteins and the adhesion of platelets from platelet-poor plasma and platelet-rich plasma, respectively, by albumin-binding surfaces under physiological shear rate (96 and 319 s −1 ) conditions. Bound proteins were quantified using enzyme-linked immunosorbent assays (ELISAs) and two-dimensional gel electrophoresis. A ratio of ∼1000:100:1 of adsorbed HSA, human immunoglobulin (HIgG) and human fibrinogen (HFib) was noted, respectively, on LP1-functionalized surfaces, and a ratio of ∼5:2:1 of the same was noted on control surfaces, as confirmed by ELISAs. The surface-adsorbed von Willebrand factor was undetectable by sensitive ELISAs. The amount of adhered platelets correlated with the ratio of adsorbed HSA/HFib. Platelet morphology was more rounded on LP1-functionalized surfaces when compared to control surfaces. The platelet adhesion response on albumin-binding surfaces can be explained by the reduction in the co-adsorption of other plasma proteins in a surface environment where there is an excess of albumin molecules, coupled with restrictions in the conformational transitions of other surface-adsorbed proteins into hemostatically active forms. (paper)

  1. Adherence of platelets to in situ albumin-binding surfaces under flow conditions: role of surface-adsorbed albumin.

    Science.gov (United States)

    Guha Thakurta, Sanjukta; Miller, Robert; Subramanian, Anuradha

    2012-08-01

    Surfaces that preferentially bind human serum albumin (HSA) were generated by grafting albumin-binding linear peptide (LP1) onto silicon surfaces. The research aim was to evaluate the adsorption pattern of proteins and the adhesion of platelets from platelet-poor plasma and platelet-rich plasma, respectively, by albumin-binding surfaces under physiological shear rate (96 and 319 s(-1)) conditions. Bound proteins were quantified using enzyme-linked immunosorbent assays (ELISAs) and two-dimensional gel electrophoresis. A ratio of ∼1000:100:1 of adsorbed HSA, human immunoglobulin (HIgG) and human fibrinogen (HFib) was noted, respectively, on LP1-functionalized surfaces, and a ratio of ∼5:2:1 of the same was noted on control surfaces, as confirmed by ELISAs. The surface-adsorbed von Willebrand factor was undetectable by sensitive ELISAs. The amount of adhered platelets correlated with the ratio of adsorbed HSA/HFib. Platelet morphology was more rounded on LP1-functionalized surfaces when compared to control surfaces. The platelet adhesion response on albumin-binding surfaces can be explained by the reduction in the co-adsorption of other plasma proteins in a surface environment where there is an excess of albumin molecules, coupled with restrictions in the conformational transitions of other surface-adsorbed proteins into hemostatically active forms.

  2. Ivermectin binding sites in human and invertebrate Cys-loop receptors

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2012-01-01

    modelling now explain how ivermectin binds to these receptors and reveal why it is selective for invertebrate members of the Cys-loop receptor family. Combining this with emerging genomic information, we are now in a position to predict species sensitivity to ivermectin and better understand the molecular...... basis of ivermectin resistance. An understanding of the molecular structure of the ivermectin binding site, which is formed at the interface of two adjacent subunits in the transmembrane domain of the receptor, should also aid the development of new lead compounds both as anthelmintics and as therapies...

  3. Mapping of the vitronectin-binding site on the urokinase receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Ploug, Michael

    2007-01-01

    a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32...... the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin....

  4. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    Science.gov (United States)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  5. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid.

    Science.gov (United States)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A; Wepasnick, Kevin A; McDonnell, Peter; Elisseeff, Jennifer H

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  6. Defining reactive sites on hydrated mineral surfaces: Rhombohedral carbonate minerals

    Science.gov (United States)

    Villegas-Jiménez, Adrián; Mucci, Alfonso; Pokrovsky, Oleg S.; Schott, Jacques

    2009-08-01

    Despite the success of surface complexation models (SCMs) to interpret the adsorptive properties of mineral surfaces, their construct is sometimes incompatible with fundamental chemical and/or physical constraints, and thus, casts doubts on the physical-chemical significance of the derived model parameters. In this paper, we address the definition of primary surface sites (i.e., adsorption units) at hydrated carbonate mineral surfaces and discuss its implications to the formulation and calibration of surface equilibria for these minerals. Given the abundance of experimental and theoretical information on the structural properties of the hydrated (10.4) cleavage calcite surface, this mineral was chosen for a detailed theoretical analysis of critical issues relevant to the definition of primary surface sites. Accordingly, a single, generic charge-neutral surface site ( tbnd CaCO 3·H 2O 0) is defined for this mineral whereupon mass-action expressions describing adsorption equilibria were formulated. The one-site scheme, analogous to previously postulated descriptions of metal oxide surfaces, allows for a simple, yet realistic, molecular representation of surface reactions and provides a generalized reference state suitable for the calculation of sorption equilibria for rhombohedral carbonate minerals via Law of Mass Action (LMA) and Gibbs Energy Minimization (GEM) approaches. The one-site scheme is extended to other rhombohedral carbonate minerals and tested against published experimental data for magnesite and dolomite in aqueous solutions. A simplified SCM based on this scheme can successfully reproduce surface charge, reasonably simulate the electrokinetic behavior of these minerals, and predict surface speciation agreeing with available spectroscopic data. According to this model, a truly amphoteric behavior is displayed by these surfaces across the pH scale but at circum-neutral pH (5.8-8.2) and relatively high ΣCO 2 (⩾1 mM), proton/bicarbonate co

  7. Adenovirus-Mediated Delivery of Decoy Hyper Binding Sites Targeting Oncogenic HMGA1 Reduces Pancreatic and Liver Cancer Cell Viability

    Directory of Open Access Journals (Sweden)

    Faizule Hassan

    2018-03-01

    Full Text Available High mobility group AT-hook 1 (HMGA1 protein is an oncogenic architectural transcription factor that plays an essential role in early development, but it is also implicated in many human cancers. Elevated levels of HMGA1 in cancer cells cause misregulation of gene expression and are associated with increased cancer cell proliferation and increased chemotherapy resistance. We have devised a strategy of using engineered viruses to deliver decoy hyper binding sites for HMGA1 to the nucleus of cancer cells with the goal of sequestering excess HMGA1 at the decoy hyper binding sites due to binding competition. Sequestration of excess HMGA1 at the decoy binding sites is intended to reduce HMGA1 binding at the naturally occurring genomic HMGA1 binding sites, which should result in normalized gene expression and restored sensitivity to chemotherapy. As proof of principle, we engineered the replication defective adenovirus serotype 5 genome to contain hyper binding sites for HMGA1 composed of six copies of an individual HMGA1 binding site, referred to as HMGA-6. A 70%–80% reduction in cell viability and increased sensitivity to gemcitabine was observed in five different pancreatic and liver cancer cell lines 72 hr after infection with replication defective engineered adenovirus serotype 5 virus containing the HMGA-6 decoy hyper binding sites. The decoy hyper binding site strategy should be general for targeting overexpression of any double-stranded DNA-binding oncogenic transcription factor responsible for cancer cell proliferation. Keywords: adenovirus, cancer therapy, oncogenic transcription factor, chemotherapy resistance, high mobility group A protein, decoy binding site, pancreatic cancer, liver cancer, HMGA1, neoadjuvant therapy

  8. Surface selective binding of nanoclay particles to polyampholyte protein chains.

    Science.gov (United States)

    Pawar, Nisha; Bohidar, H B

    2009-07-28

    Binding of nanoclay (Laponite) to gelatin-A and gelatin-B (both polyampholytes) molecules was investigated at room temperature (25 degrees C) both experimentally and theoretically. The stoichiometric binding ratio between gelatin and Laponite was found to be strongly dependent on the solution ionic strength. Large soluble complexes were formed at higher ionic strengths of the solution, a result supported by data obtained from light scattering, viscosity, and zeta potential measurements. The binding problem was theoretically modeled by choosing a suitable two-body screened Coulomb potential, U(R(+)) = (q(-)/2epsilon)[(Q(-)/R(-))e(-kR(-))-(Q(+)/R(+))e(-kR(+))], where the protein dipole has charges Q(+) and Q(-) that are located at distances R(+) and R(-) from the point Laponite charge q(-) and the dispersion liquid has dielectric constant (epsilon). U(R(+)) accounted for electrostatic interactions between a dipole (protein molecule) and an effective charge (Laponite particle) located at an angular position theta. Gelatin-A and Laponite association was facilitated by a strong attractive interaction potential that led to preferential binding of the biopolymer chains to negatively charged face of Laponite particles. In the case of gelatin-B selective surf ace patch binding dominated the process where the positively charged rim and negatively charged face of the particles were selectively bound to the oppositely charged segments of the biopolymer. The equilibrium separation (R(e)) between the protein and nanoclay particle revealed monovalent salt concentration dependence given by R(e) approximately [NaCl](alpha) where alpha = 0.6+/-0.2 for gelatin-A and alpha = 0.4+/-0.2 for gelatin-B systems. The equilibrium separations were approximately 30% less compared to the gelatin-A system implying preferential short-range ordering of the gelatin-B-nanoclay pair in the solvent.

  9. PATTERN BASED DETECTION OF POTENTIALLY DRUGGABLE BINDING SITES BY LIGAND SCREENING

    Directory of Open Access Journals (Sweden)

    Uttam Pal

    2018-03-01

    Full Text Available This article describes an innovative way of finding the potentially druggable sites on a target protein, which can be used for orthosteric and allosteric lead detection in a single virtual screening setup. Druggability estimation for an alternate binding site other than the canonical ligand-binding pocket of an enzyme is rewarding for several inherent benefits. Allostery is a direct and efficient way of regulating biomacromolecule function. The allosteric modulators can fine-tune protein mechanics. Besides, allosteric sites are evolutionarily less conserved/more diverse even in very similarly related proteins, thus, provides high degree of specificity in targeting a particular protein. Therefore, targeting of allosteric sites is gaining attention as an emerging strategy in rational drug design. However, the experimental approaches provide a limited degree of characterization of new allosteric sites. Computational approaches are useful to analyze and select potential allosteric sites for drug discovery. Here, the use of molecular docking, which has become an integral part of the drug discovery process, has been discussed to predict the druggability of novel allosteric sites as well as the active site on target proteins by ligand screening. Genetic algorithm was used for docking and the whole protein was placed in the search space. For each ligand in the library of small molecules, the genetic algorithm was run for multiple times to populate all the druggable sites in the target protein, which was then translated into two dimensional density maps or “patterns”. High density clusters were observed for lead like molecules in these pattern diagrams. Each cluster in such a pattern diagram indicated a plausible binding site and the density gave its druggability score in terms of weighted probabilities. The patterns were filtered to find the leads for each of the druggable sites on the target protein. Such a novel pattern based analysis of the

  10. Biophysical Models of Evolution: Application to Transcription Factor Binding Sites in Yeast

    Science.gov (United States)

    Manhart, Michael; Haldane, Allan; Morozov, Alexandre

    2012-02-01

    There has been growing interest in understanding the physical driving forces of evolution at the molecular scale, in particular how biophysics determines the fitness landscapes that shape the evolution of DNA and proteins. To that end we study a model of molecular evolution that explicitly incorporates the underlying biophysics. Using population genetics, we derive a steady-state distribution of monomorphic populations evolving on an arbitrary fitness landscape. Compared to previous studies, we find this result is universal for a large class of population models and fully incorporates both stochastic effects and strong natural selection. This distribution can then be used to infer the underlying fitness landscape from genomic data. This model can be applied to a variety of systems, but we focus on transcription factor binding sites, which play a crucial role in gene regulatory networks. Since these sites must be bound for successful gene regulation, we consider a simple thermodynamic model of fitness as a function of the free energy for binding a transcription factor at the site. Using empirical energy matrices and genome-wide sets of binding sites from the yeast Saccharomyces cerevisiae, we use this model to infer the role of DNA-protein interaction physics in evolution.

  11. Changes in electrostatic surface potential of Na+/K+-ATPase cytoplasmic headpiece induced by cytoplasmic ligand(s) binding.

    Science.gov (United States)

    Kubala, Martin; Grycova, Lenka; Lansky, Zdenek; Sklenovsky, Petr; Janovska, Marika; Otyepka, Michal; Teisinger, Jan

    2009-09-16

    A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.

  12. The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

    DEFF Research Database (Denmark)

    Pagh, Rasmus; Duus, Karen; Laursen, Inga

    2008-01-01

    was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding...... with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion......The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL...

  13. Study of Binding between Protein A and Immunoglobulin G Using a Surface Tension Probe

    OpenAIRE

    Yang, L.; Biswas, M. E.; Chen, P.

    2003-01-01

    Molecular interactions and binding are one of the most important and fundamental properties in the study of biochemical and biomedical systems. The understanding of such interactions and binding among biomolecules forms the basis for the design and processing of many biotechnological applications, such as bioseparation and immunoadsorption. In this study, we present a novel method to probe molecular interactions and binding based on surface tension measurement. This method complements convent...

  14. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH 2 groups (CONH 2 -SWNT) exhibited very strong interactions between the CONH 2 -SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  15. eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

    Directory of Open Access Journals (Sweden)

    Michal Brylinski

    2014-09-01

    Full Text Available Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4-9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite.

  16. A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available Kinesin-13s are microtubule (MT depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2 on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

  17. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    Science.gov (United States)

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

    Energy Technology Data Exchange (ETDEWEB)

    Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita; Karkashon, Shay; Bonanno, Jeffrey B.; Poulos, Thomas L.; Yeh, Syun-Ru (Einstein); (UCI)

    2017-11-22

    Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

  19. Mapping the Binding Site for Escitalopram and Paroxetine in the Human Serotonin Transporter Using Genetically Encoded Photo-Cross-Linkers

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Bang-Andersen, Benny

    2017-01-01

    amber codon suppression in hSERT to encode the photo-cross-linking unnatural amino acid p-azido-l-phenylalanine into the suggested high- and low-affinity binding sites. We then employ UV-induced cross-linking with azF to map the binding site of escitalopram and paroxetine, two prototypical selective...

  20. Mutations at the Qo-Site of the Cytochrome bc1 Complex Strongly Affect Oxygen Binding

    DEFF Research Database (Denmark)

    Husen, Peter; Solov'yov, Ilia A

    2017-01-01

    The homodimeric bc1 protein complex is embedded in membranes of mitochondria and photosynthetic bacteria, where it transports protons across the membrane to maintain an electrostatic potential used to drive ATP synthesis as part of the respiratory or photosynthetic pathways. The reaction cycle...... of the bc1 complex is driven by series of redox processes involving substrate molecules from the membrane, but occasional side reactions between an intermediate semiquinone substrate and molecular oxygen are suspected to be a source of toxic superoxide, which is believed to be a factor in aging. The present...... investigation employs molecular dynamics simulations to study the effect of mutations in the Qo binding sites of the bc1 complex on the ability of oxygen molecules to migrate to and bind at various locations within the complex. It is found that the mutations strongly affect the ability of oxygen to bind...

  1. Humanin binds MPP8: mapping interaction sites of the peptide and protein.

    Science.gov (United States)

    Maximov, Vadim V; Martynenko, Alina V; Arman, Inga P; Tarantul, Vyacheslav Z

    2013-05-01

    Humanin (HN), a 24-amino acid peptide encoded by the mitochondrial 16S rRNA gene, was discovered by screening a cDNA library from the occipital cortex of a patient with Alzheimer's disease (AD) for a protection factor against AD-relevant insults. Earlier, using the yeast two-hybrid system, we have identified the M-phase phosphoprotein 8 (MPP8) as a binding partner for HN. In the present work, we further confirmed interaction of HN with MPP8 in co-immunoprecipitation experiments and localized an MPP8-binding site in the region between 5 and 12 aa. of HN. We have also shown that an MPP8 fragment (residues 431-560) is sufficient to bind HN. Further studies on functional consequences of the interaction between the potential oncopetide and the oncoprotein may elucidate some aspects of the molecular mechanisms of carcinogenesis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

  2. Scavenger Receptor C-Type Lectin Binds to the Leukocyte Cell Surface Glycan Lewis By a Novel Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Feinberg, H.; Taylor, M.E.; Weis, W.I.; /Stanford U., Med. School /Imperial Coll., London

    2007-07-10

    The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

  3. Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter.

    Science.gov (United States)

    Houlihan, W J; Boja, J W; Parrino, V A; Kopajtic, T A; Kuhar, M J

    1996-12-06

    A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.

  4. Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

    Science.gov (United States)

    Anisenko, Andrey N; Knyazhanskaya, Ekaterina S; Zatsepin, Timofey S; Gottikh, Marina B

    2017-01-01

    Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  5. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  6. The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Zeng Zhizhen [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)]. E-mail: zhizhen_zeng@merck.com; Chen, T.-B. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Miller, Patricia J. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Dean, Dennis [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Tang, Y.S. [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Sur, Cyrille [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Williams, David L. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)

    2006-05-15

    We have characterized the interaction of the serotonin transporter ligand [{sup 3}H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [{sup 3}H]-DASB, a tritiated version of the widely used [{sup 11}C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K {sub d}=0.20{+-}0.04 nM). The serotonin transporter density (B {sub max}) obtained for rhesus frontal cortex was found to be 66{+-}8 fmol/mg protein using [{sup 3}H]-DASB, similar to the B {sub max} value obtained using the reference radioligand [{sup 3}H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83{+-}22 fmol/mg protein). Specific binding sites of both [{sup 3}H]-DASB and [{sup 3}H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [{sup 3}H]-citalopram binding in a competition autoradiographic study, with K {sub i} values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [{sup 3}H]-DASB and [{sup 3}H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [{sup 11}C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.

  7. Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

    Energy Technology Data Exchange (ETDEWEB)

    Kuziemko, G.M.; Stroh, M.; Stevens, R.C. [Univ. of California, Berkeley, CA (United States)]|[Lawrence Berkeley National Lab., CA (United States)

    1996-05-21

    The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 {times} 10{sup {minus}12} M for GM1 to 1.88 {times} 10{sup {minus}10} M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to K{sub d} values determined with whole-cell binding assays. Both whole-cell assays ans SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface. 34 refs., 8 figs., 2 tabs.

  8. Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study.

    Science.gov (United States)

    Kane, Lucas T; Costa, Blaise M

    2015-09-01

    The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Exploring the ATP-binding site of P2X receptors

    Directory of Open Access Journals (Sweden)

    Thierry eChataigneau

    2013-12-01

    Full Text Available P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.

  10. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...

  11. Increased number of ouabain binding sites in lymphocytes from borderline hypertensives

    DEFF Research Database (Denmark)

    Nielsen, J R; Pedersen, K E; Klitgaard, N A

    1989-01-01

    Lymphocytes were used as a cellular model for the in vitro measurements of maximal ouabain binding sites in order to assess any changes in young men at increased risk of developing essential hypertension, and to analyse whether any such changes were associated to borderline hypertension and....../or heredity. Four groups were evaluated; 28 normotensive (NTO) and 20 borderline hypertensive (BHO) offspring of hypertensives. Twelve borderline hypertensives (BH) and 28 normotensive subjects (NT) with normotensive parents. The number of ouabain binding sites were significantly increased in the borderline...... hypertensives irrespective of heredity. The borderline hypertensives were heavier than the normotensives. A stepwise multiple regression model was therefore used in order to control confoundings by body mass index (BMI) and other factors such as age, gamma glutamyl transferase, 24 h sodium excretion, serum...

  12. LTRs of Endogenous Retroviruses as a Source of Tbx6 Binding Sites

    Directory of Open Access Journals (Sweden)

    Yukuto Yasuhiko

    2017-06-01

    Full Text Available Retrotransposons are abundant in mammalian genomes and can modulate the gene expression of surrounding genes by disrupting endogenous binding sites for transcription factors (TFs or providing novel TFs binding sites within retrotransposon sequences. Here, we show that a (C/TCACACCT sequence motif in ORR1A, ORR1B, ORR1C, and ORR1D, Long Terminal Repeats (LTRs of MaLR endogenous retrovirus (ERV, is the direct target of Tbx6, an evolutionary conserved family of T-box TFs. Moreover, by comparing gene expression between control mice (Tbx6 +/− and Tbx6-deficient mice (Tbx6 −/−, we demonstrate that at least four genes, Twist2, Pitx2, Oscp1, and Nfxl1, are down-regulated with Tbx6 deficiency. These results suggest that ORR1A, ORR1B, ORR1C and ORR1D may contribute to the evolution of mammalian embryogenesis.

  13. LTRs of endogenous retroviruses as a source of Tbx6 binding sites

    Science.gov (United States)

    Yasuhiko, Yukuto; Hirabayashi, Yoko; Ono, Ryuichi

    2017-06-01

    Retrotransposons are abundant in mammalian genomes and can modulate the gene expression of surrounding genes by disrupting endogenous binding sites for transcription factors (TFs) or providing novel TFs binding sites within retrotransposon sequences. Here, we show that a (C/T)CACACCT sequence motif in ORR1A, ORR1B, ORR1C and ORR1D, Long Terminal Repeats (LTRs) of MaLR endogenous retrovirus (ERV), is the direct target of Tbx6, an evolutionary conserved family of T-box transcription factors. Moreover, by comparing gene expression between control mice (Tbx6 +/-) and Tbx6-deficient mice (Tbx6 -/-), we demonstrate that at least four genes, Twist2, Pitx2, Oscp1, and Nfxl1, are down-regulated with Tbx6 deficiency. These results suggest that ORR1A, ORR1B, ORR1C and ORR1D may contribute to the evolution of mammalian embryogenesis.

  14. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

    Science.gov (United States)

    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  15. Crystal structure of Yersinia pestis virulence factor YfeA reveals two polyspecific metal-binding sites.

    Science.gov (United States)

    Radka, Christopher D; DeLucas, Lawrence J; Wilson, Landon S; Lawrenz, Matthew B; Perry, Robert D; Aller, Stephen G

    2017-07-01

    Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that YfeA is polyspecific and can alter its substrate specificity. In minimal-media experiments, YfeA crystals grown after iron supplementation showed a threefold increase in iron fluorescence emission over the iron fluorescence emission from YfeA crystals grown from nutrient-rich conditions, and YfeA crystals grown after manganese supplementation during overexpression showed a fivefold increase in manganese fluorescence emission over the manganese fluorescence emission from YfeA crystals grown from nutrient-rich conditions. In all experiments, the YfeA crystals produced the strongest fluorescence emission from zinc and could not be manipulated otherwise. Additionally, this report documents the discovery of a novel surface metal-binding site that prefers to chelate zinc but can also bind manganese. Flexibility across YfeA crystal forms in three loops and a helix near the buried metal-binding site suggest that a structural rearrangement is required for metal loading and unloading.

  16. Identification of critical residues in loop E in the 5-HT3ASR binding site

    Directory of Open Access Journals (Sweden)

    Muthalagi Mani

    2002-06-01

    Full Text Available Abstract Background The serotonin type 3 receptor (5-HT3R is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147 to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152 also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR. Conclusion Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

  17. Polymorphisms in miRNA binding sites of nucleotide excision repair genes and colorectal cancer risk

    Czech Academy of Sciences Publication Activity Database

    Naccarati, Alessio; Pardini, Barbara; Landi, S.; Landi, D.; Slyšková, Jana; Novotný, J.; Levý, M.; Poláková, Veronika; Lipská, L.; Vodička, Pavel

    2012-01-01

    Roč. 33, č. 7 (2012), s. 1346-1351 ISSN 0143-3334 R&D Projects: GA ČR GAP304/10/1286; GA ČR GP305/09/P194 Institutional research plan: CEZ:AV0Z50390703 Keywords : DNA repair * polymorphisms * miRNA binding sites Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.635, year: 2012

  18. Association of aggression with a novel microRNA binding site polymorphism in the wolframin gene.

    Science.gov (United States)

    Kovacs-Nagy, Reka; Elek, Zsuzsanna; Szekely, Anna; Nanasi, Tibor; Sasvari-Szekely, Maria; Ronai, Zsolt

    2013-06-01

    Rare mutations in the WFS1 gene lead to Wolfram syndrome, a severe multisystem disorder with progressive neurodegeneration and diabetes mellitus causing life-threatening complications and premature death. Only a few association studies using small clinical samples tested the possible effects of common WFS1 gene variants on mood disorders and suicide, the non-clinical spectrum has not been studied yet. Self-report data on Aggression, Impulsiveness, Anxiety, and Depression were collected from a large (N = 801) non-psychiatric sample. Single nucleotide polymorphisms (SNPs) were selected to provide an adequate coverage of the entire WFS1 gene, as well as to include putative microRNA binding site polymorphisms. Molecular analysis of the assumed microRNA binding site variant was performed by an in vitro reporter-gene assay of the cloned 3' untranslated region with coexpression of miR-668. Among the 17 WFS1 SNPs, only the rs1046322, a putative microRNA (miR-668) binding site polymorphism showed significant association with psychological dimensions after correction for multiple testing: those with the homozygous form of the minor allele reported higher aggression on the Buss-Perry Aggression Questionnaire (P = 0.0005). Functional effect of the same SNP was also demonstrated in a luciferase reporter system: the minor A allele showed lower repression compared to the major G allele, if co-expressed with miR-668. To our knowledge, this is the first report describing a microRNA binding site polymorphism of the WFS1 gene and its association with human aggression based on a large, non-clinical sample. Copyright © 2013 Wiley Periodicals, Inc.

  19. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmidt, J; Luz, A

    1999-01-01

    -chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition...

  20. Characterization of the S100A1 Protein Binding Site on TRPC6 C-Terminus

    Czech Academy of Sciences Publication Activity Database

    Bílý, Jan; Gryčová, Lenka; Holendová, Blanka; Jirků, Michaela; Janoušková, Hana; Boušová, Kristýna; Teisinger, Jan

    2013-01-01

    Roč. 8, č. 5 (2013), e62677 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP301/10/1159; GA ČR(CZ) GPP205/10/P308 Institutional support: RVO:67985823 Keywords : TRP channel * circular dichoism spectroscopy * stady state fluorescence anisotropy * binding site * TRPC6 * S100A1 * calcium Subject RIV: CE - Biochemistry Impact factor: 3.534, year: 2013

  1. Kinetic analysis of inhibition of glucoamylase and active site mutants via chemoselective oxime immobilization of acarbose on SPR chip surfaces

    DEFF Research Database (Denmark)

    Sauer, Jørgen; Abou Hachem, Maher; Svensson, Birte

    2013-01-01

    We here report a quantitative study on the binding kinetics of inhibition of the enzyme glucoamylase and how individual active site amino acid mutations influence kinetics. To address this challenge, we have developed a fast and efficient method for anchoring native acarbose to gold chip surfaces...

  2. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    Science.gov (United States)

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-08

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

  3. Localization of substance P binding sites in submucous plexus of guinea pig ileum, using whole-mount autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Burcher, E.; Bornstein, J.C.

    1988-01-01

    Whole mounts of guinea pig ileum submucosa were incubated with radiolabeled tachykinins, and binding sites were visualized using autoradiography. Very dense specific binding for (/sup 125/I)-Bolton-Hunter substance P (BHSP) was observed over ganglia of the submucous plexus, with weaker binding over internodal strands. Dense specific binding was also seen over occasional strands of circular muscle, with weak binding over clumps of mucosa. Although very weak binding was seen over some large blood vessels, no binding was associated with smaller blood vessels. Localization of binding was absent in whole-mounts coincubated with 1 microM substance P, used to define nonspecific binding. Localization of BHSP-specific binding was also abolished in whole-mounts coincubated with 1 nM substance P, but not with 1 nM neurokinin B, suggesting that binding was probably to an NK-1 tachykinin receptor. In whole-mounts incubated in (/sup 125/I)-iodohistidyl neurokinin A (INKA) or (/sup 125/I)-Bolton-Hunter neurokinin B (BHNKB), no specific binding over ganglia was observed. These binding sites for BHSP are probably identical with the neuronal substance P receptors mediating mucosal ion transport.

  4. Deposition of chemically reactive and repellent sites on biosensor chips for reduced non-specific binding.

    Science.gov (United States)

    Gandhiraman, R P; Gubala, V; Le, N C H; Nam, Le Cao Hoai; Volcke, C; Doyle, C; James, B; Daniels, S; Williams, D E

    2010-08-01

    The performances of new polymeric materials with excellent optical properties and good machinability have led the biomedical diagnostics industry to develop cheap disposable biosensor platforms appropriate for point of care applications. Zeonor, a type of cycloolefin polymer (COP), is one such polymer that presents an excellent platform for biosensor chips. These polymer substrates have to be modified to have suitable physico-chemical properties for immobilizing proteins. In this work, we have demonstrated the amine functionalization of COP substrates, by plasma enhanced chemical vapour deposition (PECVD), through codeposition of ethylene diamine and 3-aminopropyltriethoxysilane precursors, for building chemistries on the plastic chip. The elemental composition, adhesion, ageing and reactivity of the plasma polymerized film were examined. The Si-O functionality present in amino silane contributed for a good interfacial adhesion of the coating to COP substrates and also acted as a network building layer for plasma polymerization. Wet chemical modification was then carried out on the amine functionalized chips to create chemically reactive isothiocyanate sites and protein repellent fluorinated sites on the same chip. The density of the reactive and repellent sites was altered by choosing appropriate mixtures of homofunctional phenyldiisothiocyanate (PDITC), pentafluoroisothiocyanate (5FITC) and phenylisothiocyanate (PITC) compounds. By tailoring the density of reactive binding sites and protein repellent sites, the non-specific binding of ssDNA has been decreased to a significant extent. Copyright 2010 Elsevier B.V. All rights reserved.

  5. Integrin activation dynamics between the RGD-binding site and the headpiece hinge.

    Science.gov (United States)

    Puklin-Faucher, Eileen; Vogel, Viola

    2009-12-25

    Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.

  6. Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge*

    Science.gov (United States)

    Puklin-Faucher, Eileen; Vogel, Viola

    2009-01-01

    Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII10-bound αVβ3 integrin headpiece how the binding pocket and interdomain βA/hybrid domain hinge on the distal end of the βA domain are allosterically linked via a hydrophobic T-junction between the middle of the α1 helix and top of the α7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca2+ in place of Mg2+ at the site adjacent to the metal ion-dependent adhesion site (“ADMIDAS”). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca2+ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated. PMID:19762919

  7. Pocketome: an encyclopedia of small-molecule binding sites in 4D.

    Science.gov (United States)

    Kufareva, Irina; Ilatovskiy, Andrey V; Abagyan, Ruben

    2012-01-01

    The importance of binding site plasticity in protein-ligand interactions is well-recognized, and so are the difficulties in predicting the nature and the degree of this plasticity by computational means. To assist in understanding the flexible protein-ligand interactions, we constructed the Pocketome, an encyclopedia of about one thousand experimentally solved conformational ensembles of druggable binding sites in proteins, grouped by location and consistent chain/cofactor composition. The multiplicity of pockets within the ensembles adds an extra, fourth dimension to the Pocketome entry data. Within each ensemble, the pockets were carefully classified by the degree of their pairwise similarity and compatibility with different ligands. The core of the Pocketome is derived regularly and automatically from the current releases of the Protein Data Bank and the Uniprot Knowledgebase; this core is complemented by entries built from manually provided seed ligand locations. The Pocketome website (www.pocketome.org) allows searching for the sites of interest, analysis of conformational clusters, important residues, binding compatibility matrices and interactive visualization of the ensembles using the ActiveICM web browser plugin. The Pocketome collection can be used to build multi-conformational docking and 3D activity models as well as to design cross-docking and virtual ligand screening benchmarks.

  8. Correlation between Virtual Screening Performance and Binding Site Descriptors of Protein Targets.

    Science.gov (United States)

    Shamsara, Jamal

    2018-01-01

    Rescoring is a simple approach that theoretically could improve the original docking results. In this study AutoDock Vina was used as a docked engine and three other scoring functions besides the original scoring function, Vina, as well as their combinations as consensus scoring functions were employed to explore the effect of rescoring on virtual screenings that had been done on diverse targets. Rescoring by DrugScore produces the most number of cases with significant changes in screening power. Thus, the DrugScore results were used to build a simple model based on two binding site descriptors that could predict possible improvement by DrugScore rescoring. Furthermore, generally the screening power of all rescoring approach as well as original AutoDock Vina docking results correlated with the Maximum Theoretical Shape Complementarity (MTSC) and Maximum Distance from Center of Mass and all Alpha spheres (MDCMA). Therefore, it was suggested that, with a more complete set of binding site descriptors, it could be possible to find robust relationship between binding site descriptors and response to certain molecular docking programs and scoring functions. The results could be helpful for future researches aiming to do a virtual screening using AutoDock Vina and/or rescoring using DrugScore.

  9. Correlation between Virtual Screening Performance and Binding Site Descriptors of Protein Targets

    Directory of Open Access Journals (Sweden)

    Jamal Shamsara

    2018-01-01

    Full Text Available Rescoring is a simple approach that theoretically could improve the original docking results. In this study AutoDock Vina was used as a docked engine and three other scoring functions besides the original scoring function, Vina, as well as their combinations as consensus scoring functions were employed to explore the effect of rescoring on virtual screenings that had been done on diverse targets. Rescoring by DrugScore produces the most number of cases with significant changes in screening power. Thus, the DrugScore results were used to build a simple model based on two binding site descriptors that could predict possible improvement by DrugScore rescoring. Furthermore, generally the screening power of all rescoring approach as well as original AutoDock Vina docking results correlated with the Maximum Theoretical Shape Complementarity (MTSC and Maximum Distance from Center of Mass and all Alpha spheres (MDCMA. Therefore, it was suggested that, with a more complete set of binding site descriptors, it could be possible to find robust relationship between binding site descriptors and response to certain molecular docking programs and scoring functions. The results could be helpful for future researches aiming to do a virtual screening using AutoDock Vina and/or rescoring using DrugScore.

  10. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    Energy Technology Data Exchange (ETDEWEB)

    Chabot, J.G.; Walker, P.; Pelletier, G.

    1986-01-01

    The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of (/sup 125/I)EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of (/sup 125/I)EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of (/sup 125/I)EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.

  11. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmidt, J; Luz, A

    1999-01-01

    ) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary...... delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta......, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant....

  12. Composite Structural Motifs of Binding Sites for Delineating Biological Functions of Proteins

    Science.gov (United States)

    Kinjo, Akira R.; Nakamura, Haruki

    2012-01-01

    Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures. PMID:22347478

  13. Using an ensemble of statistical metrics to quantify large sets of plant transcription factor binding sites.

    Science.gov (United States)

    Hosseini, Parsa; Ovcharenko, Ivan; Matthews, Benjamin F

    2013-04-11

    From initial seed germination through reproduction, plants continuously reprogram their transcriptional repertoire to facilitate growth and development. This dynamic is mediated by a diverse but inextricably-linked catalog of regulatory proteins called transcription factors (TFs). Statistically quantifying TF binding site (TFBS) abundance in promoters of differentially expressed genes can be used to identify binding site patterns in promoters that are closely related to stress-response. Output from today's transcriptomic assays necessitates statistically-oriented software to handle large promoter-sequence sets in a computationally tractable fashion. We present Marina, an open-source software for identifying over-represented TFBSs from amongst large sets of promoter sequences, using an ensemble of 7 statistical metrics and binding-site profiles. Through software comparison, we show that Marina can identify considerably more over-represented plant TFBSs compared to a popular software alternative. Marina was used to identify over-represented TFBSs in a two time-point RNA-Seq study exploring the transcriptomic interplay between soybean (Glycine max) and soybean rust (Phakopsora pachyrhizi). Marina identified numerous abundant TFBSs recognized by transcription factors that are associated with defense-response such as WRKY, HY5 and MYB2. Comparing results from Marina to that of a popular software alternative suggests that regardless of the number of promoter-sequences, Marina is able to identify significantly more over-represented TFBSs.

  14. Surface-binding through polyfunction groups of Rhodamine B on composite surface and its high performance photodegradation

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Yiqun [College of Chemistry, Nanchang University, Nanchang 330031 (China); Center of Analysis and Testing, Nanchang University, Nanchang 330047 (China); Wang, Xiaofen [College of Chemistry, Nanchang University, Nanchang 330031 (China); Gu, Yun; Guo, Lan [College of Chemistry, Nanchang University, Nanchang 330031 (China); Center of Analysis and Testing, Nanchang University, Nanchang 330047 (China); Xu, Zhaodi, E-mail: xuzhaodi@ncu.edu.cn [Center of Analysis and Testing, Nanchang University, Nanchang 330047 (China)

    2016-03-15

    Graphical abstract: The proper adsorption sites of Rh B depending on the phases of composites significantly enhance photodegradation activity under visible light. - Highlights: • The composites ZnS/In(OH){sub 3}/In{sub 2}S{sub 3} were hydrothermally prepared. • Rhodamine B dye was effectively degraded by the composite under the visible light irradiation. • The three function groups of Rhodamine B bind on the composites ZnS/In(OH){sub 3}/In{sub 2}S{sub 3}. • The proper adsorption mode and site of Rhodamine B effectively suppress the combination of carrier. • A new degradation path of Rhodamine B on ZnS/In(OH){sub 3}/In{sub 2}S{sub 3} is found. - Abstract: A kind of novel composite ZnS/In(OH){sub 3}/In{sub 2}S{sub 3} is synthesized using zinc oxide nanoplates as zinc raw material during hydrothermal process. Although the obtained samples are composited of ZnS and In(OH){sub 3} and In{sub 2}S{sub 3} phase, the samples possess different structure, morphology and optical absorption property depending on molar ratio of raw materials. Zeta potential analysis indicates different surface electrical property since various content and particle size of the phases. The equilibrium adsorption study confirms the composite ZnS/In(OH){sub 3}/In{sub 2}S{sub 3} with surface negative charge is good adsorbent for Rhodamine B (Rh B) dye. In addition, the degradation of Rh B over the samples with surface negative charge under visible light (λ ≥ 420 nm) is more effective than the samples with surface positive charge. The samples before and after adsorbing Rh B molecule are examined by FTIR spectra and Zetasizer. It is found that the three function groups of Rh B molecule, especially carboxyl group anchors to surface of the sample through electrostatic adsorption, coordination and hydrogen-bond. It contributes to rapid transformation of photogenerated electron to conduction band of In(OH){sub 3} and suppresses the recombination of photogenerated carrier. The possible

  15. Beyond the detergent effect: a binding site for sodium dodecyl sulfate (SDS) in mammalian apoferritin

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Renyu, E-mail: renyu.liu@uphs.upenn.edu; Bu, Weiming; Xi, Jin [University of Pennsylvania, Philadelphia, Pennsylvania (United States); Mortazavi, Shirin R. [Drexel University College of Medicine, Philadelphia, Pennsylvania (United States); Cheung-Lau, Jasmina C.; Dmochowski, Ivan J. [University of Pennsylvania, Philadelphia, Pennsylvania (United States); Loll, Patrick J., E-mail: renyu.liu@uphs.upenn.edu [Drexel University College of Medicine, Philadelphia, Pennsylvania (United States); University of Pennsylvania, Philadelphia, Pennsylvania (United States)

    2012-05-01

    Using X-ray crystallography and isothermal titration calorimetry, we show that sodium dodecyl sulfate (SDS) binds specifically to a pre-formed internal cavity in horse-spleen apoferritin. Although sodium dodecyl sulfate (SDS) is widely used as an anionic detergent, it can also exert specific pharmacological effects that are independent of the surfactant properties of the molecule. However, structural details of how proteins recognize SDS are scarce. Here, it is demonstrated that SDS binds specifically to a naturally occurring four-helix bundle protein: horse apoferritin. The X-ray crystal structure of the apoferritin–SDS complex was determined at a resolution of 1.9 Å and revealed that the SDS binds in an internal cavity that has previously been shown to recognize various general anesthetics. A dissociation constant of 24 ± 9 µM at 293 K was determined by isothermal titration calorimetry. SDS binds in this cavity by bending its alkyl tail into a horseshoe shape; the charged SDS head group lies in the opening of the cavity at the protein surface. This crystal structure provides insights into the protein–SDS interactions that give rise to binding and may prove useful in the design of novel SDS-like ligands for some proteins.

  16. The active site of a carbohydrate esterase displays divergent catalytic and noncatalytic binding functions.

    Directory of Open Access Journals (Sweden)

    Cedric Montanier

    2009-03-01

    Full Text Available Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of

  17. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    DEFF Research Database (Denmark)

    Yatsenko, A S; Kucherenko, M M; Pantoja, M

    2009-01-01

    BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal ......BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C......-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. RESULTS: We now find that both WW binding sites are important for maintaining full Dg...

  18. Deciphering common recognition principles of nucleoside mono/di and tri-phosphates binding in diverse proteins via structural matching of their binding sites.

    Science.gov (United States)

    Bhagavat, Raghu; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-09-01

    Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

    Directory of Open Access Journals (Sweden)

    Ju-Chen Chia

    Full Text Available Phytochelatin synthase (PCS uses the substrates glutathione (GSH, γGlu-Cys-Gly and a cadmium (Cd-bound GSH (Cd∙GS2 to produce the shortest phytochelatin product (PC2, (γGlu-Cys2-Gly through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218 by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

  20. Ab initio identification of putative human transcription factor binding sites by comparative genomics

    Directory of Open Access Journals (Sweden)

    Provero P

    2005-05-01

    Full Text Available Abstract Background Understanding transcriptional regulation of gene expression is one of the greatest challenges of modern molecular biology. A central role in this mechanism is played by transcription factors, which typically bind to specific, short DNA sequence motifs usually located in the upstream region of the regulated genes. We discuss here a simple and powerful approach for the ab initio identification of these cis-regulatory motifs. The method we present integrates several elements: human-mouse comparison, statistical analysis of genomic sequences and the concept of coregulation. We apply it to a complete scan of the human genome. Results By using the catalogue of conserved upstream sequences collected in the CORG database we construct sets of genes sharing the same overrepresented motif (short DNA sequence in their upstream regions both in human and in mouse. We perform this construction for all possible motifs from 5 to 8 nucleotides in length and then filter the resulting sets looking for two types of evidence of coregulation: first, we analyze the Gene Ontology annotation of the genes in the set, searching for statistically significant common annotations; second, we analyze the expression profiles of the genes in the set as measured by microarray experiments, searching for evidence of coexpression. The sets which pass one or both filters are conjectured to contain a significant fraction of coregulated genes, and the upstream motifs characterizing the sets are thus good candidates to be the binding sites of the TF's involved in such regulation. In this way we find various known motifs and also some new candidate binding sites. Conclusion We have discussed a new integrated algorithm for the "ab initio" identification of transcription factor binding sites in the human genome. The method is based on three ingredients: comparative genomics, overrepresentation, different types of coregulation. The method is applied to a full-scan of the

  1. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites.

    Science.gov (United States)

    Shi, Jialan; Gilbert, Gary E

    2003-04-01

    Lactadherin, a glycoprotein of the milk-fat globule membrane, contains tandem C domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We asked whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1 to 4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor VIIa-tissue factor complex efficiently whereas annexin V was less effective. Because the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitory effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150% versus 20% for 60 nM annexin V. These results indicate that lactadherin can function as a potent phospholipid-blocking anticoagulant.

  2. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Smith, L.I.

    1989-01-01

    The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with ({sup 3}H)DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both ({sup 3}H)DM and L-({sup 35}S)methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified.

  3. Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin.

    Science.gov (United States)

    Johns, Christian W; Finley, Natosha L

    2017-11-30

    Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α -helical content to CaMWt, and only modest differences in α -helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C -terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N -terminal CaM and CyaA-ACD.

  4. The chromosomal high-affinity binding sites for the Drosophila dosage compensation complex.

    Directory of Open Access Journals (Sweden)

    Tobias Straub

    2008-12-01

    Full Text Available Dosage compensation in male Drosophila relies on the X chromosome-specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC. The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called "high-affinity sites" (HAS. Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

  5. Hydrogen molecule binding to unsaturated metal sites in metal-organic frameworks studied by neutron powder diffraction and inelastic neutron scattering

    Science.gov (United States)

    Liu, Yun; Brown, Craig; Neumann, Dan; Dinca, Mircea; Long, Jeffrey; Peterson, Vanessa; Kepert, Cameron

    2007-03-01

    Metal organic framework (MOF) materials have shown considerable potential for hydrogen storage arising from very large surface areas. However, the low binding energy of hydrogen molecules limits its storage capability to very low temperatures (industrial applications. Using neutron powder diffraction (NPD), we have characterized the hydrogen adsorption sites in a selected series of MOF materials with exposed unsaturated metal ions. Direct binding between the unsaturated metal ions and hydrogen molecules is observed and responsible for the enhanced initial hydrogen adsorption enthalpy. The different metals centers in these MOFs show different binding strength and interaction distances between the hydrogen molecule and metal ions. The organic linker also affects the overall H2 binding strength. Inelastic neutron scattering spectra of H2 in these MOFs are also discussed.

  6. Kaiso Directs the Transcriptional Corepressor MTG16 to the Kaiso Binding Site in Target Promoters

    Science.gov (United States)

    Barrett, Caitlyn W.; Smith, J. Joshua; Lu, Lauren C.; Markham, Nicholas; Stengel, Kristy R.; Short, Sarah P.; Zhang, Baolin; Hunt, Aubrey A.; Fingleton, Barbara M.; Carnahan, Robert H.; Engel, Michael E.; Chen, Xi; Beauchamp, R. Daniel; Wilson, Keith T.; Hiebert, Scott W.; Reynolds, Albert B.; Williams, Christopher S.

    2012-01-01

    Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the ApcMin mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co

  7. Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme.

    Science.gov (United States)

    Aktas, Deniz F; Cook, Paul F

    2008-12-01

    Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.

  8. Biocompatible medical implant materials with binding sites for a biodegradable drug-delivery system

    Directory of Open Access Journals (Sweden)

    Al-Dubai H

    2011-10-01

    Full Text Available Haifa Al-Dubai1, Gisela Pittner1, Fritz Pittner1, Franz Gabor21Max F Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria; 2Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Life Sciences, University of Vienna, Vienna, AustriaAbstract: Feasibility studies have been carried out for development of a biocompatible coating of medical implant materials allowing the binding of biodegradable drug-delivery systems in a way that their reloading might be possible. These novel coatings, able to bind biodegradable nanoparticles, may serve in the long run as drug carriers to mediate local pharmacological activity. After biodegradation of the nanoparticles, the binding sites could be reloaded with fresh drug-delivering particles. As a suitable receptor system for the nanoparticles, antibodies are anchored. The design of the receptor is of great importance as any bio- or chemorecognitive interaction with other components circulating in the blood has to be avoided. Furthermore, the binding between receptor and the particles has to be strong enough to keep them tightly bound during their lifetime, but on the other hand allow reloading after final degradation of the particles. The nanoparticles suggested as a drug-delivery system for medical implants can be loaded with different pharmaceuticals such as antibiotics, growth factors, or immunosuppressives. This concept may enable the changing of medication, even after implantation of the medical device, if afforded by patients’ needs.Keywords: antibody immobilization, biocompatible coating, chitosan nanoparticles, drug targeting, medical device

  9. MeRNA: a Database of Metal Ion Binding Sites in RNAStructures

    Energy Technology Data Exchange (ETDEWEB)

    Stefan, Liliana R.; Zhang, Rui; Levitan, Aaron G.; Hendrix, DonnaF.; Brenner, Steven E.; Holbrook, Stephen R.

    2005-10-05

    Metal ions are essential for the folding of RNA into stable tertiary structures and for the catalytic activity of some RNA enzymes. To aid in the study of the roles of metal ions in RNA structural biology, we have created MeRNA (Metals in RNA), a comprehensive compilation of all metal binding sites identified in RNA three-dimensional structures available from the Protein Data Bank (PDB) and Nucleic Acid Database (NDB). Currently, our database contains information relating to binding of 9764 metal ions corresponding to 23 distinct elements; in 256 RNA structures. The metal ion locations were confirmed and ligands characterized using original literature references. MeRNA includes eight manually identified metal-ion binding motifs, which are described in the literature. MeRNA is searchable by PDB identifier, metal ion, method of structure determination, resolution and R-values for X-ray structure, and distance from metal to any RNA atom or to water. New structures with their respective binding motifs will be added to the database as they become available. The MeRNA database will further our understanding of the roles of metal ions in RNA folding and catalysis and have applications in structural and functional analysis, RNA design and engineering.

  10. Automatic generation of 3D motifs for classification of protein binding sites

    Directory of Open Access Journals (Sweden)

    Herzyk Pawel

    2007-08-01

    Full Text Available Abstract Background Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands. Results Our new approach was validated by generating automatically 3D patterns for the main adenine based ligands, i.e. AMP, ADP and ATP. Out of the 18 detected patterns, only one, the ADP4 pattern, is not associated with well defined structural patterns. Moreover, most of the patterns could be classified as binding site 3D motifs. Literature research revealed that the ADP4 pattern actually corresponds to structural features which show complex evolutionary links between ligases and transferases. Therefore, all of the generated patterns prove to be meaningful. Each pattern was used to query all PDB proteins which bind either purine based or guanine based ligands, in order to evaluate the classification and annotation properties of the pattern. Overall, our 3D patterns matched 31% of proteins with adenine based ligands and 95.5% of them were classified correctly. Conclusion A new metric has been introduced allowing the classification of proteins according to the similarity of atomic environment of binding sites, and a methodology has been developed to automatically produce 3D patterns from that classification. A study of proteins binding adenine based ligands showed that

  11. Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter

    International Nuclear Information System (INIS)

    Dahabieh, Matthew S.; Ooms, Marcel; Malcolm, Tom; Simon, Viviana; Sadowski, Ivan

    2011-01-01

    Transcription from the HIV-1 long terminal repeat (LTR) is mediated by numerous host transcription factors. In this study we characterized an E-box motif (RBE1) within the core promoter that was previously implicated in both transcriptional activation and repression. We show that RBE1 is a binding site for the RBF-2 transcription factor complex (USF1, USF2, and TFII-I), previously shown to bind an upstream viral element, RBE3. The RBE1 and RBE3 elements formed complexes of identical mobility and protein constituents in gel shift assays, both with Jurkat T-cell nuclear extracts and recombinant USF/TFII-I. Furthermore, both elements are regulators of HIV-1 expression; mutations in LTR-luciferase reporters and in HIV-1 molecular clones resulted in decreased transcription, virion production, and proviral expression in infected cells. Collectively, our data indicate that RBE1 is a bona fide RBF-2 binding site and that the RBE1 and RBE3 elements are necessary for mediating proper transcription from the HIV-1 LTR.

  12. Enantioselective kappa opioid binding sites on the macrophage cell line, P388d sub 1

    Energy Technology Data Exchange (ETDEWEB)

    Carr, D.J.J.; Blalock, J.E. (Univ. of Alabama, Birmingham (USA)); DeCosta, B.R.; Jacobson, A.E.; Rice, K.C. (NIDDK, NIH, Bethesda, MD (USA))

    1991-01-01

    A kappa opioid binding site has been characterized on the macrophage cell line, P388d{sub 1}, using the kappa selective affinity ligand, ({sup 3H}(1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-phrrolidinyl) cyclohexyl) benzeneacetamide ((-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl) benzeneacetamide ((-)-U-50,488) blocks ({sup 3}H)95{alpha},7{alpha},8{beta})-(-)-N-methyl-N-(7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl)benzeneacetamide (U-69,593) binding to P388d{sub 1} cells with an IC{sub 50} = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl) benzeneacetamide ((+)U-50,488) blocks ({sup 3}H)U-69,593 binding to P388d{sub 1} cells with an IC{sub 50} = 700 nM.

  13. Studies on the digitalis binding site in Na, K-ATPase

    International Nuclear Information System (INIS)

    Ahmed, K.; McParland, R.; Becker, R.; From, A.; Schimerlik, M.; Fullerton, D.S.

    1986-01-01

    Na, K-ATPase is believed to be the receptor for digitalis glycosides. The authors have previously documented that C17 side group of the cardenolide molecule is crucial to α subunit receptor binding. They have attempted to identify the structure of this binding site by labelling the enzyme with a 3 H-labelled photoactive probe localized in the C17 side group of the genin molecule. 3 H-α-subunit was purified and subjected to tryptic digestion. The digest was fractionated by gel filtration on Sephadex G-100. Fractions containing 3 H-labelled peptide were pooled and rechromatographed. The central peak fractions of 3 H-peptide were pooled, analyzed by SDS-PAGE, and subjected to amino acid sequence analysis. The tryptic peptide containing the 3 H-probe showed considerable sequence heterogeneity. Comparison of the sequence data with the published cDNA-based α-subunit sequence revealed that this peptide material was indeed a mixture of two tryptic peptides of nearly identical size containing the sequences from residue 68 through residue 146, and residues 263 through 342. The latter peptide contains the sequence ... glu tyr thr try leu glu ... speculated by Shull et al. as a possible ouabain binding site

  14. Na,K-ATPase binding sites in human erythrocytes in cirrhosis of the liver

    International Nuclear Information System (INIS)

    Schober, O.; Oetting, G.; Bossaller, C.

    1985-01-01

    The number of red blood cell ouabain binding sites, total-body potassium (TBK), serum potassium, exchangeable sodium, and serum sodium was studied in 24 patients with cirrhosis of the liver. The number of red cell ouabain binding sites, measured by equilibrium binding of 3 H-ouabain, showed a significant increase in the number of Na,K pumps in patients with cirrhosis of the liver (447+-99) as compared with a control group (281+-50, n=36). TBK was measured by counting the endogenous K-40 in a whole-body counter. TBK was 76+-10% in cirrhosis. This significant reduction in TBK was accompanied by normal serum potassium levels, and slightly decreased serum sodium levels in cirrhosis, however exchangeable sodium (Na-24) was increased in cirrhosis of the liver (55+-13 mmol/kg) compared with controls (40+-7 mmol/kg). These results support the suggestion that changes of sodium-potassium concentration at the cell membrane may regulate the synthesis of Na,K-pump molecules. (orig.) [de

  15. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Science.gov (United States)

    Martinez-Archundia, Marlet; Cordomi, Arnau; Garriga, Pere; Perez, Juan J.

    2012-01-01

    The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand. PMID:22500107

  16. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Directory of Open Access Journals (Sweden)

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  17. Prediction of protein-protein binding site by using core interface residue and support vector machine

    Directory of Open Access Journals (Sweden)

    Sun Zhonghua

    2008-12-01

    Full Text Available Abstract Background The prediction of protein-protein binding site can provide structural annotation to the protein interaction data from proteomics studies. This is very important for the biological application of the protein interaction data that is increasing rapidly. Moreover, methods for predicting protein interaction sites can also provide crucial information for improving the speed and accuracy of protein docking methods. Results In this work, we describe a binding site prediction method by designing a new residue neighbour profile and by selecting only the core-interface residues for SVM training. The residue neighbour profile includes both the sequential and the spatial neighbour residues of an interface residue, which is a more complete description of the physical and chemical characteristics surrounding the interface residue. The concept of core interface is applied in selecting the interface residues for training the SVM models, which is shown to result in better discrimination between the core interface and other residues. The best SVM model trained was tested on a test set of 50 randomly selected proteins. The sensitivity, specificity, and MCC for the prediction of the core interface residues were 60.6%, 53.4%, and 0.243, respectively. Our prediction results on this test set were compared with other three binding site prediction methods and found to perform better. Furthermore, our method was tested on the 101 unbound proteins from the protein-protein interaction benchmark v2.0. The sensitivity, specificity, and MCC of this test were 57.5%, 32.5%, and 0.168, respectively. Conclusion By improving both the descriptions of the interface residues and their surrounding environment and the training strategy, better SVM models were obtained and shown to outperform previous methods. Our tests on the unbound protein structures suggest further improvement is possible.

  18. Fast kinetic studies on the allosteric interactions between acetylcholine receptor and local anesthetic binding sites.

    Science.gov (United States)

    Heidmann, T; Changeux, J P

    1979-02-15

    Preincubation of receptor-rich membrane fragments from Torpedo marmorata with tertiary amine local anesthetics and several toxins such as histrionicotoxin, crotoxin and cerulotoxin, modifies the amplitude and time course of the relaxation processes monitored upon rapid mixing of the membrane fragments with the fluorescent agonist, Dns-C6-Cho. In particular, the amplitude of the rapid relaxation process, which is proportional to the fraction of acetylcholine receptor sites in a high-affinity state, increases; accordingly, the rate constant of the 'slow' and 'intermediate' relaxation processes also increases up to ten times (except with histrionicotoxin) whereas in a higher range of local anesthetic concentrations the rate constant of the 'rapid' relaxation process decreases. The data are accounted for by a two-state model of the acetylcholine regulator, assuming distinct binding sites for cholinergic agonists and local anesthetics and allosteric interactions between these two classes of sites; local anesthetics stabilize the regulator in a high-affinity state for agonists even in the absence of agonist, and modify the rate constants for th interconversions between the low-affinity and high-affinity states. The model accounts for the 'slow' fluorescence increase monitored upon addition of local anesthetics to a suspension of receptor-rich membranes supplemented with trace amounts of Dns-C6-Cho. The effect of local anesthetics on the apparent rate constant of the 'rapid' relaxation process can be accounted for on the basis of an additional low-affinity binding of local anesthetics to the acetylcholine receptor site. Finally the increase of the apparent rate constant of the 'intermediate' relaxation process can be simply accounted for by assuming the existence of a third state, corresponding to the 'active' state, to which local anesthetics bind and block ionic transport.

  19. Ropizine concurrently enhances and inhibits [3H] dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

    International Nuclear Information System (INIS)

    Canoll, P.D.; Smith, P.R.; and Musacchio, J.M.

    1990-01-01

    Ropizine produces a simultaneous enhancement and inhibition of [ 3 H] dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [ 3 H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances [ 3 H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)- pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of [ 3 H]DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine

  20. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    International Nuclear Information System (INIS)

    Sultatos, L.G.; Kaushik, R.

    2008-01-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B max and K d for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B max did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B max , would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K d represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K d , and dimethylphosphorylation of Ser203 decreasing K d . These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203

  1. SIgA binding to mucosal surfaces is mediated by mucin-mucin interactions.

    Directory of Open Access Journals (Sweden)

    Hannah L Gibbins

    Full Text Available The oral mucosal pellicle is a layer of absorbed salivary proteins, including secretory IgA (SIgA, bound onto the surface of oral epithelial cells and is a useful model for all mucosal surfaces. The mechanism by which SIgA concentrates on mucosal surfaces is examined here using a tissue culture model with real saliva. Salivary mucins may initiate the formation of the mucosal pellicle through interactions with membrane-bound mucins on cells. Further protein interactions with mucins may then trigger binding of other pellicle proteins. HT29 colon cell lines, which when treated with methotrexate (HT29-MTX produce a gel-forming mucin, were used to determine the importance of these mucin-mucin interactions. Binding of SIgA to cells was then compared using whole mouth saliva, parotid (mucin-free saliva and a source of purified SIgA. Greatest SIgA binding occurred when WMS was incubated with HT29-MTX expressing mucus. Since salivary MUC5B was only able to bind to cells which produced mucus and purified SIgA showed little binding to the same cells we conclude that most SIgA binding to mucosal cells occurs because SIgA forms complexes with salivary mucins which then bind to cells expressing membrane-bound mucins. This work highlights the importance of mucin interactions in the development of the mucosal pellicle.

  2. (/sup 3/H)nitrobenzylthioinosine binding as a probe for the study of adenosine uptake sites in brain

    Energy Technology Data Exchange (ETDEWEB)

    Marangos, P.J.; Patel, J.; Clark-Rosenberg, R.; Martino, A.M.

    1982-07-01

    The binding of the potent adenosine uptake inhibitor (/sup 3/H)nitrobenzylthioinosine ((/sup 3/H)NBI) to brain membrane fractions was investigated. Reversible, saturable, specific, high-affinity binding was demonstrated in both rat and human brain. The KD in both was 0.15 nM with Bmax values of 140-200 fmol/mg protein. Linear Scatchard plots were routinely obtained, indicating a homogeneous population of binding sites in brain. The highest density of binding sites was found in the caudate and hypothalamus in both species. The binding site was heat labile and trypsin sensitive. Binding was also decreased by incubation of the membranes in 0.05% Triton X-100 and by treatment with dithiothreitol and iodoacetamide. Of the numerous salt and metal ions tested, only copper and zinc had significant effects on (/sup 3/H)NBI binding. The inhibitory potencies of copper and zinc were IC50 . 160 microM and 6 mM, respectively. Subcellular distribution studies revealed a high percentage of the (/sup 3/H)NBI binding sites on synaptosomes, indicating that these sites were present in the syna