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Sample records for 125i-galanin binding sites

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

    Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of 125I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. 125I-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

  2. Plant Hormone Binding Sites

    Napier, Richard

    2004-01-01

    • Aims Receptors for plant hormones are becoming identified with increasing rapidity, although a frustrating number remain unknown. There have also been many more hormone‐binding proteins described than receptors. This Botanical Briefing summarizes what has been discovered about hormone binding sites, their discovery and descriptions, and will not dwell on receptor functions or activities except where these are relevant to understand binding.

  3. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    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)

  4. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    High (17 nM) and low (603 nM) affinity binding sites for [3]tetrahydrotrazodone ([3] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [3]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [3] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [3]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  5. Erythropoietin binding sites in human foetal tissues

    Pekonen, F.; Rosenloef, K.; Rutanen, E.-M.

    1987-01-01

    Using /sup 125/I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 10/sup 9/l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked /sup 125/I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

  6. Erythropoietin binding sites in human foetal tissues

    Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 109l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively. (author)

  7. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  8. Nickel binding sites in histone proteins

    Zoroddu, Maria Antonietta; Peana, Massimiliano Francesco; Solinas, Costantino; Medici, Serenella

    2012-01-01

    Nickel compounds are well known as human carcinogens, though the molecular events that are responsible for this are not well understood. It has been proposed that a crucial element in the mechanism of carcinogenesis is the binding of Ni(II) ions within the cell nucleus. It is known that DNA polymer binds Ni(II) only weakly, leaving the proteins of the cell nucleus as the likely Ni(II) targets. Being histone proteins the most abundant among them, they can be considered the primary sites fo...

  9. SiteComp: a server for ligand binding site analysis in protein structures

    Lin, Yingjie; Yoo, Seungyeul; Sanchez, Roberto

    2012-01-01

    Motivation: Computational characterization of ligand-binding sites in proteins provides preliminary information for functional annotation, protein design and ligand optimization. SiteComp implements binding site analysis for comparison of binding sites, evaluation of residue contribution to binding sites and identification of sub-sites with distinct molecular interaction properties.

  10. Oxytocin binding sites in bovine mammary tissue

    Zhao, Xin.

    1989-01-01

    Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressin binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.

  11. Comparison of Transcription Factor Binding Site Models

    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.

  12. STUDY OF ESTROGEN BINDING SITE ON HUMAN EJACULATED SPERMATOZOA

    CHUJin-Shong; WANGYi-Fei

    1989-01-01

    The specific estrogen binding site for 17β-estradiol has been investigated on human spermatozoa by electron microscopec autoradiography. The results show that the binding sites were distributed over the surface of human spermatozoa: acrosomal cap, equatorial

  13. Grafting of protein-protein binding sites

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  14. Characteristics of human erythrocyte insulin binding sites.

    Okada, Yoshio

    1981-01-01

    Insulin and human erythrocyte cell membrane interactions were studied with respect to binding and dissociation. The per cent of specific binding of 125I-labeled insulin to erythrocytes was directly proportional to the cell concentration. The optimum pH for binding was 8.1. The initial binding rate was directly proportional to, and the steady state insulin binding was reversely proportional to, the incubation temperature. The per cent of specific binding of 125I-labeled insulin was 12.10 +/- 1...

  15. Negative Example Aided Transcription Factor Binding Site Search

    Lee, Chih; Huang, Chun-Hsi

    2011-01-01

    Computational approaches to transcription factor binding site identification have been actively researched for the past decade. Negative examples have long been utilized in de novo motif discovery and have been shown useful in transcription factor binding site search as well. However, understanding of the roles of negative examples in binding site search is still very limited. We propose the 2-centroid and optimal discriminating vector methods, taking into account negative examples. Cross-val...

  16. LASAGNA: A novel algorithm for transcription factor binding site alignment

    Lee, Chih; Huang, Chun-Hsi

    2013-01-01

    Background Scientists routinely scan DNA sequences for transcription factor (TF) binding sites (TFBSs). Most of the available tools rely on position-specific scoring matrices (PSSMs) constructed from aligned binding sites. Because of the resolutions of assays used to obtain TFBSs, databases such as TRANSFAC, ORegAnno and PAZAR store unaligned variable-length DNA segments containing binding sites of a TF. These DNA segments need to be aligned to build a PSSM. While the TRANSFAC database provid...

  17. Searching for transcription factor binding sites in vector spaces

    Lee Chih; Huang Chun-Hsi

    2012-01-01

    Abstract Background Computational approaches to transcription factor binding site identification have been actively researched in the past decade. Learning from known binding sites, new binding sites of a transcription factor in unannotated sequences can be identified. A number of search methods have been introduced over the years. However, one can rarely find one single method that performs the best on all the transcription factors. Instead, to identify the best method for a particular trans...

  18. Whole-genome cartography of estrogen receptor alpha binding sites.

    Chin-Yo Lin

    2007-06-01

    Full Text Available Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript, suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs, 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha

  19. Predicted metal binding sites for phytoremediation

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-01-01

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do no...

  20. Identification and characterization of anion binding sites in RNA

    Kieft, Jeffrey S.; Chase, Elaine; Costantino, David A.; Golden, Barbara L. (Purdue); (Colorado)

    2010-05-24

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.

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

    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.

  2. Temperature and pressure adaptation of the binding site of acetylcholinesterase.

    Hochachka, P W

    1974-12-01

    1. Studies with a carbon substrate analogue, 3,3-dimethylbutyl acetate, indicate that the hydrophobic contribution to binding at the anionic site of acetylcholinesterase is strongly disrupted at low temperatures and high pressures. 2. Animals living in different physical environments circumvent this problem by adjusting the enthalpic and entropic contributions to binding. 3. An extreme example of this adaptational strategy is supplied by brain acetylcholinesterase extracted from an abyssal fish living at 2 degrees C and up to several hundred atmospheres of pressure. This acetylcholinesterase appears to have a smaller hydrophobic binding region in the anionic site, playing a measurably decreased role in ligand binding. PMID:4462739

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

    These studies show that nicotine binds to the rat brain P2 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 (+)-[3H]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 (+)-[3H]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

  4. Microbes bind complement inhibitor factor H via a common site.

    Meri, T; Amdahl, H; Lehtinen, M J; Hyvärinen, S; McDowell, J V; Bhattacharjee, A; Meri, S; Marconi, R; Goldman, A; Jokiranta, T S

    2013-01-01

    To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site." PMID:23637600

  5. PeptiSite: a structural database of peptide binding sites in 4D

    Acharya, Chayan; Kufareva, Irina; Ilatovskiy, Andrey V.; Abagyan, Ruben

    2014-01-01

    We developed PeptiSite, a comprehensive and reliable database of biologically and structurally characterized peptide-binding sites, in which each site is represented by an ensemble of its complexes with protein, peptide and small molecule partners. The unique features of the database include (1) the ensemble site representation that provides a fourth dimension to the otherwise three dimensional data, (2) comprehensive characterization of the binding site architecture that may consist of a mul...

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

    V Joachim Haupt

    Full Text Available 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

  7. Identification, characterization, and developmental regulation of embryonic benzodiazepine binding sites

    We report the identification and characterization of 2 classes of benzodiazepine binding sites in the embryonic chick CNS. Binding was examined by competition and saturation binding experiments, using as radioligands 3H-flunitrazepam, a classical benzodiazepine anxiolytic, and 3H-Ro5-4864, a convulsant benzodiazepine. The results demonstrate that high-affinity (KD = 2.3 nM) 3H-flunitrazepam binding sites (site-A) are present by embryonic day 5 (Hamburger and Hamilton stage 27) and increase throughout development (Bmax = 0.3 and 1.3 pmol/mg protein in 7 and 20 d brain membranes, respectively). When 7 or 20 d brain membranes are photoaffinity-labeled with 3H-flunitrazepam and ultraviolet light, the radioactivity migrates as 2 bands on SDS-PAGE, consistent with Mrs of 48,000 and 51,000. GABA potentiates 3H-flunitrazepam binding at both 7 and 20 d of development, indicating that site-A is coupled to receptors for GABA early in development. Importantly, we have also identified a novel site (site-B) that binds classical benzodiazepine agonists with low affinity (micromolar) but displays high affinity for Ro5-4864 (KD = 41 nM). Site-B displays characteristics expected for a functional receptor, including stereospecificity and sensitivity to inactivation by heat and protease treatment. Saturation binding studies employing 3H-Ro5-4864 indicate that the levels of site-B are similar in 7 and 20 d brain (ca. 2.5 pmol/mg protein). The function of site-B is not known, but its preponderance in 7 d brain, relative to site-A, suggests that it might be important during early embryonic development

  8. Reliable prediction of transcription factor binding sites by phylogenetic verification

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H.

    2005-01-01

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is ...

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

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

    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

  11. Chloride binding site of neurotransmitter sodium symporters

    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...

  12. Calculation of binding constants and concentration of binding sites in a reaction of a ligand with a heterogeneous system of binding sites

    A method is presented for the calculation of association constants and the concentration of binding sites in a reaction of a ligand with a heterogeneous system of binding sites. The Scatchard plot for such a system is curvelinear and the method employs previously established relationships between the parameters of the limiting slopes to such a curve and the above mentioned association constants and concentrations of binding sites. The special case of a system with two different and non-interacting groups of binding sites was solved. The expressions thus obtained were used to characterize the reaction of a polypeptide neurotoxin with its specific binding sites in a membranal preparation from insect central nervous system. Moreover it is evident from these expressions that the widely accepted method to analyze such system, by an intuitive generalization of the method applicable to homogeneous systems, is erroneous and should be avoided. (author)

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

    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 [3H] bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/106 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 [3H] bremazocine with an IC50 value = 0.57μM. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, [D-Pen2, D-Pen5] enkephalin and β-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC50 = 60μM, that was similar to naloxone. 32 references, 3 figures, 2 tables

  14. Human chorionic ganodotropin binding sites in the human endometrium

    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 Zn2+ effected a remarkable increase in [125I]hCG binding to the endometrium, whereas Mn2+ 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 [125I]hCG binding to endometrium. 14 refs., 3 figs

  15. Domain-based small molecule binding site annotation

    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. 3H-spiroperidol binding sites in blood platelets

    3H-spiroperidol, an antagonist of dopamine receptors in brain (striatum), was found to bind to human and rat platelet membrane preparations. The binding was rapid, reversible, saturable and specific. Unlabelled haloperidol displaced the specifically bound 3H-spiroperidol. Binding equilibrium was attained in 15 min at pH 7.4 and 37 degrees C. Scatchard analysis of 3H-spiroperidol binding revealed a single population of binding site with Kd of 7.6 nM in rat platelet membrane and Kd of 15 nM in human platelet membrane. Unlabelled 5-hydroxytryptamine produced no significant effect on 3H-spiroperidol binding to rat or human blood platelet membranes in the presence or absence of haloperidol. Some dopaminergic agents, known to inhibit spiroperidol binding in corpus striatum, also inhibited the same in rat and human blood platelet membranes under in vitro conditions. This study suggests the presence of specific 3H-spiroperidol binding sites in blood platelets

  17. Identification and characterization of anion binding sites in RNA.

    Kieft, Jeffrey S; Chase, Elaine; Costantino, David A; Golden, Barbara L

    2010-06-01

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions. PMID:20410239

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

    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

  19. Penicillin-binding site on the Escherichia coli cell envelope

    The binding of 35S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

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

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

    1983-07-01

    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 /sup 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 /sup 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.

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

    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 125I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125I-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

  2. Binding-site assessment by virtual fragment screening.

    Niu Huang

    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.

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

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

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless......, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with...... therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to...

  4. Agonist binding to high-affinity dopamine sites

    Tedesco, J.L.

    1985-01-01

    The authors have characterized the dopamine D/sub 3/ site and its binding requirements. The dopamine D/sub 3/ site in calf caudate crude homogenate has a site density of 214-230 fmoles/mg. protein by both /sup 3/H-apomorphine (/sup 3/H-AOP) and /sup 3/H-dopamine (/sup 3/H-DA) Scatchard analysis of specific binding (SB). Stereospecific subsets of /sup 3/H-APO and /sup 3/H-DA sites were defined by the use of agonist and antagonist enantiomer-pairs as a rigorous test for D/sub 3/ site heterogeneity. IC/sub 50/ values for both /sup 3/H-APO and /sup 3/H-DA SB sites were assessed for 55 agonist ligands and an excellent correlation was obtained. The authors conclude that both /sup 3/H-ligands label the same D/sub 3/ site. The D/sub 3/ site affinities of 105 dopamine-agonist ligands, in particular 2-aminotetralins,, aporphines and flexible dopamine analogues were measured. Low D/sub 3/-site affinities of N-quaternary analogues confirm the need for a lone pair. Subadditivity of substituents' effects in semi-flexible DA analogues confirms their postulate that sidechain conformation is the critical determinant of affinity. They conclude that there are at least two high-affinity ligand conformations of the DA sidechain pharmacophore. These binding requirements are presented as two interface-Geometry tetrahedral models of the double H-bond interface between the D/sub 3/ site and the ideal ligand.

  5. Thymocyte plasma membrane: the location of specific glucocorticoid binding sites

    In modern molecular endocrinology it is now possible to determine the localization of receptors for biologically active substances with the aid of ligands, with high affinity for the receptor, immobilized on polymers. The purpose of this paper is to study the ability of hydrocortisone (HC), immobilized on polyvinylpyrrolidone (PVP-HC), to reduce binding of tritium-HC by thymocytes of adrenalectomized rats. It is determined that specific binding sites for HC on rat thymocytes are also accessible for PVP-HC, which, due to the fact that this immobilized version of HC does not penetrate into the cell, leads to the conclusion that the binding sites for HC itself are located in the plasma membrane

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

    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...

  7. Structures of quinone binding sites in bc complexes: Functional implications

    Near-atomic resolution structures are becoming available for the respiratory chain enzyme known as ubiquinol:cytochrome c oxidoreductase or the cytochrome bc1 complex. Here we examine our current structure for the chicken bc1 complex to see what it can tell us about the mode of binding and mechanism of reaction of quinone at the two active sites

  8. Reliable prediction of transcription factor binding sites by phylogenetic verification.

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H

    2005-11-22

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is capable of accurately detecting TF binding sites, it also enhances our ability to predict the cis-regulatory modules. We applied this method on the published chromatin immunoprecipitation (ChIP)-chip data in Saccharomyces cerevisiae and found that its sensitivity and specificity are 9% and 14% higher than those of two recent methods. We also recovered almost all of the previously verified TF binding sites and made predictions on the cis-regulatory elements that govern the tight regulation of ribosomal protein genes in 13 eukaryote species (2 plants, 4 yeasts, 2 worms, 2 insects, and 3 mammals). These results give insights to the transcriptional regulation in eukaryotic organisms. PMID:16286651

  9. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

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

    Abha S Bais; Steffen Grossmann; Martin Vingron

    2007-08-01

    Identifying transcription factor binding sites (TFBSs) is essential to elucidate putative regulatory mechanisms. A common strategy is to combine cross-species conservation with single sequence TFBS annotation to yield ``conserved TFBSs”. Most current methods in this field adopt a multi-step approach that segregates the two aspects. Again, it is widely accepted that the evolutionary dynamics of binding sites differ from those of the surrounding sequence. Hence, it is desirable to have an approach that explicitly takes this factor into account. Although a plethora of approaches have been proposed for the prediction of conserved TFBSs, very few explicitly model TFBS evolutionary properties, while additionally being multi-step. Recently, we introduced a novel approach to simultaneously align and annotate conserved TFBSs in a pair of sequences. Building upon the standard Smith-Waterman algorithm for local alignments, SimAnn introduces additional states for profiles to output extended alignments or annotated alignments. That is, alignments with parts annotated as gaplessly aligned TFBSs (pair-profile hits) are generated. Moreover, the pair-profile related parameters are derived in a sound statistical framework. In this article, we extend this approach to explicitly incorporate evolution of binding sites in the SimAnn framework. We demonstrate the extension in the theoretical derivations through two position-specific evolutionary models, previously used for modelling TFBS evolution. In a simulated setting, we provide a proof of concept that the approach works given the underlying assumptions, as compared to the original work. Finally, using a real dataset of experimentally verified binding sites in human-mouse sequence pairs, we compare the new approach (eSimAnn) to an existing multi-step tool that also considers TFBS evolution. Although it is widely accepted that binding sites evolve differently from the surrounding sequences, most comparative TFBS identification

  11. Structural Fingerprints of Transcription Factor Binding Site Regions

    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.

  12. SuperSite: dictionary of metabolite and drug binding sites in proteins

    Bauer, Raphael André; Günther, Stefan; Jansen, Dominic; Heeger, Carolin; Thaben, Paul Florian; Preissner, Robert

    2008-01-01

    The increasing structural information about target-bound compounds provide a rich basis to study the binding mechanisms of metabolites and drugs. SuperSite is a database, which combines the structural information with various tools for the analysis of molecular recognition. The main data is made up of 8000 metabolites including 1300 drugs, bound to about 290 000 different receptor binding sites. The analysis tools include features, like the highlighting of evolutionary conserved receptor resi...

  13. Architecture of the sugar binding sites in carbohydrate binding proteins--a computer modeling study.

    Rao, V S; Lam, K; Qasba, P K

    1998-11-01

    Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity. PMID:9849627

  14. Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

    Herrero, Salvador; González-Cabrera, Joel; Tabashnik, Bruce E; Ferré, Juan

    2001-01-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At lea...

  15. Binding site of MraZ transcription factor in Mollicutes.

    Fisunov, G Y; Evsyutina, D V; Semashko, T A; Arzamasov, A A; Manuvera, V A; Letarov, A V; Govorun, V M

    2016-06-01

    Mollicutes (mycoplasmas) feature a significant loss of known regulators of gene expression. Here, we identified the recognition site of the MraZ-family regulator of Mycoplasma gallisepticum, which is conserved in many species of different clades within class Mollicutes. The MraZ binding site is AAAGTG[T/G], in the promoter of mraZ gene it forms a series of direct repeats with a structure (AAAGTG[T/G]N3)k, where k = 3 most frequently. MraZ binds to a single repeat as an octamer complex. MraZ can also bind a single binding site or a series of repeats with different spacer lengths (2-4 nt); thus, it may play a role in the regulation of multiple operons in Mollicutes. In M. gallisepticum, MraZ acts as a transcriptional activator. The overexpression of MraZ leads to moderate filamentation of cells and the formation of aggregates, likely as a result of incomplete cytokinesis. PMID:26945841

  16. 14C-glucose binding assay of the glucose transporter binding sites in muscular cell membrane

    A method of determining the binding sites of glucose transporter in rat muscular cell membrane was introduced. The crude products of cell membrane form the skeletal muscle of control and insulin treated rats were prepared, and then fractionated in sucrose gradient. Both plasma membrane and microsome membrane were incubated with D-[U-14C] glucose respectively for the measurement of radioactivity and Scatchard plot analysis. It was found that the binding sites of glucose transporter in plasma membrane and intracellular membrane were 5.6 nmol 14C-glucose/mg protein and 8.7 nmol 14C-glucose-mg protein respectively at basic state. Insulin treatment in experimental groups caused approximately 146% increase in plasma membrane fraction and 88% decrease in intracellular membrane fraction. Moreover, the kinetic data of Scatchard plot curve were similar to those of the [3H]-cytochalasin B binding assay. D-[U-14C] glucose binding assay of glucose transporter binding sites in muscular cell membrane is simple, easy and practicable. The D-[U-14C] glucose is commercially available

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

    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.

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

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

    2012-01-01

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off......-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...

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

    Xiang Zhou

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

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

    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

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

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area 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-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

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

    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. PMID:23762824

  3. Binding characterization, synthesis and biological evaluation of RXRα antagonists targeting the coactivator binding site.

    Xu, Dingyu; Guo, Shangjie; Chen, Ziwen; Bao, Yuzhou; Huang, Fengyu; Xu, Dan; Zhang, Xindao; Zeng, Zhiping; Zhou, Hu; Zhang, Xiaokun; Su, Ying

    2016-08-15

    Previously we identified the first retinoid X receptor-alpha (RXRα) modulators that regulate the RXRα biological function via binding to the coregulator-binding site. Here we report the characterization of the interactions between the hit molecule and RXRα through computational modeling, mutagenesis, SAR and biological evaluation. In addition, we reported studies of additional new compounds and identified a molecule that mediated the NF-κB pathway by inhibiting the TNFα-induced IκBα degradation and p65 nuclear translocation. PMID:27450787

  4. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    Hu, Xiaohu [ORNL; Norris, Adrianne [University of Tennessee, Knoxville (UTK); Baudry, Jerome Y [ORNL; Serpersu, Engin H [University of Tennessee, Knoxville (UTK)

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

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

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial [3H]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 [3H]diazepam binding are those that are active in displacing [3H]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

  6. Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site.

    Oikonomakos, N G; Schnier, J B; Zographos, S E; Skamnaki, V T; Tsitsanou, K E; Johnson, L N

    2000-11-01

    Flavopiridol (L86-8275) ((-)-cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl] -4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC(50) = 15.5 microm. The inhibition is synergistic with glucose resulting in a reduction of IC(50) for flavopiridol to 2.3 microm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-A resolution, and refined to crystallographic R values of 0.216 (R(free) = 0.247), 0.189 (R(free) = 0.219), and 0.195 (R(free) = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe(285) and Tyr(613). Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes. PMID:10924512

  7. A Unitary Anesthetic-Binding Site at High Resolution

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

    2009-01-01

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA 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.

  8. A Unitary Anesthetic Binding Site at High Resolution

    Vedula, L. Sangeetha; Brannigan, Grace; Economou, Nicoleta J.; Xi, Jin; Hall, Michael A.; Liu, Renyu; Rossi, Matthew J.; Dailey, William P.; Grasty, Kimberly C.; Klein, Michael L.; Eckenhoff, Roderic G.; Loll, Patrick J.; (Drexel-MED); (UPENN)

    2009-10-21

    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.

  9. A Unitary Anesthetic Binding Site at High Resolution

    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.

  10. PeptiSite: a structural database of peptide binding sites in 4D.

    Acharya, Chayan; Kufareva, Irina; Ilatovskiy, Andrey V; Abagyan, Ruben

    2014-03-21

    We developed PeptiSite, a comprehensive and reliable database of biologically and structurally characterized peptide-binding sites, in which each site is represented by an ensemble of its complexes with protein, peptide and small molecule partners. The unique features of the database include: (1) the ensemble site representation that provides a fourth dimension to the otherwise three dimensional data, (2) comprehensive characterization of the binding site architecture that may consist of a multimeric protein assembly with cofactors and metal ions and (3) analysis of consensus interaction motifs within the ensembles and identification of conserved determinants of these interactions. Currently the database contains 585 proteins with 650 peptide-binding sites. http://peptisite.ucsd.edu/ link allows searching for the sites of interest and interactive visualization of the ensembles using the ActiveICM web-browser plugin. This structural database for protein-peptide interactions enables understanding of structural principles of these interactions and may assist the development of an efficient peptide docking benchmark. PMID:24406170

  11. Photoaffinity labelling of t-RNA binding sites

    For the photoaffinity labelling of E.coli ribosomes in the region of peptidyl transferase, an analogue to the substrate peptidyl-tRNA-ethyl-2-diazomalalonyl-Phe-tRNAsup(Phe) was synthesized. UV irradiation of the reversible complex with 70S ribosomes and poly(U) led to the formation of a covalent bond between N-acyl-Phe-tRNA and 23S-rRNA. The irreversibly bound N-acyl-phenylalanyl group may be transferred to puromycin in a reaction catalyzed by peptidyl transferase, in the presence of the Phe-tRNA, it forms products of a peptide synthesis covalently bound to 23S-RNA. The 23S-rRNA sequence thus labelled, which has not yet been identified, should therefore be in the active centre of the peptidyl transferase or in its near neighbourhood. An analysis of the reaction product showed that the N-acyl-Phe-tRNA is bound specifically to one or more sites of a 3'-terminal 18S fragment of the 23S-RNA. An attempt to prove the existence of further tRNA interaction with ribosonal substrate binding sites led to the discovery of a poly(U2,G)-stimulated, UV-inducible irreversible binding of valin-specific tRNA (E.coli) to 16S-rRNA in one or several tRNA decoding sites. A preliminary analysis of the T1 fragments of tRNAsup(Val) after binding to 16S-rRNA indicates that the DHU loop of tRNA takes part in this photoreaction. (orig.)

  12. Basis for Half-Site Ligand Binding in Yeast NAD+-Specific Isocitrate Dehydrogenase†

    Lin, An-Ping; McAlister-Henn, Lee

    2011-01-01

    Yeast NAD+-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD+ binding sites, and four AMP binding sites, only half of the sites for each ligand are measurable in binding assays. Based on a potential interaction between side chains of Cys-150 residues in IDH2 subunits in eac...

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

    Quader Saad; Huang Chun-Hsi

    2012-01-01

    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 pape...

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

    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.

  15. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    Clifford, Jacob

    2015-01-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through Position Weight Matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain a...

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

    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...

  17. Effects of cytosine methylation on transcription factor binding sites

    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.

  18. Atrial natriuretic factor binding sites in experimental congestive heart failure

    A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor [(Ser99, Tyr126)ANF] binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF [des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2] (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease

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

    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.

  20. NMR Mapping of the IFNAR1-EC binding site on IFNα2 reveals allosteric changes in the IFNAR2-EC binding site

    Akabayov, Sabine Ruth; Biron, Zohar; Lamken, Peter; Piehler, Jacob; Anglister, Jacob

    2010-01-01

    All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits IFNAR1 and IFNAR2. In this study we mapped the surface of IFNα2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the [1H,15N]-TROSY-HSQC cross peaks of IFNα2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNα2 with IFNAR2-EC. The NMR study on the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 out of 165 IFNα2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNα2 binding site for IFNAR2-EC indicating allosteric communication between the binding sites for the two receptor subunits. PMID:20047337

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

    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

  2. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.

    Singh, Satinder K; Yamashita, Atsuko; Gouaux, Eric

    2007-08-23

    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 A 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 design of

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

    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

  4. TIM-4 structures identify a Metal Ion-dependent Ligand Binding Site where phosphatidylserine binds

    Santiago, Cesar; Ballesteros, Angela; Martinez-Muñoz, Laura; Mellado, Mario; Kaplan, Gerardo G.; Freeman, Gordon J.; Casasnovas, José M.

    2007-01-01

    The T-cell immunoglobulin and mucin domain (TIM) proteins are important regulators of T cell responses. They have been linked to autoimmunity and cancer. Structures of the murine TIM-4 identified a Metal Ion-dependent Ligand Binding Site (MILIBS) in the immunoglobulin (Ig) domain of the TIM family. The characteristic CC’ loop of the TIM domain and the hydrophobic FG loop shaped a narrow cavity where acidic compounds penetrate and coordinate to a metal ion bound to conserved residues in the TI...

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

    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.

    1988-11-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for /sup 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.

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

    Previous work from our laboratory demonstrated specific binding sites for 3H-RRR-alpha-tocopherol (3H-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 3H-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 3H-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

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

    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.

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

    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.

  9. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  10. Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

    QIAO,Yan-Hong; LIN,Hai; LIN,Hua-Kuan

    2007-01-01

    A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity,and also displayed dramatic color changes from colorless to yellow in the presence of TBAF (5 × 10-5 mol/L). The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO- while a little change with H2PO-4 and OH-. Receptor 1 has almost not affinity abilities to Cl-, Br- and I-. The binding ability of receptor 1to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular (AcO-), tetrahedral (H2PO-4 ) and linear (OH-) anions maybe result from their geometry configuration.

  11. Functional impact of HIV coreceptor-binding site mutations

    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. XAS and Pulsed EPR Studies of the Copper Binding Site in Riboflavin Binding Protein

    Smith,S.; Bencze, K.; Wasiukanis, K.; Benore-Parsons, T.; Stemmler, T.

    2008-01-01

    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 Angstroms, 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 CuO3N 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.

  13. Nuclear estradiol-binding sites in human breast cancer.

    Vandewalle, B; Peyrat, J P; Bonneterre, J; Hecquet, B; Dewailly, D; Lefebvre, J

    1983-09-01

    The binding of estradiol to nuclear fractions extracted from human breast carcinomatous tissue was demonstrated. The material, which was extracted with KCl, had an approximate molecular weight of 37,000 and bound estradiol with both high and low affinity (Kd congruent to 1 nM, type A receptors; Kd congruent to 30 nM, type B receptors) as calculated according to the method of Scatchard. Competition studies indicated that both components were specific for estradiol, and among the 134 tumors studied the receptors were found to be linked in almost all cases. Thirty-six % of the tumors were nuclear receptor positive. Cytoplasmic estradiol and progesterone receptors were also measured. Among the cytoplasmic tumors positive for cytoplasmic and progesterone receptors, 37% were devoid of both types of nuclear receptors; this may explain the failure of endocrine therapy in some cases. The determination of nuclear binding sites in human breast tumors appeared to be an interesting criterion for the assessment of estradiol-dependent cell growth. PMID:6683589

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

    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.

  15. An aprotinin binding site localized in the hormone binding domain of the estrogen receptor from calf uterus.

    Nigro, V; Medici, N; Abbondanza, C; Minucci, S; Moncharmont, B; Molinari, A M; Puca, G A

    1990-07-31

    It has been proposed that the estrogen receptor bears proteolytic activity responsible for its own transformation. This activity was inhibited by aprotinin. Incubation of transformed ER with aprotinin modified the proteolytic digestion of the hormone binding subunit by proteinase K. The smallest hormone-binding fragment of the ER, obtained by tryptic digestion, was still able to bind to aprotinin. These results suggest that aprotinin interacts with ER and the hormone-binding domain of ER is endowed with a specific aprotinin-binding site. PMID:1696480

  16. ncDNA and drift drive binding site accumulation

    Ruths Troy

    2012-08-01

    Full Text Available Abstract Background The amount of transcription factor binding sites (TFBS in an organism’s genome positively correlates with the complexity of the regulatory network of the organism. However, the manner by which TFBS arise and accumulate in genomes and the effects of regulatory network complexity on the organism’s fitness are far from being known. The availability of TFBS data from many organisms provides an opportunity to explore these issues, particularly from an evolutionary perspective. Results We analyzed TFBS data from five model organisms – E. coli K12, S. cerevisiae, C. elegans, D. melanogaster, A. thaliana – and found a positive correlation between the amount of non-coding DNA (ncDNA in the organism’s genome and regulatory complexity. Based on this finding, we hypothesize that the amount of ncDNA, combined with the population size, can explain the patterns of regulatory complexity across organisms. To test this hypothesis, we devised a genome-based regulatory pathway model and subjected it to the forces of evolution through population genetic simulations. The results support our hypothesis, showing neutral evolutionary forces alone can explain TFBS patterns, and that selection on the regulatory network function does not alter this finding. Conclusions The cis-regulome is not a clean functional network crafted by adaptive forces alone, but instead a data source filled with the noise of non-adaptive forces. From a regulatory perspective, this evolutionary noise manifests as complexity on both the binding site and pathway level, which has significant implications on many directions in microbiology, genetics, and synthetic biology.

  17. Identification of the Escherichia coli ADP-glucose synthetase inhibitor binding site(s)

    The photoaffinity labeling agent 8-azido adenylate (AMP) is an inhibitor site specific probe of the E. coli ADPG synthetase. In the absence of light, 8-azido AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), [2-3H]8-azido AMP specifically and covalently incorporates into the enzyme. Photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADP-glucose (ADPG) provides nearly 100% protection from 8-azido AMP photoinactivation, while the substrate AMP provides approximately 50% protection and the inhibitor AMP provides approximately 30% protection. These three adenylate allosteric effects of E. coli ADPG synthetase also protect it from photoincorporation of 8-azido AMP. The reaction site(s) of [2-3H]-azido AMP with the enzyme was identified by reverse phase HPLC isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease generated peptides containing the labeled analog. This site is the same as the major binding region of the substrate site specific probe, 8-azido ADP-[14C]glucose. Conformational analysis of this region predicts that it is a part of a Rossmann fold, the super-secondary structure found in many adenine nucleotide binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido AMP were also identified. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure

  18. High-affinity dextromethorphan binding sites in guinea pig brain. II. Competition experiments.

    Craviso, G L; Musacchio, J M

    1983-05-01

    Binding of dextromethorphan (DM) to guinea pig brain is stereoselective, since levomethorphan is 20 times weaker than DM in competing for DM sites. In general, opiate agonists and antagonists as well as their corresponding dextrorotatory isomers are weak competitors for tritiated dextromethorphan ([3H]DM) binding sites and display IC50 values in the micromolar range. In contrast, several non-narcotic, centrally acting antitussives are inhibitory in the nanomolar range (IC50 values for caramiphen, carbetapentane, dimethoxanate, and pipazethate are 25 nM, 9 nM, 41 nM, and 190 nM, respectively). Other antitussives, such as levopropoxyphene, chlophedianol, and fominoben, have poor affinity for DM sites whereas the antitussive noscapine enhances DM binding by increasing the affinity of DM for its central binding sites. Additional competition studies indicate that there is no correlation of DM binding with any of the known or putative neurotransmitters in the central nervous system. DM binding is also not related to tricyclic antidepressant binding sites or biogenic amine uptake sites. However, certain phenothiazine neuroleptics and typical and atypical antidepressants inhibit binding with IC50 values in the nanomolar range. Moreover, the anticonvulsant drug diphenylhydantoin enhances DM binding in a manner similar to that of noscapine. Preliminary experiments utilizing acid extracts of brain have not demonstrated the presence of an endogenous ligand for DM sites. The binding characteristics of DM sites studied in rat and mouse brain indicate that the relative potencies of several antitussives to inhibit specific DM binding vary according to species. High-affinity, saturable, and stereoselective [3H]DM binding sites are present in liver homogenates, but several differences have been found for these peripheral binding sites and those described for brain. Although the nature of central DM binding sites is not known, the potent interaction of several classes of centrally

  19. Osteopontin: A uranium phosphorylated binding-site characterization

    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 UO22+/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)

  20. Mapping convulsants’ binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites

    Kalueff, A.V.

    2006-01-01

    Gamma aminobutyric acid (GABA) type A receptors play a key role in brain inhibitory neurotransmission, and are ligand-activated chloride channels blocked by numerous convulsant ligands. Here we summarize data on binding of picrotoxin, tetrazoles, β-lactams, bicyclophosphates, butyrolactones and neurotoxic pesticides to GABA-A ionophore, and discuss functional and structural overlapping of their binding sites. The paper reviews data on convulsants’ binding sensitivity to different point mutati...

  1. MicroRNA binding sites in C. elegans 3' UTRs.

    Liu, Chaochun; Rennie, William A; Mallick, Bibekanand; Kanoria, Shaveta; Long, Dang; Wolenc, Adam; Carmack, C Steven; Ding, Ye

    2014-01-01

    MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Since the discovery of lin-4, the founding member of the miRNA family, over 360 miRNAs have been identified for Caenorhabditis elegans (C. elegans). Prediction and validation of targets are essential for elucidation of regulatory functions of these miRNAs. For C. elegans, crosslinking immunoprecipitation (CLIP) has been successfully performed for the identification of target mRNA sequences bound by Argonaute protein ALG-1. In addition, reliable annotation of the 3' untranslated regions (3' UTRs) as well as developmental stage-specific expression profiles for both miRNAs and 3' UTR isoforms are available. By utilizing these data, we developed statistical models and bioinformatics tools for both transcriptome-scale and developmental stage-specific predictions of miRNA binding sites in C. elegans 3' UTRs. In performance evaluation via cross validation on the ALG-1 CLIP data, the models were found to offer major improvements over established algorithms for predicting both seed sites and seedless sites. In particular, our top-ranked predictions have a substantially higher true positive rate, suggesting a much higher likelihood of positive experimental validation. A gene ontology analysis of stage-specific predictions suggests that miRNAs are involved in dynamic regulation of biological functions during C. elegans development. In particular, miRNAs preferentially target genes related to development, cell cycle, trafficking, and cell signaling processes. A database for both transcriptome-scale and stage-specific predictions and software for implementing the prediction models are available through the Sfold web server at http://sfold.wadsworth.org. PMID:24827614

  2. Engineering of binding affinity at metal ion binding sites for the stabilization of proteins: Subtilisin as a test case

    A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65/degrees/C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range of 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log K/sub a/ = 1.49. A series of refined X-ray crystal structures of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein. X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 /Angstrom/, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory. Moreover, when both Asp changes were introduced together, the binding affinity for Ca2+ was found to be increased about 6-fold over that for the wild-type protein, suggesting an independent and nearly additive effect on the total electrostatic potential at this locus

  3. Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.

    Herrero, S; González-Cabrera, J; Tabashnik, B E; Ferré, J

    2001-12-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far. PMID:11722929

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

    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....

  5. The hepcidin-binding site on ferroportin is evolutionarily conserved

    De Domenico, Ivana; Nemeth, Elizabeta; Nelson, Jenifer M.; Phillips, John D.; Ajioka, Richard S.; Kay, Michael S.; Kushner, James P.; Ganz, Tomas; Ward, Diane M.; Kaplan, Jerry

    2008-01-01

    Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell surface ferroportin. The bind...

  6. Energy-dependent fitness: A quantitative model for the evolution of yeast transcription factor binding sites

    Mustonen, Ville; Kinney, Justin; Callan, Curtis G.; Lässig, Michael

    2008-01-01

    We present a genomewide cross-species analysis of regulation for broad-acting transcription factors in yeast. Our model for binding site evolution is founded on biophysics: the binding energy between transcription factor and site is a quantitative phenotype of regulatory function, and selection is given by a fitness landscape that depends on this phenotype. The model quantifies conservation, as well as loss and gain, of functional binding sites in a coherent way. Its predictions are supported...

  7. Multiplicity of carbohydrate-binding sites in -prism fold lectins: occurrence and possible evolutionary implications

    Alok Sharma; Divya Chandran; Desh D Singh; M Vijayan

    2007-09-01

    The -prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, -prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the -prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a -prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of -prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the -prism II fold, is related to the role of plant lectins in defence.

  8. Ligand binding studies in the mouse olfactory bulb: identification and characterisation of a L-[3H]carnosine binding site

    Binding sites for the dipeptide L-carnosine (β-alanyl-t-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]- carnosine was saturable, reversible and stereospecific and had a Ksub(d) of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the totoal binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibted by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interfered with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. (Auth.)

  9. Mutations and binding sites of human transcription factors

    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.

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

    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.

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

    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: ► Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. ► Involvement of a static quenching component in an overall dynamic quenching process. ► Ability of quercetin and rutin to change the binding constants of 6-MP–BSA complex. ► Binding of 6-MP to BSA through entropy-driven hydrophobic interactions

  12. Knowledge-based annotation of small molecule binding sites in proteins

    Panchenko Anna R

    2010-07-01

    Full Text Available Abstract Background The study of protein-small molecule interactions is vital for understanding protein function and for practical applications in drug discovery. To benefit from the rapidly increasing structural data, it is essential to improve the tools that enable large scale binding site prediction with greater emphasis on their biological validity. Results We have developed a new method for the annotation of protein-small molecule binding sites, using inference by homology, which allows us to extend annotation onto protein sequences without experimental data available. To ensure biological relevance of binding sites, our method clusters similar binding sites found in homologous protein structures based on their sequence and structure conservation. Binding sites which appear evolutionarily conserved among non-redundant sets of homologous proteins are given higher priority. After binding sites are clustered, position specific score matrices (PSSMs are constructed from the corresponding binding site alignments. Together with other measures, the PSSMs are subsequently used to rank binding sites to assess how well they match the query and to better gauge their biological relevance. The method also facilitates a succinct and informative representation of observed and inferred binding sites from homologs with known three-dimensional structures, thereby providing the means to analyze conservation and diversity of binding modes. Furthermore, the chemical properties of small molecules bound to the inferred binding sites can be used as a starting point in small molecule virtual screening. The method was validated by comparison to other binding site prediction methods and to a collection of manually curated binding site annotations. We show that our method achieves a sensitivity of 72% at predicting biologically relevant binding sites and can accurately discriminate those sites that bind biological small molecules from non-biological ones. Conclusions

  13. Identification of clustered YY1 binding sites in Imprinting Control Regions

    Kim, J D; Hinz, A; Bergmann, A; Huang, J; Ovcharenko, I; Stubbs, L; Kim, J

    2006-04-19

    Mammalian genomic imprinting is regulated by Imprinting Control Regions (ICRs) that are usually associated with tandem arrays of transcription factor binding sites. In the current study, the sequence features derived from a tandem array of YY1 binding sites of Peg3-DMR (differentially methylated region) led us to identify three additional clustered YY1 binding sites, which are also localized within the DMRs of Xist, Tsix, and Nespas. These regions have been shown to play a critical role as ICRs for the regulation of surrounding genes. These ICRs have maintained a tandem array of YY1 binding sites during mammalian evolution. The in vivo binding of YY1 to these regions is allele-specific and only to the unmethylated active alleles. Promoter/enhancer assays suggest that a tandem array of YY1 binding sites function as a potential orientation-dependent enhancer. Insulator assays revealed that the enhancer-blocking activity is detected only in the YY1 binding sites of Peg3-DMR but not in the YY1 binding sites of other DMRs. Overall, our identification of three additional clustered YY1 binding sites in imprinted domains suggests a significant role for YY1 in mammalian genomic imprinting.

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

    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, [3H] 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 [3H] [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

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

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

    2015-01-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch...

  16. Active site and laminarin binding in glycoside hydrolase family 55.

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  17. Using circular permutation analysis to redefine the R17 coat protein binding site.

    Gott, J M; Pan, T; LeCuyer, K A; Uhlenbeck, O C

    1993-12-14

    The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used. PMID:7504949

  18. Evidence for a non-opioid sigma binding site din the guinea-pig myenteric plexus

    The presence of a binding site to (+)-(3H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site and a low affinity site. Morphine and naloxone 10-4M were unable to displace (+)-(3H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig

  19. Evidence for a non-opioid sigma binding site din the guinea-pig myenteric plexus

    Roman, F.; Pascaud, X.; Vauche, D.; Junien, J.

    1988-01-01

    The presence of a binding site to (+)-(/sup 3/H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site and a low affinity site. Morphine and naloxone 10/sup -4/M were unable to displace (+)-(/sup 3/H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.

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

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L; Shi, Lei; Gracia, Luis; Raniszewska, Klaudia; Newman, Amy Hauck; Javitch, Jonathan A.; Weinstein, Harel; Gether, Ulrik; Loland, Claus J

    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 LeuT. 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 dopami...

  1. PREDICTION OF ANTIGENIC AND BINDING SITES OF NEUROTOXIN 23 OF SCORPION (LYCHASMUCRONACTUS SP.)

    Bharati K Thosare; Ingale, Arun G

    2015-01-01

    Identification of antigenic and binding site of protein is highly desirable for the design of vaccines and immunodiagnostics. The present exercise deals with a prediction of antigenic as well as binding sites of neurotoxin 23 of Lychasmucronactus. This species of scorpion having diverse molecules of toxic peptide, the peptide neurotoxin 23 is 96 amino acids long of which 23 to 96 specifically code for neurotoxin. The total of 27 such different ligand binding residue were identifie...

  2. A systematic, large-scale comparison of transcription factor binding site models

    Hombach, Daniela; Schwarz, Jana Marie; Peter N. Robinson; Schuelke, Markus; Seelow, Dominik

    2016-01-01

    Background The modelling of gene regulation is a major challenge in biomedical research. This process is dominated by transcription factors (TFs) and mutations in their binding sites (TFBSs) may cause the misregulation of genes, eventually leading to disease. The consequences of DNA variants on TF binding are modelled in silico using binding matrices, but it remains unclear whether these are capable of accurately representing in vivo binding. In this study, we present a systematic comparison ...

  3. Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA

    Veprintsev, Dmitry B.; Fersht, Alan R.

    2008-01-01

    The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution...

  4. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  5. Identification of pyrazosulfuron-ethyl binding affinity and binding site subdomain IIA in human serum albumin by spectroscopic methods

    Ding, Fei; Liu, Wei; Zhang, Xi; Wu, Li-Jun; Zhang, Li; Sun, Ying

    2010-03-01

    Pyrazosulfuron-ethyl (PY) is a sulfonylurea herbicide developed by DuPont which has been widely used for weed control in cereals. The determination of PY binding affinity and binding site in human serum albumin (HSA) by spectroscopic methods is the subject of this work. From the fluorescence emission, circular dichroism and three-dimensional fluorescence results, the interaction of PY with HSA caused secondary structure changes in the protein. Fluorescence data demonstrated that the quenching of HSA fluorescence by PY was the result of the formation of HSA-PY complex at 1:1 molar ratio, a static mechanism was confirmed to lead to the fluorescence quenching. Hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results show that hydrophobic patches are the major sites for PY binding on HSA. The thermodynamic parameters Δ H° and Δ S° were calculated to be -36.32 kJ mol -1 and -35.91 J mol -1 K -1, which illustrated van der Waals forces and hydrogen bonds interactions were the dominant intermolecular force in stabilizing the complex. Also, site marker competitive experiments showed that the binding of PY to HSA took place primarily in subdomain IIA (Sudlow's site I). What presented in this paper binding research enriches our knowledge of the interaction between sulfonylurea herbicides and the physiologically important protein HSA.

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

    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

  7. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel.

    Joseph, Thomas T; Mincer, Joshua S

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  8. A biophysical model for analysis of transcription factor interaction and binding site arrangement from genome-wide binding data.

    Xin He

    Full Text Available BACKGROUND: How transcription factors (TFs interact with cis-regulatory sequences and interact with each other is a fundamental, but not well understood, aspect of gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational method to address this question, relying on the established biophysical principles. This method, STAP (sequence to affinity prediction, takes into account all combinations and configurations of strong and weak binding sites to analyze large scale transcription factor (TF-DNA binding data to discover cooperative interactions among TFs, infer sequence rules of interaction and predict TF target genes in new conditions with no TF-DNA binding data. The distinctions between STAP and other statistical approaches for analyzing cis-regulatory sequences include the utility of physical principles and the treatment of the DNA binding data as quantitative representation of binding strengths. Applying this method to the ChIP-seq data of 12 TFs in mouse embryonic stem (ES cells, we found that the strength of TF-DNA binding could be significantly modulated by cooperative interactions among TFs with adjacent binding sites. However, further analysis on five putatively interacting TF pairs suggests that such interactions may be relatively insensitive to the distance and orientation of binding sites. Testing a set of putative Nanog motifs, STAP showed that a novel Nanog motif could better explain the ChIP-seq data than previously published ones. We then experimentally tested and verified the new Nanog motif. A series of comparisons showed that STAP has more predictive power than several state-of-the-art methods for cis-regulatory sequence analysis. We took advantage of this power to study the evolution of TF-target relationship in Drosophila. By learning the TF-DNA interaction models from the ChIP-chip data of D. melanogaster (Mel and applying them to the genome of D. pseudoobscura (Pse, we found that only about half of the

  9. Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

    Crankshaw, D.; Gaspar, V.; Pliska, V. (McMaster Univ., Hamilton, Ontario, (Canada))

    1990-01-01

    The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.

  10. Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

    Zhuang, X; Wang, Q; Wang, B; Zhong, T; Cao, Y; Li, K; Yin, J

    2014-06-01

    The scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a predominant underground pest in the northern parts of China, and its larvae (grubs) cause great economic losses because of its wide range of host plants and covert habitats. Environmentally friendly strategies for controlling adults would have novel and broad potential applications. One potential pest management measure is the regulation of olfactory chemoreception to control target insect pests. In the process of olfactory recognition, odorant-binding proteins (OBPs) are believed to carry hydrophobic odorants from the environment to the surface of olfactory receptor neurons. To obtain a better understanding of the relationship between OBP structures and their ligands, homology modelling and molecular docking have been conducted on the interaction between HoblOBP1 and hexyl benzoate in the present study. Based on the results, site-directed mutagenesis and binding experiments were combined to describe the binding sites of HoblOBP1 and to explore its ligand-binding mechanism. After homology modelling of HoblOBP1, it was found that the three-dimensional structure of HoblOBP1 consists of six α-helices and three disulphide bridges that connect the helices, and the hydrophobic pockets are both composed of five helices. Based on the docking study, we found that van der Waals interactions and hydrophobic interactions are both important in the bonding between HoblOBP1 and hexyl benzoate. Intramolecular residues formed the hydrogen bonds in the C terminus of the protein and the bonds are crucial for the ligand-binding specificity. Finally, MET48, ILE80 and TYR111 are binding sites predicted for HoblOBP1. Using site-directed mutagenesis and fluorescence assays, it was found that ligands could not be recognized by mutant of Tyr111. A possible explanation is that the compound could not be recognized by the mutant, and remains in the binding cavity because of the loss of the intramolecular

  11. Impact of Binding Site Comparisons on Medicinal Chemistry and Rational Molecular Design.

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

    Modern rational drug design not only deals with the search for ligands binding to interesting and promising validated targets but also aims to identify the function and ligands of yet uncharacterized proteins having impact on different diseases. Additionally, it contributes to the design of inhibitors with distinct selectivity patterns and the prediction of possible off-target effects. The identification of similarities between binding sites of various proteins is a useful approach to cope with those challenges. The main scope of this perspective is to describe applications of different protein binding site comparison approaches to outline their applicability and impact on molecular design. The article deals with various substantial application domains and provides some outstanding examples to show how various binding site comparison methods can be applied to promote in silico drug design workflows. In addition, we will also briefly introduce the fundamental principles of different protein binding site comparison methods. PMID:27046190

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

    Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

    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. PMID:27318092

  13. An NMR-Based Structural Rationale for Contrasting Stoichiometry and Ligand Binding Site(s) in Fatty Acid-binding Proteins†

    He, Yan; Estephan, Rima; Yang, Xiaomin; Vela, Adriana; Wang, Hsin; Bernard, Cédric; Stark, Ruth E.

    2011-01-01

    Liver fatty acid-binding protein (LFABP) is a 14-kDa cytosolic polypeptide, differing from other family members in number of ligand binding sites, diversity of bound ligands, and transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional 1H-15N NMR signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without solving the protein-ligand complex structures...

  14. Evidence for two distinct binding sites for tau on microtubules

    Makrides, Victoria; Massie, Michelle R.; Feinstein, Stuart C.; Lew, John

    2004-01-01

    The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low- to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo exchange with the bulk medium readily, suggesting that the tau-microtubule interaction is essentially irreversible. Given this dilemma, we investigated the mechanism of interaction between tau and microtubules in kinetic detail. Stopped-flow kinetic analysis reveals moderate binding affinity between tau and preassembled microtubules and rapid dissociation/association kinetics. In contrast, when microtubules are generated by copolymerization of tubulin and tau, a distinct population of microtubule-bound tau is observed, the binding of which seems irreversible. We propose that reversible binding occurs between tau and the surface of preassembled microtubules, whereas irreversible binding results when tau is coassembled with tubulin into a tau-microtubule copolymer. Because the latter is expected to be physiologically relevant, its characterization is of central importance. PMID:15096589

  15. Differential modulation by cations of sigma and phencyclidine binding sites in rat brain

    The present investigation attempted to differentiate haloperidol-sensitive sigma sites (sigma H) from phencyclidine (PCP) binding sites in rat brain membranes. We studied the effects of several cations at physiologically relevant concentrations on the binding of radioligands selective for sigma H sites ([3H]haloperidol, [3H](+)3-PPP**), and [3H](+)SKF10,047, or for PCP sites ([3H]PCP and [3H]TCP). The PCP sites displayed a markedly greater sensitivity to cations than sigma H sites. This property was reflected by a greater extent of inhibition of the binding of PCP-selective relative to sigma H-selective ligands at a given cation concentration, as well as by lower IC50's and by steeper slopes of the cation dose-response curves. Divalent cations were approximately 100 times more potent than monovalent cations. All cations were inhibitory, except Sr2+ and Ba2+ which, at micromolar concentrations, enhanced PCP binding but not sigma H binding. Thus, PCP-selective sites appeared to be distinct from sigma H sites with regards to several aspects of cation modulation. This is consistent with the view that PCP and sigma H sites are distinct molecular entities. Further, the marked cation sensitivity of the PCP site is consistent with the current hypothesis according to which the PCP site is linked to the N-methyl-D-aspartate (NMDA) receptor-cation channel complex

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

    Mashiur Rahman; Farzana Prianka; Mohammad Shohel; Md. Abdul Mazid

    2014-01-01

    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 a...

  17. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    Long, Katherine S.; Vester, Birte

    2012-01-01

    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...... evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design...

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

    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.

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

    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.

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

    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 45Ca. All the calcium-binding sites are located in the NH2-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

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

    Bennick, A. (Univ. of Toronto, Ontario); McLaughlin, A.C.; Grey, A.A.; Madapallimattam, G.

    1981-05-25

    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 /sup 45/Ca. All the calcium-binding sites are located in the NH/sub 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 ..mu..M and are responsible for the binding of 2.6 mol of Ca/mol of peptide. The corresponding figures for Type II sites are 780 ..mu..M 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 ..mu..M. 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 ..beta..-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.

  2. Activation of brown adipose tissue mitochondrial GDP binding sites

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of [3H]-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time

  3. Activation of brown adipose tissue mitochondrial GDP binding sites

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  4. SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

    VANDERLINDEN, MPG; DEHAAN, L; DIDEBERG, O; KECK, W

    1994-01-01

    Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47),

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

    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...... bridging oxygen, which is part of either a water molecule or a hydroxide ion. Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions. By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined...

  6. Saturable triiodothyronine-binding sites in the pituitary nuclei of salmonid teleost fish

    High-affinity, limited-capacity, 3,5,3'-triiodo-L-thyronine (T3)-binding sites were established by in vitro saturation analysis in cell nuclei of the pituitary gland of arctic charr. The sites were extracted from the purified nuclei using 0.4 M NaCl and incubated with [125I]T3 in the presence of 0.2 M NaCl. T3 saturable binding attained equilibrium after 18-24 hr of incubation at 4 degrees. The association constant ranged from 6.7 to 20.1 liters.mol-1 x 10(9), indicating a T3 affinity greater than that for T3-binding sites in rainbow trout liver. The maximal binding capacity ranged from 0.93 to 2.05 10(-13) mol.mg DNA-1, representing a mean site abundance corresponding to 60% of that for nuclei from trout liver. Thyroxine (T4) completely displaced [125I]T3 in the pituitary nuclei of arctic charr and T3 completely displaced [125I]T4 in the pituitary nuclei of rainbow trout, suggesting that in salmonids both T4 and T3 bind to the same single class of sites. However, the site affinity for T4 was approximately 20-50x less than that for T3. The possible roles of these sites in pituitary function as well as their relationship to other nuclear T3-binding sites in salmonid fish are discussed

  7. Computational investigation of stoichiometric effects, binding site heterogeneities, and selectivities of molecularly imprinted polymers.

    Terracina, Jacob J; Bergkvist, Magnus; Sharfstein, Susan T

    2016-06-01

    A series of quantum mechanical (QM) computational optimizations of molecularly imprinted polymer (MIP) systems were used to determine optimal monomer-to-target ratios. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (3-7 molecules) and larger-scale models (15-35 molecules). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to quantify the heterogeneity of these sites. The more fully surrounded sites had greater binding energies. No discretization of binding modes was seen, furthering arguments for continuous affinity distribution models. Molecular mechanical (MM) docking was then used to measure the selectivities of the QM-optimized binding sites. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. Here we present a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Modeling schemes were designed such that no computing clusters or other specialized modeling equipment would be required. Improving the in silico analysis of MIP system properties will ultimately allow for the production of more sensitive and selective polymers. PMID:27207254

  8. Experimental and theoretical characterization of the high-affinity cation binding site of the purple membrane

    Pardo, Leonardo; Sepulcre Sánchez, Francesc; Cladera Cerdà, Josep Bartomeu; Duñach, Mireia; Labarta, A.; Tejada, J.; Padrós Morell, Esteve

    1998-01-01

    Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff b...

  9. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans

    Weyer, Sven; Pääbo, Svante

    2016-01-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  10. Quantitative autoradiography of [125I] apamin binding sites in the central nervous system.

    Janicki, P K; Horvath, E; Seibold, G; Habermann, E

    1984-01-01

    The binding sites for [125I] 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. PMID:6335967

  11. Quantitative autoradiography of [125I] apamin binding sites in the central nervous system

    The binding sites for [125I] 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. (author)

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

    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.

  13. Bacterial Surface Display of Metal-Binding Sites

    Kotrba, P.; Rulíšek, Lubomír; Ruml, T.

    Dordrecht: Springer, 2011 - (Kotrba, P.; Macková, M.; Macek, T.), s. 249-283 ISBN 978-94-007-0442-8 Grant ostatní: GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506 Keywords : bioremediation * biosorption * metal-binding peptide * cell-surface display Subject RIV: EI - Biotechnology ; Bionics

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

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

    2014-01-01

    Phosphorus is a ubiquitous element of the cell, which is found throughout numerous key molecules related to cell structure, energy and information storage and transfer, and a diverse array of other cellular functions. In this work, we adopt an approach often used for characterizing metal binding ...

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

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

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L; Shi, Lei; Gracia, Luis; Raniszewska, Klaudia; Newman, Amy Hauck; Javitch, Jonathan A; Weinstein, Harel; Gether, Ulrik; Løland, Claus Juul

    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...

  17. Internal binding sites for MSH: Analyses in wild-type and variant Cloudman melanoma cells

    Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient and centrifuged; fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: (1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. (2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. (3) Most of the internal binding sites were not as dense as fully melanized melanosomes. (4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. (5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) (6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone

  18. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

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

    In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125I-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)

  20. Current Understanding of the Binding Sites, Capacity, Affinity, and Biological Significance of Metals in Melanin

    Hong, Lian; Simon, John D.

    2007-01-01

    Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of u...

  1. In Silico Investigation of the Neurotensin Receptor 1 Binding Site

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W.;

    2016-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurol. disorders and the promotion of cancer cells. Recently, a high-resoln. x-ray crystal...... structure of NTSR1 in complex with NTS8-13 has been detd., providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small mol. antagonist has previously been used extensively as a tool compd. to study NTSR1 receptor signaling properties. To investigate...... the binding mode of SR48692 and other small mol. compds. to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were obsd., despite...

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

    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.

  3. Evidence for two distinct binding sites for tau on microtubules

    Makrides, Victoria; Massie, Michelle R.; Feinstein, Stuart C.; Lew, John

    2004-01-01

    The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low- to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo ex...

  4. Microbes Bind Complement Inhibitor Factor H via a Common Site

    Meri, T.; Amdahl, H.; Lehtinen, M. J.; Hyvärinen, S.; McDowell, J.V.; Bhattacharjee, A.; Meri, S.; Marconi, R.; Goldman, A; Jokiranta, T. S.

    2013-01-01

    To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic ...

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

    Ramos, Ana P.; Goncalves, Rogeria R.; Serra, Osvaldo A. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil); Zaniquelli, Maria Elisabete D. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil)], E-mail: medzaniquelli@ffclrp.usp.br; Wong, Kenneth [Laboratorio de Fisico-Quimica, Centro de Pesquisas de Paulinia, Rhodia Brasil, Paulinia, Sao Paulo (Brazil)

    2007-12-15

    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{sup 3+}/Na{sup +} or K{sup +} 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.

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

    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: Eu3+/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

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

    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.

  8. Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

    Chun-Hai Dong; Wei-Ping Tang; Jia-Yao Liu

    2013-01-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin,and is directly involved in the depolymerization of actin filaments.To better understand the actin binding sites of the Arabidopsis thaliana L.AtADF1,we generated mutants of AtADF1 and investigated their functions in vitro and in vivo.Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G-and F-actin binding.The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A,R137/A) form another actin binding site that is important for F-actin binding.Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L.plants overexpressing these mutants,we analyzed how these mutant proteins regulate actin organization and affect seedling growth.Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional,unless the affinity foractin monomers is also affected.The G-actin binding activity of the ADF plays an essential role in actin binding,depolymerization of actin polymers,and therefore in the control of actin organization.

  9. Quantitative autoradiography of 3H-nomifensine binding sites in rat brain

    The distribution of 3H-nomifensine binding sites in the rat brain has been studied by quantitative autoradiography. The binding of 3H-nomifensine to caudate putamen sections was saturable, specific, of a highly affinity (Kd = 56 nM) and sodium-dependent. The dopamine uptake inhibitors benztropine, nomifensine, cocaine, bupropion and amfonelic acid were the most potent competitors of 3H-nomifensine binding to striatal sections. The highest levels of (benztropine-displaceable) 3H-nomifensine binding sites were found in the caudate-putamen, the olfactory tubercle and the nucleus accumbens. 6-Hydroxy-dopamine-induced lesion of the ascending dopaminergic bundle resulted in a marked decrease in the 3H-ligand binding in these areas. Moderately high concentrations of the 3H-ligand were observed in the bed nucleus of the stria terminalis, the anteroventral thalamic nucleus, the cingulate cortex, the lateral septum, the hippocampus, the amygdala, the zona incerta and some hypothalamic nuclei. There were low levels of binding sites in the habenula, the dorsolateral geniculate body, the substantia nigra, the ventral tegmental area and the periaqueductal gray matter. These autoradiographic data are consistent with the hypothesis that 3H-nomifensine binds primarily to the presynaptic uptake site for dopamine but also labels the norepinephrine uptake site. 33 references, 2 figures, 1 table

  10. Quantitative autoradiography of /sup 3/H-nomifensine binding sites in rat brain

    Scatton, B.; Dubois, A.; Dubocovich, M.L.; Zahniser, N.R.; Fage, D.

    1985-03-04

    The distribution of /sup 3/H-nomifensine binding sites in the rat brain has been studied by quantitative autoradiography. The binding of /sup 3/H-nomifensine to caudate putamen sections was saturable, specific, of a highly affinity (Kd = 56 nM) and sodium-dependent. The dopamine uptake inhibitors benztropine, nomifensine, cocaine, bupropion and amfonelic acid were the most potent competitors of /sup 3/H-nomifensine binding to striatal sections. The highest levels of (benztropine-displaceable) /sup 3/H-nomifensine binding sites were found in the caudate-putamen, the olfactory tubercle and the nucleus accumbens. 6-Hydroxy-dopamine-induced lesion of the ascending dopaminergic bundle resulted in a marked decrease in the /sup 3/H-ligand binding in these areas. Moderately high concentrations of the /sup 3/H-ligand were observed in the bed nucleus of the stria terminalis, the anteroventral thalamic nucleus, the cingulate cortex, the lateral septum, the hippocampus, the amygdala, the zona incerta and some hypothalamic nuclei. There were low levels of binding sites in the habenula, the dorsolateral geniculate body, the substantia nigra, the ventral tegmental area and the periaqueductal gray matter. These autoradiographic data are consistent with the hypothesis that /sup 3/H-nomifensine binds primarily to the presynaptic uptake site for dopamine but also labels the norepinephrine uptake site. 33 references, 2 figures, 1 table.

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

    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 Na125 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)

  12. Protective Action of Resveratrol in Human Skin: Possible Involvement of Specific Receptor Binding Sites

    Stéphane Bastianetto; Yvan Dumont; Albert Duranton; Freya Vercauteren; Lionel Breton; Rémi Quirion

    2010-01-01

    BACKGROUND: Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238-245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol. METHODS A...

  13. DETERMINANTS OF LIGAND BINDING AFFINITY AND COOPERATIVITY AT THE GLUT1 ENDOFACIAL SITE

    Robichaud, Trista; Appleyard, Antony N.; Herbert, Richard B.; Henderson, Peter J. F.; Carruthers, Anthony

    2011-01-01

    Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport (Cloherty, E. K., Levine, K. B., & Carruthers, A. (2001). The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry, 40(51), 15549–15561). This result is consistent with t...

  14. Partial enterectomy decreases somatostatin-binding sites in residual intestine of rabbits

    Colás Escudero, Begoña; Bodega Magro, Guillermo; Sanz, M.; Prieto Villapún, Juan Carlos; Arilla Ferreiro, Eduardo

    1988-01-01

    Three weeks after partial enterectomy in the rabbit there was an increased somatostatin concentration and a decreased number of somatostatin-binding sites (without changes in the corresponding affinity values) in the cytosol of the residual intestinal tissue, except in the terminal ileum and the colon. Five weeks after surgery both the somatostatin concentration and the number of somatostatin-binding sites returned towards control values. These results suggest that an increase in bowel ...

  15. Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system

  16. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

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

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.; Robey, F.A.

    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 and...... of 25 mu M, while the IC50 of AP-(27-38)-peptide and AP-(33-38)-peptide are 10 mu M and 2 mu M, respectively, The understanding of the structure and function of active AP peptides will be useful for development of amyloid-targeted diagnostics and therapeutics....

  18. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    Kovalevskaya, N.V.; Bokhovchuk, F.M.; Vuister, G.W.

    2012-01-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-t

  19. Ca2+ binding sites in calmodulin and troponin C alter interhelical angle movements.

    Goto, Kunihiko; Toyama, Akira; Takeuchi, Hideo; Takayama, Kazuyoshi; Saito, Tsutomu; Iwamoto, Masatoshi; Yeh, Jay Z; Narahashi, Toshio

    2004-03-12

    Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism. PMID:15013750

  20. Localization of binding sites for purified Escherichia coli P fimbriae in the human kidney.

    Korhonen, T K; Virkola, R; Holthöfer, H

    1986-01-01

    Binding sites in the human kidney for purified P fimbriae of pyelonephritogenic Escherichia coli were determined. The purified KS71A (F7(1)) fimbriae bound only to epithelial elements of the kidney, i.e., to the apical aspect of proximal and distal tubular cells, as well as to the apical and cytoplasmic sites of collecting ducts. In addition, binding was seen at the vascular endothelium throughout the kidney and at the parietal epithelium of the glomeruli. The binding was specifically inhibit...

  1. Solution measurement of DNA curvature in papillomavirus E2 binding sites

    Zimmerman, Jeff M.; Maher, L. James

    2003-01-01

    ‘Indirect readout’ refers to the proposal that proteins can recognize the intrinsic three-dimensional shape or flexibility of a DNA binding sequence apart from direct protein contact with DNA base pairs. The differing affinities of human papillomavirus (HPV) E2 proteins for different E2 binding sites have been proposed to reflect indirect readout. DNA bending has been observed in X-ray structures of E2 protein–DNA complexes. X-ray structures of three different E2 DNA binding sites revealed di...

  2. Molecular simulations of Taxawallin I inside classical taxol binding site of β-tubulin.

    Khan, Inamullah; Nisar, Muhammad; Ahmad, Manzoor; Shah, Hamidullah; Iqbal, Zafar; Saeed, Muhammad; Halimi, Syed Muhammad Ashhad; Kaleem, Waqar Ahmad; Qayum, Mughal; Aman, Akhter; Abdullah, Syed Muhammad

    2011-03-01

    A new taxoid Taxawallin I (1) along with two known taxoids (2-3) were isolated from methanolic bark extract of Taxus wallichiana Zucc. Structural characterization was confirmed by mass and NMR spectral techniques. Taxawallin I exhibited significant in-vitro anticancer activity against HepG2, A498, NCI-H226 and MDR 2780AD cancer lines. Tubulin binding assay was performed to assess its tubulin binding activity. Molecular docking analysis was performed to study the potential binding mode inside the taxol binding site of β-tubulin. PMID:20969934

  3. Crystallographic location of two Zn2+ binding sites in the avian cytochrome bc1 complex

    The chicken mitochondrial ubiquinol cytochrome c oxidoreductase (bc1 complex) is inhibited by Zn2+ ions, but with higher Ki (∼3 microm) than the corresponding bovine enzyme. When equilibrated with mother liquor containing 200 mM ZnCl2 for 7 days, the crystalline chicken bc1 complex specifically binds Zn2+ at 4 sites representing two sites on each monomer in the dimer. These two sites are close to the stigmatellin-binding site, taken to be center Qo of the Q-cycle mechanism, and are candidates for the inhibitory site. One binding site is actually in the hydrophobic channel between the Qo site and the bulk lipid phase, and may interfere with quinone binding. The other is in a hydrophilic area between cytochromes b and c1, and might interfere with the egress of protons from the Qo site to the intermembrane aqueous medium. No zinc was bound near the putative proteolytic active site of subunits 1 and 2 (homologous to mitochondrial processing peptidase) under these conditions

  4. Identification of neomycin B-binding site in T box antiterminator model RNA.

    Anupam, Rajaneesh; Denapoli, Leyna; Muchenditsi, Abigael; Hines, Jennifer V

    2008-04-15

    The T box transcription antitermination mechanism regulates the expression of unique genes in many Gram-positive bacteria by responding, in a magnesium-dependent manner, to uncharged cognate tRNA base pairing with an antiterminator RNA element and other regions of the 5'-untranslated region. Model T box antiterminator RNA is known to bind aminoglycosides, ligands that typically bind RNA in divalent metal ion-binding sites. In this study, enzymatic footprinting and spectroscopic assays were used to identify and characterize the binding site of neomycin B to an antiterminator model RNA. Neomycin B binds the antiterminator bulge nucleotides in an electrostatic-dependent manner and displaces 3-4 monovalent cations, indicating that the antiterminator likely contains a divalent metal ion-binding site. Neomycin B facilitates rather than inhibits tRNA binding indicating that bulge-targeted inhibitors that bind the antiterminator via non-electrostatic interactions may be the more optimal candidates for antiterminator-targeted ligand design. PMID:18329274

  5. In vivo labelling in several rat tissues of 'peripheral type' benzodiazepine binding sites

    'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of [3H]PK 11195 and [3H]RO5-4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 > PK 11211 > RO5-4864 > diazepam > dipyridamole > clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However, some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make [3H]PK 11195 the most suitable ligand for this kind of studies. (Auth.)

  6. Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa

    Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of 125I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites

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

    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 Kd between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31P and 1H 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 Kd 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

  8. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).

    Van Nostrand, Eric L; Pratt, Gabriel A; Shishkin, Alexander A; Gelboin-Burkhart, Chelsea; Fang, Mark Y; Sundararaman, Balaji; Blue, Steven M; Nguyen, Thai B; Surka, Christine; Elkins, Keri; Stanton, Rebecca; Rigo, Frank; Guttman, Mitchell; Yeo, Gene W

    2016-06-01

    As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity. PMID:27018577

  9. Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

    Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

    2008-11-21

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS. PMID:18820259

  10. DNA-MATRIX: a tool for constructing transcription factor binding sites Weight matrix

    Chandra Prakash Singh,

    2009-12-01

    Full Text Available Despite considerable effort to date, DNA transcription factor binding sites prediction in whole genome remains a challenge for the researchers. Currently the genome wide transcription factor binding sites prediction tools required either direct pattern sequence or weight matrix. Although there are known transcription factor binding sites pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a DNA-MATRIX tool for searching putative transcription factor binding sites in genomic sequences. DNA-MATRIX uses the simple heuristic approach for weight matrix construction, which can be transformed into different formats as per the requirement of researcher’s for further genome wide prediction and therefore provides the possibility to identify the conserved known DNA binding sites in the coregulated genes and also to search for a great variety of different regulatory binding patterns. The user may construct and save specific weight or frequency matrices in different formats derived through user selected set of known motif sequences.

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

    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.

  12. Subtilases and metal binding - the weak binding site of sutilisins revisited

    Dohnálek, Jan; MacAuley, K.; Brzozowski, A. M.; Borchert, T. V.; Wilson, K. S.

    2007-01-01

    Roč. 14, č. 1 (2007), s. 28. ISSN 1211-5894. [Discussions in Structural Molecular Biology and Bioinformatics /6./. 29.03.2007-31.03.2007, Nové Hrady] R&D Projects: GA MŠk 1K05008 Institutional research plan: CEZ:AV0Z40500505 Keywords : subtilisin-like proteases * metal binding Subject RIV: CF - Physical ; Theoretical Chemistry

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

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-01-01

    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. PMID:27092480

  14. Identification of candidate transcription factor binding sites in the cattle genome

    A resource that provides candidate transcription factor binding sites does not currently exist for cattle. Such data is necessary, as predicted sites may serve as excellent starting locations for future 'omics studies to develop transcriptional regulation hypotheses. In order to generate this resour...

  15. Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

    125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7

  16. Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

    Frost, S.J.; Raja, R.H.; Weigel, P.H. (Univ. of Texas Medical Branch, Galveston (USA))

    1990-11-13

    125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4{degrees}C increased greater than 10-fold at pH 5.0 as compared to pH 7.

  17. Autoradiographic localization of (/sup 125/I)-angiotensin II binding sites in the rat adrenal gland

    Healy, D.P.; Maciejewski, A.R.; Printz, M.P.

    1985-03-01

    To gain greater insight into sites of action of circulating angiotensin II (Ang II) within the adrenal, we have localized the (/sup 125/I)-Ang II binding site using in vitro autoradiography. Autoradiograms were generated either by apposition of isotope-sensitive film or with emulsion-coated coverslips to slide-mounted adrenal sections labeled in vitro with 1.0 nM (/sup 125/I)-Ang II. Analysis of the autoradiograms showed that Ang II binding sites were concentrated in a thin band in the outer cortex (over the cells of the zona glomerulosa) and in the adrenal medulla, which at higher power was seen as dense patches. Few sites were evident in the inner cortex. The existence of Ang II binding sites in the adrenal medulla was confirmed by conventional homogenate binding techniques which revealed a single class of high affinity Ang II binding site (K/sub d/ . 0.7nM, B/sub max/ . 168.7 fmol/mg). These results suggest that the adrenal medulla may be a target for direct receptor-mediated actions of Ang II.

  18. Regression applied to protein binding site prediction and comparison with classification

    Gala Jean-Luc

    2009-09-01

    Full Text Available Abstract Background The structural genomics centers provide hundreds of protein structures of unknown function. Therefore, developing methods enabling the determination of a protein function automatically is imperative. The determination of a protein function can be achieved by studying the network of its physical interactions. In this context, identifying a potential binding site between proteins is of primary interest. In the literature, methods for predicting a potential binding site location generally are based on classification tools. The aim of this paper is to show that regression tools are more efficient than classification tools for patches based binding site predictors. For this purpose, we developed a patches based binding site localization method usable with either regression or classification tools. Results We compared predictive performances of regression tools with performances of machine learning classifiers. Using leave-one-out cross-validation, we showed that regression tools provide better predictions than classification ones. Among regression tools, Multilayer Perceptron ranked highest in the quality of predictions. We compared also the predictive performance of our patches based method using Multilayer Perceptron with the performance of three other methods usable through a web server. Our method performed similarly to the other methods. Conclusion Regression is more efficient than classification when applied to our binding site localization method. When it is possible, using regression instead of classification for other existing binding site predictors will probably improve results. Furthermore, the method presented in this work is flexible because the size of the predicted binding site is adjustable. This adaptability is useful when either false positive or negative rates have to be limited.

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

    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.

  20. Transcriptional stimulation via SC site of Bombyx sericin-1 gene through an interaction with a DNA binding protein SGF-3.

    Matsuno, K.; Takiya, S; Hui, C C; Suzuki, T.; Fukuta, M.; Ueno, K.; Suzuki, Y

    1990-01-01

    Three protein binding sites have been identified in the upstream region of the sericin-1 gene. Two of them, SA and SC sites, have been known as putative cis-acting elements. Using synthetic oligonucleotides of these binding sites, it was found that silk gland factor-1 (SGF-1) binds to the SA site, and silk gland factor-3 (SGF-3) binds to the SC site but not to a mutated SC site, SCM. Tissue distribution of the two factors was different. SGF-3 is present abundantly in the middle silk gland (MS...

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

    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.

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

    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

  3. Putative hAPN receptor binding sites in SARS_CoV spike protein

    YUXiao-Jing; LUOCheng; LinJian-Cheng; HAOPei; HEYou-Yu; GUOZong-Ming; QINLei; SUJiong; LIUBo-Shu; HUANGYin; NANPeng; LIChuan-Song; XIONGBin; LUOXiao-Min; ZHAOGuo-Ping; PEIGang; CHENKai-Xian; SHENXu; SHENJian-Hua; ZOUJian-Ping; HEWei-Zhong; SHITie-Liu; ZHONGYang; JIANGHua-Liang; LIYi-Xue

    2003-01-01

    AIM:To obtain the information of ligand-receptor binding between thd S protein of SARS_CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites sites in the S protein of SARS_CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS_CoV in validating the bioinformatics predictions. RESULTS:Possible binding sites in the SARS_CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS_CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION:CD13 may be a possible receptor of the SARS_CoV S protein which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

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

    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.

  5. A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

    Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 X g particulate fraction. Binding of [8-14C] - benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at ph 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (Ksub(d)) of 33+- 6 n.M. The number of binding sites found was 1,100+-120 fmol g-1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [i R]Z, [9 R] A. (author)

  6. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

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

    The mechanism by which delta9 tetrahydrocannabinol (delta9THC), 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-delta8THC (TMA) is a positively charged analog of delta-8THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [3H]-5'-trimethylammonium-delta-8THC ([3H]TMA) to rat neuronal membranes. [3H]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 [3H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

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

    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.

  9. Impact of disruption of secondary binding site S2 on dopamine transporter function.

    Zhen, Juan; Reith, Maarten E A

    2016-09-01

    The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm. PMID

  10. A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins.

    He, Yan; Estephan, Rima; Yang, Xiaomin; Vela, Adriana; Wang, Hsin; Bernard, Cédric; Stark, Ruth E

    2011-03-01

    Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family. PMID:21226535

  11. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue.

    Chen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E; Brighty, Gabriel J; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S; Hulce, Jonathan J; Cravatt, Benjamin F; Saez, Enrique; Powers, Evan T; Wilson, Ian A; Sharpless, K Barry; Kelly, Jeffery W

    2016-06-15

    Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function. PMID:27191344

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

    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.

  13. Isolation of active site and antibody-binding fragments of human erythrocyte transglutaminase

    Catalytically active human erythrocyte transglutaminase (TGase) was purified using an immunoaffinity column prepared from a monoclonal antibody to guinea pig liver TGase. The enzyme activity was completely inhibited by incorporation of iodo[14C]acetamide to the level of 1 mole per 1 mole of TGase. The 14C-labeled TGase was digested with cyanogen bromide, subjected to HPLC, and four pure peptides were isolated with molecular weights ranging from 3-22 KDa. Only one of the peptides was radiolabeled and characterized as an active site peptide of 10 KDa. Another peptide of 18 KDa was identified as a monoclonal antibody-binding domain of TGase. Although the active site and the antibody-binding domain were present on different cyanogen bromide fragments, the mouse anti-TGase inhibited 100% of TGase activity. The results suggest that the antibody-binding site is not located on the enzyme active site sequence, but that the three dimensional space configuration of the antigen-antibody complex hinders substrate binding to the active site. The radiolabeled active site cysteine residue was not found in the N-terminal 21 amino acids of the 10 KDa peptide. Additional fragments of the active site peptide are currently being analyzed

  14. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, 3H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a 3H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of 3H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A4, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each

  15. Mercury Binding Sites in Thiol-Functionalized Mesostructured Silica

    Thiol-functionalized mesostructured silica with anhydrous compositions of (SiO2)1-x(LSiO1.5)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(SBut)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.

  16. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  17. Comparison of Different Ranking Methods in Protein-Ligand Binding Site Prediction

    Gao, Jun; Liu, Qi; Kang, Hong; Cao, Zhiwei; Zhu, Ruixin

    2012-01-01

    In recent years, although many ligand-binding site prediction methods have been developed, there has still been a great demand to improve the prediction accuracy and compare different prediction algorithms to evaluate their performances. In this work, in order to improve the performance of the protein-ligand binding site prediction method presented in our former study, a comparison of different binding site ranking lists was studied. Four kinds of properties, i.e., pocket size, distance from the protein centroid, sequence conservation and the number of hydrophobic residues, have been chosen as the corresponding ranking criterion respectively. Our studies show that the sequence conservation information helps to rank the real pockets with the most successful accuracy compared to others. At the same time, the pocket size and the distance of binding site from the protein centroid are also found to be helpful. In addition, a multi-view ranking aggregation method, which combines the information among those four properties, was further applied in our study. The results show that a better performance can be achieved by the aggregation of the complementary properties in the prediction of ligand-binding sites. PMID:22942732

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

    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.

  19. Effects of sodium on cell surface and intracellular TH-naloxone binding sites

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

    1987-07-27

    The binding of the opiate antagonist TH-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, TH-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.

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

    The binding of the opiate antagonist 3H-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, 3H-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

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

    H. Lortat-Jacob

    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.

  2. Discovering structural motifs using a structural alphabet: Application to magnesium-binding sites

    Lim Carmay

    2007-03-01

    Full Text Available Abstract Background For many metalloproteins, sequence motifs characteristic of metal-binding sites have not been found or are so short that they would not be expected to be metal-specific. Striking examples of such metalloproteins are those containing Mg2+, one of the most versatile metal cofactors in cellular biochemistry. Even when Mg2+-proteins share insufficient sequence homology to identify Mg2+-specific sequence motifs, they may still share similarity in the Mg2+-binding site structure. However, no structural motifs characteristic of Mg2+-binding sites have been reported. Thus, our aims are (i to develop a general method for discovering structural patterns/motifs characteristic of ligand-binding sites, given the 3D protein structures, and (ii to apply it to Mg2+-proteins sharing 2+-structural motifs are identified as recurring structural patterns. Results The structural alphabet-based motif discovery method has revealed the structural preference of Mg2+-binding sites for certain local/secondary structures: compared to all residues in the Mg2+-proteins, both first and second-shell Mg2+-ligands prefer loops to helices. Even when the Mg2+-proteins share no significant sequence homology, some of them share a similar Mg2+-binding site structure: 4 Mg2+-structural motifs, comprising 21% of the binding sites, were found. In particular, one of the Mg2+-structural motifs found maps to a specific functional group, namely, hydrolases. Furthermore, 2 of the motifs were not found in non metalloproteins or in Ca2+-binding proteins. The structural motifs discovered thus capture some essential biochemical and/or evolutionary properties, and hence may be useful for discovering proteins where Mg2+ plays an important biological role. Conclusion The structural motif discovery method presented herein is general and can be applied to any set of proteins with known 3D structures. This new method is timely considering the increasing number of structures for

  3. Outer membrane protein binding sites of complement component 3 during opsonization of Haemophilus influenzae.

    Hetherington, S V; Patrick, C C; Hansen, E J

    1993-01-01

    Complement component 3 (C3) binding to Haemophilus influenzae type b (Hib) is an important step in host defense against invasive disease, but the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sites for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose. Immunobl...

  4. A Novel Alignment-Free Method for Comparing Transcription Factor Binding Site Motifs

    Minli Xu; Zhengchang Su

    2010-01-01

    BACKGROUND: Transcription factor binding site (TFBS) motifs can be accurately represented by position frequency matrices (PFM) or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between ...

  5. Novel Phosphotidylinositol 4,5-Bisphosphate Binding Sites on Focal Adhesion Kinase

    Jun Feng; Blake Mertz

    2015-01-01

    Focal adhesion kinase (FAK) is a protein tyrosine kinase that is ubiquitously expressed, recruited to focal adhesions, and engages in a variety of cellular signaling pathways. Diverse cellular responses, such as cell migration, proliferation, and survival, are regulated by FAK. Prior to activation, FAK adopts an autoinhibited conformation in which the FERM domain binds the kinase domain, blocking access to the activation loop and substrate binding site. Activation of FAK occurs through confor...

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

    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....

  7. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;

    2007-01-01

    have dramatically increased potencies, more than three orders of magnitude higher than the corresponding monomers. Dimer (R,R)-2a was cocrystallized with the GluR2-S1S2J construct, and an X-ray crystallographic analysis showed (R,R)-2a to bridge two identical binding pockets on two neighboring GluR2...... subunits. Thus, this is biostructural evidence of a homomeric dimer bridging two identical receptor-binding sites....

  8. Functional identification of catalytic metal ion binding sites within RNA.

    James L Hougland

    2005-09-01

    Full Text Available The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s. In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

  9. A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

    Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise; Sander, Tommy; Balle, Thomas

    2013-01-01

    -gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode...... similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues a1R66, b2T202, a1T129, b2E155, b2Y205 and the backbone of b2S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy...

  10. Molecular docking characterizes substrate-binding sites and efflux modulation mechanisms within P-glycoprotein.

    Ferreira, Ricardo J; Ferreira, Maria-José U; dos Santos, Daniel J V A

    2013-07-22

    P-Glycoprotein (Pgp) is one of the best characterized ABC transporters, often involved in the multidrug-resistance phenotype overexpressed by several cancer cell lines. Experimental studies contributed to important knowledge concerning substrate polyspecificity, efflux mechanism, and drug-binding sites. This information is, however, scattered through different perspectives, not existing a unifying model for the knowledge available for this transporter. Using a previously refined structure of murine Pgp, three putative drug-binding sites were hereby characterized by means of molecular docking. The modulator site (M-site) is characterized by cross interactions between both Pgp halves herein defined for the first time, having an important role in impairing conformational changes leading to substrate efflux. Two other binding sites, located next to the inner leaflet of the lipid bilayer, were identified as the substrate-binding H and R sites by matching docking and experimental results. A new classification model with the ability to discriminate substrates from modulators is also proposed, integrating a vast number of theoretical and experimental data. PMID:23802684

  11. Characterization of specific binding sites for [3H](d)-N-allylnormetazocine in rat brain membranes

    Binding of [3H](d)-N-allylnormetazocine ([3H](d)-NANM) to rat brain membranes is stereospecific, reversible, and saturable (Bmax . 260 fmol/mg of protein) and manifests moderately high affinity (Kd . 20 nM). The rank order of potency among opioidbenzomorphans and phencyclidine (PCP) analogs for competition for [3H](d)-NANM-binding sites is as follows: (d)-NANM . PCP-3-OH greater than (d)-cyclazocine greater than N-ethylphenylcyclohexylamine greater than PCP greater than (l)-cyclazocine . dextrorphan greater than (d/l)-ethylketocyclazocine greater than (d/l)-bremazocine greater than (1)-NANM greater than 1-phenylcyclohexylamine greater than levorphanol. Other opioid ligands, relatively selective for each of the types of opioid binding sites other than sigma, such as morphine (mu), H-Tyr-D-Ala(Me)Phe-NH-CH2-OH (mu), D-Ala2-D-Leu5-enkephalin (delta), tifluadom (kappa), and U 50488 (kappa) as well as etorphine and naloxone were all unable to compete with [3H](d)-NANM for specific binding even at a concentration of 1 microM. Regional distribution studies of [3H](d)-NANM-binding sites show high density in the hippocampus, thalamus, hypothalamus, and amygdala and low density in cerebellum and nonfrontal neocortex membranes of the rat brain. These binding sites are very sensitive to protein-modifying enzymes and reagents such as trypsin and N-ethylmaleimide and to heat denaturation. These results provide direct biochemical evidence for the existence of distinct (d)-NANM-binding sites in rat brain

  12. Rational design of a protein that binds integrin αvβ3 outside the ligand binding site

    Turaga, Ravi Chakra; Yin, Lu; Yang, Jenny J.; Lee, Hsiauwei; Ivanov, Ivaylo; Yan, Chunli; Yang, Hua; Grossniklaus, Hans E.; Wang, Siming; Ma, Cheng; Sun, Li; Liu, Zhi-Ren

    2016-01-01

    Integrin αvβ3 expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin αvβ3 outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3. ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature. Toxicity analyses demonstrate that ProAgio is not toxic to mice. Our study reports a new integrin-targeting agent with a unique mechanism of action, and provides a template for the development of integrin-targeting therapeutics. PMID:27241473

  13. Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes.

    Sandhya, B; Hegde, Ashwini H; Kalanur, Shankara S; Katrahalli, Umesha; Seetharamappa, J

    2011-04-01

    The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the Förster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP. PMID:21215548

  14. Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain

    The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [3H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation.The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  15. Wnts grasp the WIF domain of Wnt Inhibitory Factor 1 at two distinct binding sites.

    Kerekes, Krisztina; Bányai, László; Patthy, László

    2015-10-01

    Wnts have a structure resembling a hand with "thumb" and "index" fingers that grasp the cysteine rich domains of Frizzled receptors at two distinct binding sites. In the present work we show that the WIF domain of Wnt Inhibitory Factor 1 is also bound by Wnts at two sites. Using C-terminal domains of Wnt5a and Wnt7a and arginine-scanning mutagenesis of the WIF domain we demonstrate that, whereas the N-terminal, lipid-modified "thumb" of Wnts interacts with the alkyl-binding site of the WIF domain, the C-terminal domain of Wnts (Wnt-CTD) binds to a surface on the opposite side of the WIF domain. PMID:26342861

  16. Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in vitro

    Cooney, Michael; Czernuszewicz, Graznya; Postel, Edith H.; Flint, S. Jane; Hogan, Michael E.

    1988-07-01

    A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

  17. PREDICTION OF ANTIGENIC AND BINDING SITES OF NEUROTOXIN 23 OF SCORPION (LYCHASMUCRONACTUS SP.

    Bharati K Thosare

    2015-07-01

    Full Text Available Identification of antigenic and binding site of protein is highly desirable for the design of vaccines and immunodiagnostics. The present exercise deals with a prediction of antigenic as well as binding sites of neurotoxin 23 of Lychasmucronactus. This species of scorpion having diverse molecules of toxic peptide, the peptide neurotoxin 23 is 96 amino acids long of which 23 to 96 specifically code for neurotoxin. The total of 27 such different ligand binding residue were identified by ConSurf and Raptor X server. The web tool Ellipro which implements Modeller and Jmol viewer, predicted and visualized the linear and discontinuous antibody epitopes ofneurotoxin 23 protein sequence.Thus the information discussed here provides a clue for understanding antigenic site and molecular function of neurotoxin 23.

  18. Ivermectin binding sites in human and invertebrate Cys-loop receptors.

    Lynagh, Timothy; Lynch, Joseph W

    2012-08-01

    Ivermectin is a gold standard antiparasitic drug that has been used successfully to treat billions of humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular 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 for a wide variety of human neurological disorders. PMID:22677714

  19. Receptor binding site-deleted foot-and-mouth disease (FMD) virus protects cattle from FMD.

    McKenna, T S; Lubroth, J; Rieder, E; Baxt, B; Mason, P W

    1995-01-01

    Binding of foot-and-mouth disease virus (FMDV) to cells requires an arginine-glycine-aspartic acid (RGD) sequence in the capsid protein VP1. We have genetically engineered an FMDV in which these three amino acids have been deleted, producing a virus particle which is unable to bind to cells. Cattle vaccinated with these receptor binding site-deleted virions were protected from disease when challenged with a virulent virus, demonstrating that these RGD-deleted viruses could serve as the basis ...

  20. Sodium-dependent reorganization of the sugar-binding site of SGLT1

    Hirayama, Bruce A; Loo, Donald D F; Díez-Sampedro, Ana;

    2007-01-01

    The sodium-dependent glucose cotransporter SGLT1 undergoes a series of voltage- and ligand-induced conformational changes that underlie the cotransport mechanism. In this study we describe how the binding of external Na changes the conformation of the sugar-binding domain, exposing residues that...... involved in transport. Arranging the four TMHs to account for Na-dependent accessibility and potential for sugar interaction allows us to propose a testable model for the SGLT1 sugar binding site. Udgivelsesdato: 2007-Nov-20...

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

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

    2010-09-28

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

  2. Disruption of NAD~+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    Manali; Phadke; Natalia; Krynetskaia; Anurag; Mishra; Carlos; Barrero; Salim; Merali; Scott; A; Gothe; Evgeny; Krynetskiy

    2015-01-01

    AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.

  3. Computational prediction of cAMP receptor protein (CRP) binding sites in cyanobacterial genomes

    Xu, Minli; Su, Zhengchang

    2009-01-01

    Background Cyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The loss of CRPs in these species

  4. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

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

    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

  6. Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators

    Karapetyan, Sargis; Buchler, Nicolas E.

    2015-12-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

  7. Identification of Covalent Binding Sites Targeting Cysteines Based on Computational Approaches.

    Zhang, Yanmin; Zhang, Danfeng; Tian, Haozhong; Jiao, Yu; Shi, Zhihao; Ran, Ting; Liu, Haichun; Lu, Shuai; Xu, Anyang; Qiao, Xin; Pan, Jing; Yin, Lingfeng; Zhou, Weineng; Lu, Tao; Chen, Yadong

    2016-09-01

    Covalent drugs have attracted increasing attention in recent years due to good inhibitory activity and selectivity. Targeting noncatalytic cysteines with irreversible inhibitors is a powerful approach for enhancing pharmacological potency and selectivity because cysteines can form covalent bonds with inhibitors through their nucleophilic thiol groups. However, most human kinases have multiple noncatalytic cysteines within the active site; to accurately predict which cysteine is most likely to form covalent bonds is of great importance but remains a challenge when designing irreversible inhibitors. In this work, FTMap was first applied to check its ability in predicting covalent binding site defined as the region where covalent bonds are formed between cysteines and irreversible inhibitors. Results show that it has excellent performance in detecting the hot spots within the binding pocket, and its hydrogen bond interaction frequency analysis could give us some interesting instructions for identification of covalent binding cysteines. Furthermore, we proposed a simple but useful covalent fragment probing approach and showed that it successfully predicted the covalent binding site of seven targets. By adopting a distance-based method, we observed that the closer the nucleophiles of covalent warheads are to the thiol group of a cysteine, the higher the possibility that a cysteine is prone to form a covalent bond. We believe that the combination of FTMap and our distance-based covalent fragment probing method can become a useful tool in detecting the covalent binding site of these targets. PMID:27483186

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

    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.

  9. The nucleotide-binding site of Aquifex aeolicus LpxC

    Buetow, Lori; Dawson, Alice; Hunter, William N.

    2006-01-01

    The structure of recombinant Aquifex aeolicus UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP has been determined to a resolution of 2.2 Å. Previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, UDP-(3-O-hydroxymyristoyl)-N-­acetylglucosamine, but not that of the nucleotide. The uracil-binding site is constructed from amino acids that are highly conserved across species. Hydrophobic associations with the Phe155 and ...

  10. Increased number of ouabain binding sites in lymphocytes from borderline hypertensives

    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...... triglyceride, and serum cholesterol, which may influence the number of ouabain binding sites. Only BMI entered the stepwise model. These results indicate the presence of an increased number of sodium-potassium pumps in lymphocytes from borderline hypertensives. This difference may be attributed to the blood...

  11. Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

    Møller, Charlotte; Sprenger, Richard R.; Stürup, Stefan; Højrup, Peter

    2011-01-01

    fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges...... were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached...

  12. Characterization of Genomic Vitamin D Receptor Binding Sites through Chromatin Looping and Opening

    Seuter, Sabine; Neme, Antonio; Carlberg, Carsten

    2014-01-01

    The vitamin D receptor (VDR) is a transcription factor that mediates the genomic effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide there are several thousand binding sites and hundreds of primary 1,25(OH)2D3 target genes, but their functional relation is largely elusive. In this study, we used ChIA-PET data of the transcription factor CTCF in combination with VDR ChIP-seq data, in order to map chromatin domains containing VDR binding sites. In total, we found 1,599 such VDR cont...

  13. Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution.

    Sedlyarov, Vitaly; Fallmann, Jörg; Ebner, Florian; Huemer, Jakob; Sneezum, Lucy; Ivin, Masa; Kreiner, Kristina; Tanzer, Andrea; Vogl, Claus; Hofacker, Ivo; Kovarik, Pavel

    2016-01-01

    Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation-associated mRNA decay remains to be established. Using time-resolved high-resolution RNA binding analysis of the mRNA-destabilizing protein tristetraprolin (TTP), an inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non-destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP-controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA-stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co-regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome-wide atlas (http://ttp-atlas.univie.ac.at) of cis-acting elements controlling mRNA decay in inflammation. PMID:27178967

  14. Characterization of pancreatic somatostatin binding sites with a 125I-somatostatin 28 analog

    Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S28): 125I-[Leu8,DTrp22,Tyr25]S28. The binding was highly dependent on calcium ions. In 0.2 mM free Ca2+ medium, binding at 37 degrees C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (KD = 0.05 +/- 0.01 nM, Bmax = 157 +/- 33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca2+ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA1,2[AzaAla4-5,DTrp8, Phe12-13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using 125I-[Leu8,DTrp22,Tyr25]S28 as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand 125I-[Tyr11]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors

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

    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.

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

    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.

  17. Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

    Yang, Jingsong; Campobasso, Nino; Biju, Mangatt P.; Fisher, Kelly; Pan, Xiao-Qing; Cottom, Josh; Galbraith, Sarah; Ho, Thau; Zhang, Hong; Hong, Xuan; Ward, Paris; Hofmann, Glenn; Siegfried, Brett; Zappacosta, Francesca; Washio, Yoshiaki; Cao, Ping; Qu, Junya; Bertrand, Sophie; Wang, Da-Yuan; Head, Martha S.; Li, Hu; Moores, Sheri; Lai, Zhihong; Johanson, Kyung; Burton, George; Erickson-Miller, Connie; Simpson, Graham; Tummino, Peter; Copeland, Robert A.; Oliff, Allen (GSKPA)

    2014-10-02

    c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the {alpha}I helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the {alpha}I helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

  18. Spatial determinants of the alfalfa mosaic virus coat protein binding site.

    Laforest, Siana M; Gehrke, Lee

    2004-01-01

    The biological functions of RNA-protein complexes are, for the most part, poorly defined. Here, we describe experiments that are aimed at understanding the functional significance of alfalfa mosaic virus RNA-coat protein binding, an interaction that parallels the initiation of viral RNA replication. Peptides representing the RNA-binding domain of the viral coat protein are biologically active in initiating replication and bind to a 39-nt 3'-terminal RNA with a stoichiometry of two peptides: 1 RNA. To begin to understand how RNA-peptide interactions induce RNA conformational changes and initiate replication, the AMV RNA fragment was experimentally manipulated by increasing the interhelical spacing, by interrupting the apparent nucleotide symmetry, and by extending the binding site. In general, both asymmetric and symmetric insertions between two proposed hairpins diminished binding, whereas 5' and 3' extensions had minimal effects. Exchanging the positions of the binding site hairpins resulted in only a moderate decrease in peptide binding affinity without changing the hydroxyl radical footprint protection pattern. To assess biological relevance in viral RNA replication, the nucleotide changes were transferred into infectious genomic RNA clones. RNA mutations that disrupted coat protein binding also prevented viral RNA replication without diminishing coat protein mRNA (RNA 4) translation. These results, coupled with the highly conserved nature of the AUGC865-868 sequence, suggest that the distance separating the two proposed hairpins is a critical binding determinant. The data may indicate that the 5' and 3' hairpins interact with one of the bound peptides to nucleate the observed RNA conformational changes. PMID:14681584

  19. A reexamination of information theory-based methods for DNA-binding site identification

    O'Neill Michael C

    2009-02-01

    Full Text Available Abstract Background Searching for transcription factor binding sites in genome sequences is still an open problem in bioinformatics. Despite substantial progress, search methods based on information theory remain a standard in the field, even though the full validity of their underlying assumptions has only been tested in artificial settings. Here we use newly available data on transcription factors from different bacterial genomes to make a more thorough assessment of information theory-based search methods. Results Our results reveal that conventional benchmarking against artificial sequence data leads frequently to overestimation of search efficiency. In addition, we find that sequence information by itself is often inadequate and therefore must be complemented by other cues, such as curvature, in real genomes. Furthermore, results on skewed genomes show that methods integrating skew information, such as Relative Entropy, are not effective because their assumptions may not hold in real genomes. The evidence suggests that binding sites tend to evolve towards genomic skew, rather than against it, and to maintain their information content through increased conservation. Based on these results, we identify several misconceptions on information theory as applied to binding sites, such as negative entropy, and we propose a revised paradigm to explain the observed results. Conclusion We conclude that, among information theory-based methods, the most unassuming search methods perform, on average, better than any other alternatives, since heuristic corrections to these methods are prone to fail when working on real data. A reexamination of information content in binding sites reveals that information content is a compound measure of search and binding affinity requirements, a fact that has important repercussions for our understanding of binding site evolution.

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

    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

  1. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  2. Tritium-bradykinin binding site localization in guinea pig urinary system

    Bradykinin (BK) causes vasodilation and increases free water and sodium excretion in the kidney and stimulates smooth muscle contraction in the ureter and bladder. Several proposed sites of action for BK include the renal medullary collecting duct, renal blood vessels and the ureter and bladder smooth muscle. This study employs 3H-BK autoradiography to localize the sites of BK action. 3H-BK binding sites in the kidney are localized in the medullary interstitium where BK may produce prostaglandins which mediate its blood flow, natriuretic and diuretic effects. 3H-BK binding sites in the ureter and bladder are localized in the lamina propria below the basal epithelial layer and absent over the muscle layers suggesting an indirect action on urinary tract smooth muscle

  3. Auto-FACE: an NMR based binding site mapping program for fast chemical exchange protein-ligand systems.

    Janarthanan Krishnamoorthy

    Full Text Available BACKGROUND: Nuclear Magnetic Resonance (NMR spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQCexperiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately. METHODOLOGY/PRINCIPAL FINDINGS: We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues.Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBcl(XL and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3.0 x 10(4 calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12.8 x 10(4. Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker

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

    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.

  5. Locating the binding sites of antioxidants resveratrol, genistein and curcumin with tRNA.

    N'soukpoé-Kossi, C N; Bourassa, P; Mandeville, J S; Bekale, L; Bariyanga, J; Tajmir-Riahi, H A

    2015-09-01

    We located the binding sites of antioxidants resveratrol, genistein and curcumin on tRNA in aqueous solution at physiological conditions using constant tRNA concentration and various polyphenol contents. FTIR, UV-visible, CD spectroscopic methods and molecular modeling were used to determine polyphenol binding sites, the binding constant and the effects of polyphenol complexation on tRNA conformation and particle formation. Structural analysis showed that polyphenols bind tRNA via G-C and A-U base pairs through hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of K(res-tRNA)=8.95(±0.80)×10(3) M(-1), K(gen-tRNA)=3.07(±0.5)×10(3) M(-1) and K(cur-tRNA)=1.55(±0.3)×10(4) M(-1). Molecular modeling showed the participation of several nucleobases in polyphenol-tRNA adduct formation with free binding energy of -4.43 for resveratrol, -4.26 kcal/mol for genistein and -4.84 kcal/mol for curcumin, indicating that the interaction process is spontaneous at room temperature. While tRNA remains in A-family structure, major biopolymer aggregation and particle formation occurred at high polyphenol contents. PMID:26093317

  6. rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines.

    Chukwudi, Chinwe U

    2016-08-01

    The tetracycline antibiotics are known to be effective in the treatment of both infectious and noninfectious disease conditions. The 16S rRNA binding mechanism currently held for the antibacterial action of the tetracyclines does not explain their activity against viruses, protozoa that lack mitochondria, and noninfectious conditions. Also, the mechanism by which the tetracyclines selectively inhibit microbial protein synthesis against host eukaryotic protein synthesis despite conservation of ribosome structure and functions is still questionable. Many studies have investigated the binding of the tetracyclines to the 16S rRNA using the small ribosomal subunit of different bacterial species, but there seems to be no agreement between various reports on the exact binding site on the 16S rRNA. The wide range of activity of the tetracyclines against a broad spectrum of bacterial pathogens, viruses, protozoa, and helminths, as well as noninfectious conditions, indicates a more generalized effect on RNA. In the light of recent evidence that the tetracyclines bind to various synthetic double-stranded RNAs (dsRNAs) of random base sequences, suggesting that the double-stranded structures may play a more important role in the binding of the tetracyclines to RNA than the specific base pairs, as earlier speculated, it is imperative to consider possible alternative binding modes or sites that could help explain the mechanisms of action of the tetracyclines against various pathogens and disease conditions. PMID:27246781

  7. Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

    This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 40C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 40C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 40C dissociated from MVM when the temperature was raised to 370C, but reassociated when the temperature was again brought to 40C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined

  8. Structural Studies of GABAA Receptor Binding Sites: Which Experimental Structure Tells us What?

    Puthenkalam, Roshan; Hieckel, Marcel; Simeone, Xenia; Suwattanasophon, Chonticha; Feldbauer, Roman V; Ecker, Gerhard F; Ernst, Margot

    2016-01-01

    Atomic resolution structures of cys-loop receptors, including one of a γ-aminobutyric acid type A receptor (GABAA receptor) subtype, allow amazing insights into the structural features and conformational changes that these pentameric ligand-gated ion channels (pLGICs) display. Here we present a comprehensive analysis of more than 30 cys-loop receptor structures of homologous proteins that revealed several allosteric binding sites not previously described in GABAA receptors. These novel binding sites were examined in GABAA receptor homology models and assessed as putative candidate sites for allosteric ligands. Four so far undescribed putative ligand binding sites were proposed for follow up studies based on their presence in the GABAA receptor homology models. A comprehensive analysis of conserved structural features in GABAA and glycine receptors (GlyRs), the glutamate gated ion channel, the bacterial homologs Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus GLIC, and the serotonin type 3 (5-HT3) receptor was performed. The conserved features were integrated into a master alignment that led to improved homology models. The large fragment of the intracellular domain that is present in the structure of the 5-HT3 receptor was utilized to generate GABAA receptor models with a corresponding intracellular domain fragment. Results of mutational and photoaffinity ligand studies in GABAA receptors were analyzed in the light of the model structures. This led to an assignment of candidate ligands to two proposed novel pockets, candidate binding sites for furosemide and neurosteroids in the trans-membrane domain were identified. The homology models can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo-EM microscopic structures of the closest homologs that were solved in different conformational

  9. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

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

    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.

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

    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.

  12. H274Y's Effect on Oseltamivir Resistance: What Happens Before the Drug Enters the Binding Site.

    Yusuf, Muhammad; Mohamed, Nornisah; Mohamad, Suriyati; Janezic, Dusanka; Damodaran, K V; Wahab, Habibah A

    2016-01-25

    Increased reports of oseltamivir (OTV)-resistant strains of the influenza virus, such as the H274Y mutation on its neuraminidase (NA), have created some cause for concern. Many studies have been conducted in the attempt to uncover the mechanism of OTV resistance in H274Y NA. However, most of the reported studies on H274Y focused only on the drug-bound system, so the direct effects of the mutation on NA itself prior to drug binding still remain unclear. Therefore, molecular dynamics simulations of NA in apo form, followed by principal component analysis and interaction energy calculations, were performed to investigate the structural changes of the NA binding site as a result of the H274Y mutation. It was observed that the disruption of the NA binding site due to the H274Y mutation was initiated by the repulsive effect of Y274 on the 250-loop, which in turn altered the hydrogen-bonding network around residue 274. The rotated W295 side chain caused the upward movement of the 340-loop. Consequently, sliding box docking results suggested that the binding pathway of OTV was compromised because of the disruption of this binding site. This study also highlighted the importance of the functional group at C6 of the sialic acid mimicry. It is hoped that these results will improve the understanding of OTV resistance and shed some light on the design of a novel anti-influenza drug. PMID:26703840

  13. Identification of the third binding site of arsenic in human arsenic (III methyltransferase.

    Xiangli Li

    Full Text Available Arsenic (III methyltransferase (AS3MT catalyzes the process of arsenic methylation. Each arsenite (iAs(3+ binds to three cysteine residues, methylarsenite (MMA(3+ binds to two, and dimethylarsenite (DMA(3+ binds to one. However, only two As-binding sites (Cys156 and Cys206 have been confirmed on human AS3MT (hAS3MT. The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs(3+ and active in the methylation of MMA(3+. The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S(C32 is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S(C61 and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S(C61 and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.

  14. Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

    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.

  15. The Human p73 Promoter: Characterization and Identification of Functional E2F Binding Sites

    Ratnam S. Seelan

    2002-01-01

    Full Text Available p73, a member of the p53 family, is overexpressed in many cancers. To understand the mechanism(s underlying this overexpression, we have undertaken a detailed characterization of the human p73 promoter. The promoter is strongly activated in cells expressing exogenous E2F1 and suppressed by exogenous Rb. At least three functional E2F binding sites, located immediately upstream of exon 1 (at-284,-155 and-132 mediate this induction. 5' serially deleted promoter constructs and constructs harboring mutated E2F sites were analyzed for their response to exogenously expressed E2F1 or Rb to establish functionality of these sites. Authenticity of E2F sites was further confirmed by electrophoretic mobility shift assay (EMSA using E2F1 /DP1 heterodimers synthesized in vitro, followed by competition assays with unlabeled wild-type or mutant oligonucleotides and supershift analysis using anti-E2F1 antibodies. In vivo binding of E2F1 to the p73 promoter was demonstrated using nuclear extracts prepared from E2F1-inducible Saos2 cells. The region conferring the highest promoter activity was found to reside between-113 to-217 of the p73 gene. Two of the three functional E2F sites (at-155 and-132 reside within this region. Our results suggest that regulation of p73 expression is primarily mediated through binding of E2 F1 to target sites at-155 and-132.

  16. Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

    Derebe, Mehabaw G.; Sauer, David B.; Zeng, Weizhong; Alam, Amer; Shi, Ning; Jiang, Youxing (UTSMC); (ETH Zurich)

    2015-11-30

    Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K{sup +} channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K{sup +} selective, whereas those with two or three are nonselective and permeate Na{sup +} and K{sup +} equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K{sup +} channels is essential for highly selective and efficient permeation of K{sup +} ions.

  17. Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins.

    Sandhya, B; Hegde, Ashwini H; Seetharamappa, J

    2013-05-01

    The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV-blood protein were determined from fluorescence quenching studies. Stern-Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow's site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11% in free HHb to 38.34% in STV-HHb, and from 66.44% in free HSA to 52.26% in STV-HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV-HHb and STV-HSA, respectively based on Föster's non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8%. PMID:23275205

  18. Specific binding sites for proadrenomedullin N-terminal 20 peptide (PAMP) in the rat.

    Iwasaki, H; Hirata, Y; Iwashina, M; Sato, K; Marumo, F

    1996-07-01

    Adrenomedullin (AM), a potent and novel vasodilator 52-residue peptide originally isolated from pheochromocytoma, is processed from a precursor molecule (preproAM) in which another unique 20-residue sequence, termed proadrenomedullin N-terminal 20 peptide (PAMP), exists. Using [125I Tyr0] rat PAMP as a radioligand, we have examined PAMP binding sites in various rat tissues and cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding sites for rat PAMP, although very low, were widely distributed in various rat tissues examined. The relatively more abundant sites were present in aorta and adrenal glands, followed by lung, kidney, brain, spleen, and heart. An equilibrium binding study using cultured rat VSMC revealed the presence of a single class of high-affinity [dissociation constant (Kd): 3.5 x 10(-8) M] binding sites for rat PAMP with a maximal binding capacity of 4.5 x 10(6) sites per cell. Binding studies revealed that synthetic rat PAMP(1-19)-NH2 was about 10-fold less potent, and rat PAMP(1-20)-OH and human PAMP were about 20-fold less potent than rat PAMP(1-20)-NH2. SDS-polyacylamide gel electrophoresis after affinity-labeling of membranes from various rat tissues (aorta, adrenal glands, lung) and VSMC revealed a distinct labeled band with the apparent molecular mass of 90 kDa, which was diminished by excess unlabeled rat PAMP. A nonhydrolyzable GTP analog (GTP-gammaS) dose-dependently reduced binding of [125I] rat PAMP to VSMC membranes, while ATP-gammaS had no effect. Neither cyclic AMP nor inositol-1,4,5-triphosphate formation was affected by rat PAMP in rat VSMC. The present study demonstrates for the first time that PAMP receptors are widely distributed in various rat tissues, among which aorta and adrenal glands have the most abundant sites. Our data suggest that PAMP receptors are functionally coupled to G-proteins, although its signal transduction remains obscure. The present study also shows that amidation of C-terminal residue

  19. The role of DNA binding sites and slow unbinding kinetics in titration-based oscillators

    Karapetyan, Sargis

    2015-01-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ul...

  20. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    Yatsenko, A S; Kucherenko, M M; Pantoja, M;

    2009-01-01

    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...

  1. The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

    Mazin, A V; Kowalczykowski, S C

    1998-01-01

    RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product...

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

    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.

  3. External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine

    Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of [3H]NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content of the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane

  4. PDNAsite: Identification of DNA-binding Site from Protein Sequence by Incorporating Spatial and Sequence Context.

    Zhou, Jiyun; Xu, Ruifeng; He, Yulan; Lu, Qin; Wang, Hongpeng; Kong, Bing

    2016-01-01

    Protein-DNA interactions are involved in many fundamental biological processes essential for cellular function. Most of the existing computational approaches employed only the sequence context of the target residue for its prediction. In the present study, for each target residue, we applied both the spatial context and the sequence context to construct the feature space. Subsequently, Latent Semantic Analysis (LSA) was applied to remove the redundancies in the feature space. Finally, a predictor (PDNAsite) was developed through the integration of the support vector machines (SVM) classifier and ensemble learning. Results on the PDNA-62 and the PDNA-224 datasets demonstrate that features extracted from spatial context provide more information than those from sequence context and the combination of them gives more performance gain. An analysis of the number of binding sites in the spatial context of the target site indicates that the interactions between binding sites next to each other are important for protein-DNA recognition and their binding ability. The comparison between our proposed PDNAsite method and the existing methods indicate that PDNAsite outperforms most of the existing methods and is a useful tool for DNA-binding site identification. A web-server of our predictor (http://hlt.hitsz.edu.cn:8080/PDNAsite/) is made available for free public accessible to the biological research community. PMID:27282833

  5. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    Lund, Anders Henrik; Schmidt, J; Luz, A;

    1999-01-01

    Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) 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 to...... findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly...

  6. External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine

    Agbanyo, F.R.; Cass, C.E.; Paterson, A.R.

    1988-03-01

    Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of (/sup 3/H)NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content of the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane.

  7. Characterisation of the zebrafish serotonin transporter functionally links TM10 to the ligand binding site

    Severinsen, Kasper; Müller, Heidi Kaastrup; Wiborg, Ove;

    2008-01-01

    [(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...

  8. Longer peptide can be accommodated in the MHC class I binding site by a protrusion mechanism

    Stryhn, A; Pedersen, L O; Holm, A; Buus, S

    2000-01-01

    and C termini of a bound peptide interact through hydrogen bonding networks to conserved residues at either end of the class I binding site. Accordingly, it is thought that the termini are fixed and that only minor variations in peptide size are possible through a central bulging mechanism. We find...

  9. Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

    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. Recognition of binding sites and targeting of drugs on nucleic acids

    Šíp, Miroslav

    České Budějovice : Kopp Publishing, 2002 - (Berger, J.), s. 84-85 [Conference on Cell Biology /4./. České Budějovice (CZ), 09.09.2002-11.09.2002] Institutional research plan: CEZ:AV0Z5051902 Keywords : nucleic acid * binding sites Subject RIV: BO - Biophysics

  11. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  12. Synthesis of Zn-MOF incorporating titanium-hydrides as active sites binding H2 molecules

    Kim, Jongsik; Ok Kim, Dong; Wook Kim, Dong; Sagong, Kil

    2015-10-01

    This paper describes the synthetic effort for a Zn-MOF imparting Ti-H as a preferential binding site potentially capturing H2 molecules via Kubas-type interaction. The formation mechanism of Ti-H innate to the final material was potentially demonstrated to follow a radical dissociation rather than a β-hydrogen elimination and a C-H reductive elimination.

  13. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  14. Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem

    Eftekhari, Sajedeh; Roberts, Rhonda; Chen, Tsing-Bau;

    2016-01-01

    -like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3)H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary...

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

    Troels T Marstrand

    Full Text Available 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-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 promoter sequences. Controlling all aspects of our input data we are able to identify the optimal statistics across multiple threshold values and for sequence sets containing different distributions of transcription factor binding sites. CONCLUSIONS: We show that our implementation is significantly faster than more naïve scanning algorithms when searching with many weight matrices in large sequence sets. When comparing the various statistics, we show that those based on binomial over-representation and Fisher's exact test performs almost equally good and better than the others. An online server is available at http://servers.binf.ku.dk/asap/.

  16. Preliminary study of the metal binding site of an anti-DTPA-indium antibody by equilibrium binding immunoassays and immobilized metal ion affinity chromatography.

    Boden, V; Colin, C; Barbet, J; Le Doussal, J M; Vijayalakshmi, M

    1995-01-01

    Creating metal coordination sites by modifying an existing enzyme or by eliciting antibodies against metal chelate haptens is of great interest in biotechnology to create enzyme catalysts with novel specificities. Here, we investigate the metal binding potential of a monoclonal antibody raised against a DTPA-In(III) hapten (mAb 734). We study its relative binding efficiency to metals of biological relevance by equilibrium binding immunoassays and immobilized metal ion affinity chromatography, two approaches which can give complementary information regarding composition and/or structure of the metal binding site(s). Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) binding was compared to In(III). All of them were shown to displace indium, but their affinity for mAb 734 decreased by 100-fold compared to indium. Competitive metal binding immunoassays between Zn(II) and In(III) revealed an unusual behavior by Zn(II) which remains to be explained. Moreover, IMAC allowed us to predict the metal binding amino acids involved in the antibody paratope. The antibody metal binding site was shown to contain at least two histidine residues in a cluster, and the presence of aspartic and glutamic acid as well as cysteine residues could not be excluded. Thus, simple competition studies allows us to obtain some partial information on the metal binding structural features of this anti-metal chelate antibody and to guide our screening of its catalytic potential. PMID:7578356

  17. Conformational Sampling and Binding Site Assessment of Suppression of Tumorigenicity 2 Ectodomain.

    Chao-Yie Yang

    Full Text Available Suppression of Tumorigenicity 2 (ST2, a member of the interleukin-1 receptor (IL-1R family, activates type 2 immune responses to pathogens and tissue damage via binding to IL-33. Dysregulated responses contribute to asthma, graft-versus-host and autoinflammatory diseases and disorders. To study ST2 structure for inhibitor development, we performed the principal component (PC analysis on the crystal structures of IL1-1R1, IL1-1R2, ST2 and the refined ST2 ectodomain (ST2ECD models, constructed from previously reported small-angle X-ray scattering data. The analysis facilitates mapping of the ST2ECD conformations to PC subspace for characterizing structural changes. Extensive coverage of ST2ECD conformations was then obtained using the accelerated molecular dynamics simulations started with the IL-33 bound ST2ECD structure as instructed by their projected locations on the PC subspace. Cluster analysis of all conformations further determined representative conformations of ST2ECD ensemble in solution. Alignment of the representative conformations with the ST2/IL-33 structure showed that the D3 domain of ST2ECD (containing D1-D3 domains in most conformations exhibits no clashes with IL-33 in the crystal structure. Our experimental binding data informed that the D1-D2 domain of ST2ECD contributes predominantly to the interaction between ST2ECD and IL-33 underscoring the importance of the D1-D2 domain in binding. Computational binding site assessment revealed one third of the total detected binding sites in the representative conformations may be suitable for binding to potent small molecules. Locations of these sites include the D1-D2 domain ST2ECD and modulation sites conformed to ST2ECD conformations. Our study provides structural models and analyses of ST2ECD that could be useful for inhibitor discovery.

  18. Analysis of the Binding Sites of Porcine Sialoadhesin Receptor with PRRSV

    Yibo Jiang

    2013-12-01

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV can infect pigs and cause enormous economic losses to the pig industry worldwide. Porcine sialoadhesin (pSN and CD163 have been identified as key viral receptors on porcine alveolar macrophages (PAM, a main target cell infected by PRRSV. In this study, the protein structures of amino acids 1–119 from the pSN and cSN (cattle sialoadhesin N-termini (excluding the 19-amino acid signal peptide were modeled via homology modeling based on mSN (mouse sialoadhesin template structures using bioinformatics tools. Subsequently, pSN and cSN homology structures were superposed onto the mSN protein structure to predict the binding sites of pSN. As a validation experiment, the SN N-terminus (including the wild-type and site-directed-mutant-types of pSN and cSN was cloned and expressed as a SN-GFP chimera protein. The binding activity between SN and PRRSV was confirmed by WB (Western blotting, FAR-WB (far Western blotting, ELISA (enzyme-linked immunosorbent assay and immunofluorescence assay. We found that the S107 amino acid residue in the pSN N-terminal played a crucial role in forming a special cavity, as well as a hydrogen bond for enhancing PRRSV binding during PRRSV infection. S107 may be glycosylated during PRRSV infection and may also be involved in forming the cavity for binding PRRSV along with other sites, including W2, Y44, S45, R97, R105, W106 and V109. Additionally, S107 might also be important for pSN binding with PRRSV. However, the function of these binding sites must be confirmed by further studies.

  19. Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.

    Raquel Castellanos

    Full Text Available Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome. TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5'-AGGTGTGAAGGTGTGA-3'. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq to further elucidate the

  20. De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.

    Mohamed Sabry Hamza

    Full Text Available BACKGROUND: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma. PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET to map PPARgamma binding sites in 3T3-L1 preadipocyte cells. METHODOLOGY/PRINCIPAL FINDINGS: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down

  1. Solubilization and characterization of haloperidol-sensitive (+)-[3H]SKF-10,047 binding sites (sigma sites) from rat liver membranes

    The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-[3H]SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-[3H]SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-[3H]SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition, the binding of 20 nM [3H]progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-[3H]SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-[3H]SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex

  2. STarMirDB: A database of microRNA binding sites.

    Rennie, William; Kanoria, Shaveta; Liu, Chaochun; Mallick, Bibekanand; Long, Dang; Wolenc, Adam; Carmack, C Steven; Lu, Jun; Ding, Ye

    2016-06-01

    microRNAs (miRNAs) are an abundant class of small endogenous non-coding RNAs (ncRNAs) of ∼22 nucleotides (nts) in length. These small regulatory molecules are involved in diverse developmental, physiological and pathological processes. miRNAs target mRNAs (mRNAs) for translational repression and/or mRNA degradation. Predictions of miRNA binding sites facilitate experimental validation of miRNA targets. Models developed with data from CLIP studies have been used for predictions of miRNA binding sites in the whole transcriptomes of human, mouse and worm. The prediction results have been assembled into STarMirDB, a new database of miRNA binding sites available at http://sfold.wadsworth.org/starmirDB.php . STarMirDB can be searched by miRNAs or mRNAs separately or in combination. The search results are categorized into seed and seedless sites in 3' UTR, CDS and 5' UTR. For each predicted site, STarMirDB provides a comprehensive list of sequence, thermodynamic and target structural features that are known to influence miRNA: target interaction. A high resolution PDF diagram of the conformation of the miRNA:target hybrid is also available for visualization and publication. The results of a database search are available through both an interactive viewer and downloadable text files. PMID:27144897

  3. A structural-based strategy for recognition of transcription factor binding sites.

    Beisi Xu

    Full Text Available Scanning through genomes for potential transcription factor binding sites (TFBSs is becoming increasingly important in this post-genomic era. The position weight matrix (PWM is the standard representation of TFBSs utilized when scanning through sequences for potential binding sites. However, many transcription factor (TF motifs are short and highly degenerate, and methods utilizing PWMs to scan for sites are plagued by false positives. Furthermore, many important TFs do not have well-characterized PWMs, making identification of potential binding sites even more difficult. One approach to the identification of sites for these TFs has been to use the 3D structure of the TF to predict the DNA structure around the TF and then to generate a PWM from the predicted 3D complex structure. However, this approach is dependent on the similarity of the predicted structure to the native structure. We introduce here a novel approach to identify TFBSs utilizing structure information that can be applied to TFs without characterized PWMs, as long as a 3D complex structure (TF/DNA exists. This approach utilizes an energy function that is uniquely trained on each structure. Our approach leads to increased prediction accuracy and robustness compared with those using a more general energy function. The software is freely available upon request.

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

    Patel, Hershna; Kukol, Andreas

    2016-01-01

    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. PMID:27553084

  5. HPV 16 E2 binding sites 1 and 2 become more methylated than E2 binding site 4 during cervical carcinogenesis.

    Leung, Tsin-Wah; Liu, Stephanie S; Leung, Rebecca C Y; Chu, Mandy M Y; Cheung, Annie N Y; Ngan, Hextan Y S

    2015-06-01

    E2 protein binding to the four E2 binding sites (E2BSs) at the long control region of Human Papillomavirus (HPV) 16/18 genome may exert either transcriptional activation/repression on E6 and E7 oncoproteins. Methylation status at the E2BSs may affect the relative binding of E2 protein to them. In this study, methylation percentage at E2BS 1, 2 (promoter-proximal), and 4 (promoter-distal) were assessed by pyrosequencing and compared among HPV 16/18-positive cervical cancer, high-grade, and low-grade Cervical Intraepithelial Neoplasia, Atypical Squamous Cells of Undetermined Significance, and normal cervical epithelium. HPV 16 E2BS1&2 were more methylated than HPV 16 E2BS4 in cervical cancer whereas in cervical premalignant lesions and normal epithelium, HPV 16 E2BS1&2 were less methylated than HPV 16 E2BS4. HPV 18 E2BS1&2 remained more methylated than E2BS4 in all histological groups. HPV 16 E2BS1&2 methylation increased from high-grade lesions to cervical cancer (P E2 protein to E2BS4. Increasing methylation at HPV 16/18 E2BSs are potentially useful adjunctive molecular markers for predicting progression from low-grade to high-grade cervical premalignant lesions and from high-grade lesions to cervical cancer. PMID:25648229

  6. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    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.

  7. Pharmacological characterization of axonally transported (125I)-alpha-bungatoxin binding sites in rat sciatic nerve

    The authors attempt to label the putative receptors as they are axonally transported in peripheral nerves. With the use of an innovative autoradiographic technique, this approach as enabled the investigation of the pharmacological properties of the toxin-binding site interaction. The tissue sections from adult male rat sciatic nerves were incubated for 60 min at room temperature in phosphate buffer saline containing 2 nM I 125-alpha-BuTX with or without displacer. A bright field micrograph as well as dark field autoradiograph is illustrated of a ligated (12 hr.) rat sciatic nerve section incubated with I 125-alpha-BuTX. If one presumes that axonally transported I 125-alpha-BuTX binding sites correspond to receptors whose destination is the presynaptic membrane, then the data presented in this study may provide a pharmacological basis for differentiating pre- and postsynaptic sites of action of cholinergic drugs on the mammalian neuromuscular junction

  8. Metal ion binding sites of bacteriorhodopsin. Laser-induced lanthanide luminescence study

    Laser-excited luminescence lifetimes of lanthanide ions bound to bacteriorhodopsin have been measured in deionized membranes. The luminescence titration curve, as well as the binding curve of apomembrane (retinal-free) with Eu3+, has shown that the removal of the retinal does not significantly affect the affinity of Eu3+ for the two high affinity sites of bacteriorhodopsin. The D2O effects on decay rate constants indicate that Eu3+ bound to the high affinity sites of native membrane or apomembrane is coordinated by about six ligands in the first coordination sphere. Tb3+ is shown to be coordinated by four ligands. The data indicate that metal ions bind to the protein with a specific geometry. From intermetal energy transfer experiments using Eu3+-Pr3+, Tb3+-Ho3+, and Tb3+-Er3+, the distance between the two high affinity sites is estimated to be 7-8 A

  9. The Tetrodotoxin Binding Site Is within the Outer Vestibule of the Sodium Channel

    Gregory M. Lipkind

    2010-02-01

    Full Text Available Tetrodotoxin and saxitoxin are small, compact asymmetrical marine toxins that block voltage-gated Na channels with high affinity and specificity. They enter the channel pore’s outer vestibule and bind to multiple residues that control permeation. Radiolabeled toxins were key contributors to channel protein purification and subsequent cloning. They also helped identify critical structural elements called P loops. Spacial organization of their mutation-identified interaction sites in molecular models has generated a molecular image of the TTX binding site in the outer vestibule and the critical permeation and selectivity features of this region. One site in the channel’s domain I P loop determines affinity differences in mammalian isoforms.

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

    Long, Katherine S; Porse, Bo T

    2003-01-01

    The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m(5)U747 and C2611/C2612, in domains II and V, respectively......, 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...

  11. The propagation of binding interactions to remote sites in proteins: analysis of the binding of the monoclonal antibody D1.3 to lysozyme.

    Freire, E

    1999-08-31

    The interaction of a ligand with a protein occurs at a local site (the binding site) and involves only a few residues; however, the effects of that interaction are often propagated to remote locations. The chain of events initiated by binding provides the basis for fundamental biological phenomena such as allosterism, signal transduction, and structural-stability modification. In this paper, a structure-based statistical thermodynamic approach is presented and used to predict the propagation of the stabilization effects triggered by the binding of the monoclonal antibody D1.3 to hen egg white lysozyme. Previously, Williams et al. [Williams, D. C., Benjamin, D. C., Poljak, R. J. & Rule, G. S. (1996) J. Mol. Biol. 257, 866-876] showed that the binding of this antibody affects the stability of hen egg white lysozyme and that the binding effects propagate to a selected number of residues at remote locations from the binding epitope. In this paper, we show that this phenomenon can be predicted from structure. The formalism presented here permits the identification of the structural path followed by cooperative interactions that originate at the binding site. It is shown that an important condition for the propagation of binding effects to distal regions is the presence of a significant fraction of residues with low structural stability in the uncomplexed binding site. A survey of protein structures indicates that many binding sites have a dual character and are defined by regions of high and low structural stabilities. The low-stability regions might be involved in the transmission of binding information to other regions in the protein. PMID:10468572

  12. Identification of inhibitor binding site in human sirtuin 2 using molecular docking and dynamics simulations.

    Sakkiah, Sugunadevi; Arooj, Mahreen; Kumar, Manian Rajesh; Eom, Soo Hyun; Lee, Keun Woo

    2013-01-01

    The ability to identify the site of a protein that can bind with high affinity to small, drug-like compounds has been an important goal in drug design. Sirtuin 2 (SIRT2), histone deacetylase protein family, plays a central role in the regulation of various pathways. Hence, identification of drug for SIRT2 has attracted great interest in the drug discovery community. To elucidate the molecular basis of the small molecules interactions to inhibit the SIRT2 function we employed the molecular docking, molecular dynamics simulations, and the molecular mechanism Poisson-Boltzmann/surface area (MM-PBSA) calculations. Five well know inhibitors such as suramin, mol-6, sirtinol, 67, and nf675 were selected to establish the nature of the binding mode of the inhibitors in the SIRT2 active site. The molecular docking and dynamics simulations results revealed that the hydrogen bonds between Arg97 and Gln167 are crucial to inhibit the function of SIRT2. In addition, the MM-PBSA calculations revealed that binding of inhibitors to SIRT2 is mainly driven by van der Waals/non-polar interactions. Although the five inhibitors are very different in structure, shape, and electrostatic potential, they are able to fit in the same binding pocket. These findings from this study provide insights to elucidate the binding pattern of SIRT2 inhibitors and help in the rational structure-based design of novel SIRT2 inhibitors with improved potency and better resistance profile. PMID:23382805

  13. Identification of inhibitor binding site in human sirtuin 2 using molecular docking and dynamics simulations.

    Sugunadevi Sakkiah

    Full Text Available The ability to identify the site of a protein that can bind with high affinity to small, drug-like compounds has been an important goal in drug design. Sirtuin 2 (SIRT2, histone deacetylase protein family, plays a central role in the regulation of various pathways. Hence, identification of drug for SIRT2 has attracted great interest in the drug discovery community. To elucidate the molecular basis of the small molecules interactions to inhibit the SIRT2 function we employed the molecular docking, molecular dynamics simulations, and the molecular mechanism Poisson-Boltzmann/surface area (MM-PBSA calculations. Five well know inhibitors such as suramin, mol-6, sirtinol, 67, and nf675 were selected to establish the nature of the binding mode of the inhibitors in the SIRT2 active site. The molecular docking and dynamics simulations results revealed that the hydrogen bonds between Arg97 and Gln167 are crucial to inhibit the function of SIRT2. In addition, the MM-PBSA calculations revealed that binding of inhibitors to SIRT2 is mainly driven by van der Waals/non-polar interactions. Although the five inhibitors are very different in structure, shape, and electrostatic potential, they are able to fit in the same binding pocket. These findings from this study provide insights to elucidate the binding pattern of SIRT2 inhibitors and help in the rational structure-based design of novel SIRT2 inhibitors with improved potency and better resistance profile.

  14. In vivo receptor binding of opioid drugs at the mu site

    The in vivo receptor binding of a series of opioid drugs was investigated in intact rats after s.c. administration of [3H]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

  15. Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase.

    Gribovskaja, Irena; Brownlow, Kaleb C; Dennis, Sam J; Rosko, Andrew J; Marletta, Michael A; Stevens-Truss, Regina

    2005-05-24

    Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation. PMID:15896003

  16. Optimizing the GATA-3 position weight matrix to improve the identification of novel binding sites

    Nandi Soumyadeep

    2012-08-01

    Full Text Available Abstract Background The identifying of binding sites for transcription factors is a key component of gene regulatory network analysis. This is often done using position-weight matrices (PWMs. Because of the importance of in silico mapping of tentative binding sites, we previously developed an approach for PWM optimization that substantially improves the accuracy of such mapping. Results The present work implements the optimization algorithm applied to the existing PWM for GATA-3 transcription factor and builds a new di-nucleotide PWM. The existing available PWM is based on experimental data adopted from Jaspar. The optimized PWM substantially improves the sensitivity and specificity of the TF mapping compared to the conventional applications. The refined PWM also facilitates in silico identification of novel binding sites that are supported by experimental data. We also describe uncommon positioning of binding motifs for several T-cell lineage specific factors in human promoters. Conclusion Our proposed di-nucleotide PWM approach outperforms the conventional mono-nucleotide PWM approach with respect to GATA-3. Therefore our new di-nucleotide PWM provides new insight into plausible transcriptional regulatory interactions in human promoters.

  17. Identification of small molecule binding sites within proteins using phage display technology.

    Rodi, D. J.; Agoston, G. E.; Manon, R.; Lapcevich, R.; Green, S. J.; Makowski, L.; Biosciences Division; EntreMed Inc.; Florida State Univ.

    2001-11-01

    Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

  18. Antibody remodeling: a general solution to the design of a metal-coordination site in an antibody binding pocket.

    Roberts, V A; Iverson, B L; Iverson, S A; Benkovic, S J; Lerner, R A; Getzoff, E D; Tainer, J A

    1990-01-01

    To develop a general approach to designing cofactor-binding sites for catalytic antibodies, we characterized structural patterns in the binding sites of antibodies and zinc enzymes. Superposition of eight sets of antibody light- and heavy-chain variable domains identified structurally conserved sites within the sequence-variable complementarity determining regions. The pattern for catalytic zinc sites included two ligands close in sequence, a sequence-distant ligand, and a main-chain hydrogen...

  19. Binding site for the adenosyl group of coenzyme B12 in diol dehydrase

    The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible

  20. Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm.

    Li, Na; Wang, Jing; Han, Heyou; Huang, Liang; Shao, Feng; Li, Xuepu

    2014-02-15

    Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa-QD probes compared to CrylAc-QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa-QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins. PMID:24252542

  1. Novel Phosphotidylinositol 4,5-Bisphosphate Binding Sites on Focal Adhesion Kinase.

    Jun Feng

    Full Text Available Focal adhesion kinase (FAK is a protein tyrosine kinase that is ubiquitously expressed, recruited to focal adhesions, and engages in a variety of cellular signaling pathways. Diverse cellular responses, such as cell migration, proliferation, and survival, are regulated by FAK. Prior to activation, FAK adopts an autoinhibited conformation in which the FERM domain binds the kinase domain, blocking access to the activation loop and substrate binding site. Activation of FAK occurs through conformational change, and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2 are known to facilitate this process. PIP2 binding alters the autoinhibited conformation of the FERM and kinase domains and subsequently exposes the activation loop to phosphorylation. However, the detailed molecular mechanism of PIP2 binding and its role in FAK activation remain unclear. In this study, we conducted coarse-grained molecular dynamics simulations to investigate the binding of FAK to PIP2. Our simulations identified novel areas of basic residues in the kinase domain of FAK that potentially undergo transient binding to PIP2 through electrostatic attractions. Our investigation provides a molecular picture of PIP2-initiated FAK activation and introduces promising new pathways for future studies of FAK regulation.

  2. Identification and Analysis of Papillomavirus E2 Protein Binding Sites in the Human Genome

    Võsa, Liisi; Sudakov, Aleksander; Remm, Maido; Ustav, Mart; Kurg, Reet

    2012-01-01

    Papillomavirus E2 protein is required for the replication and maintenance of viral genomes and transcriptional regulation of viral genes. E2 functions through sequence-specific binding to 12-bp DNA motifs—E2 binding sites (E2BS)—in the virus genome. Papillomaviruses are able to establish persistent infection in their host and have developed a long-term relationship with the host cell in order to guarantee the propagation of the virus. In this study, we have analyzed the occurrence and functio...

  3. Identification of the rheumatoid arthritis shared epitope binding site on calreticulin.

    Song Ling

    Full Text Available BACKGROUND: The rheumatoid arthritis (RA shared epitope (SE, a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRbeta chain. The molecular mechanisms by which the SE affects susceptibility to--and severity of--RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT. PRINCIPAL FINDINGS: Surface plasmon resonance (SPR experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido-2-(p-azido-benzamido hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217-224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu(217 and Glu(223--and to a lesser extent residue Asp(220--in cell-free SPR-based binding and signal transduction assays. SIGNIFICANCE: We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

  4. Characterization of [3H]leukotriene D4 binding sites in guinea-pig ventricular myocardium

    [3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30 degrees C. The specific [3H] LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30 degrees C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 +/- 2.1 nM and a maximum number of binding sites of 850 +/- 91 fmol/mg of protein. Competition binding studies with [3H]LTD4, synthetic 5S, 6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific [3H]LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific [3H]LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the [3H]LTD4 binding to 368 +/- 61 fmol/mg of protein with minimal effects on the apparent Kd

  5. The propagation of binding interactions to remote sites in proteins: Analysis of the binding of the monoclonal antibody D1.3 to lysozyme

    Freire, Ernesto

    1999-01-01

    The interaction of a ligand with a protein occurs at a local site (the binding site) and involves only a few residues; however, the effects of that interaction are often propagated to remote locations. The chain of events initiated by binding provides the basis for fundamental biological phenomena such as allosterism, signal transduction, and structural-stability modification. In this paper, a structure-based statistical thermodynamic approach is presented and used...

  6. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods

    The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (Ka) between MG and BSA at four different temperatures were obtained to be 3.734 x 104, 3.264 x 104, 2.718 x 104, and 2.164 x 104 L mol-1, respectively. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -27.25 kJ mol-1 and -11.23 J mol-1 K-1, indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to Foerster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the α-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules

  7. Mutational Mapping and Modeling of the Binding Site for (S)-Citalopram in the Human Serotonin Transporter

    Andersen, Jacob; Olsen, Lars; Hansen, Kasper B.;

    2010-01-01

    , and (S)-citalopram, which are competitive inhibitors of the transport function. Knowledge of the molecular details of the antidepressant binding sites in SERT has been limited due to lack of structural data on SERT. Here, we present a characterization of the (S)-citalopram binding pocket in human SERT...... (hSERT) using mutational and computational approaches. Comparative modeling and ligand docking reveal that (S)-citalopram fits into the hSERT substrate binding pocket, where (S)-citalopram can adopt a number of different binding orientations. We find, however, that only one of these binding modes is...... functionally relevant from studying the effects of 64 point mutations around the putative substrate binding site. The mutational mapping also identify novel hSERT residues that are crucial for (S)-citalopram binding. The model defines the molecular determinants for (S)-citalopram binding to hSERT and...

  8. Target molecular weights for red cell band 3 stilbene and mercurial binding sites

    Radiation inactivation was used to measure the target sizes for binding of disulfonic stilbene anion transport inhibitor 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) and mercurial water transport inhibitor p-chloromercuribenzene sulfonate (pCMBS) to human erythrocytes. The measured target size for erythrocyte ghost acetylcholinesterase was 78 +/- 3 kDa. DBDS binding to ghost membranes was measured by a fluorescence enhancement technique. Radiation (0-26 Mrad) had no effect on total membrane protein and DBDS binding affinity, whereas DBDS binding stoichiometry decreased exponentially with radiation dose, giving a target size of 59 +/- 4 kDa. H2-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2-DIDS, 5 microM) blocked greater than 95% of DBDS binding at all radiation doses. pCMBS binding was measured from the time course of tryptophan fluorescence quenching in ghosts treated with the sulfhydryl reagent N-ethylmaleimide (NEM). Radiation did not affect the kinetics of tryptophan quenching, whereas the total amplitude of the fluorescence signal inactivated with radiation with a target size of 31 +/- 6 kDa. These results support the notion that DBDS and pCMBS bind to the transmembrane domain of erythrocyte band 3 in NEM-treated ghosts and demonstrate that radiation inactivation may probe a target significantly smaller than a covalently linked protein subunit. The small target size for the band 3 stilbene binding site may correspond to the intramembrane domain of the band 3 monomer (52 kDa), which is physically distinct from the cytoplasmic domain (42 kDa)

  9. eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

    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.

  10. Characterization of specific binding sites for PAF in the iris and ciliary body of rabbit

    The protective effect exerted by BN 52021 a specific PAF-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAF in rabbit iris and ciliary body. Two classes of PAF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes: a high affinity site Kd1 congruent to 4.9 +/- 0.47 nM, Bmax1 congruent to 3.17 +/- 0.50 pmoles/mg protein, a low affinity sites Kd2 congruent to 11.6 +/- 0.33 nM, Bmax2 congruent to 12.46 +/- 2.3 pmoles/mg protein for iris. The specific binding was not affected by lyso-PAF the biologically inactive precursor and metabolite of PAF, up to 10(-6) M; inhibition by unlabelled PAF demonstrated a biphasic curve partially antagonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAF in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021

  11. Endogenous progesterone and its cellular binding sites in wheat exposed to drought stress.

    Janeczko, Anna; Oklešťková, Jana; Siwek, Agata; Dziurka, Michał; Pociecha, Ewa; Kocurek, Maciej; Novák, Ondřej

    2013-11-01

    Progesterone is a basic hormone that regulates the metabolism in mammals. The presence of this compound has also been found in certain plants. It is believed that progesterone can regulate growth processes and resistance to stress, however, its precise role in plants remains unknown. The research conducted in this study was aimed at analyzing the content of endogenous progesterone and its cellular binding sites in the leaves of spring wheat exposed to drought. Changes were studied in two cultivars of wheat - a cultivar sensitive to drought (Katoda) and tolerant cultivar (Monsun). Plants had undergone periodic droughts during the seedling stage or in the phase of heading. The occurrence of free progesterone as well as its conjugated forms was observed in wheat studied. The amount of progesterone ranged from 0.2 to 5.8pmolgFW(-1) and was dependent on the cultivar, age of the plants, stage of development and fluctuated as a result of the exposure to drought. Cv. Katoda responded to a water deficit by lowering the amount of progesterone and cv. Monsun by increasing its level. Progesterone in plants grown in limited water conditions occurred primarily in a free form. While in the optimal watering conditions, some of its pool was found in the form of conjugates. In the spring wheat the occurrence of binding sites for progesterone was detected in cell membranes, cytoplasm and nuclei in the range of 10-36fmol/mg of protein. The wheat cultivars tested, Monsun and Katoda, differ in their concentration of cellular binding sites for progesterone. This number varied in the individual fractions during different stages of plant development and due to the effect of drought stress. The number of binding sites for progesterone located in the membrane fraction of seedlings and flag leaves increased significantly under drought in the cv. Katoda (35-46%), but did not change in the cv. Monsun. Whereas the number of cytoplasmic progesterone binding sites increased during the drought in

  12. A new histochemical method using human placenta alkaline phosphatase for demonstrating concanavalin A binding sites on cell surfaces

    Kanzaki,Yoshito

    1975-12-01

    Full Text Available Human placenta alkaline phosphatase (HP-ALP, a glycoprotein, was stained histochemically for the purpose of examining the concanavalin A (Con A binding sites on the cell surface. HP-ALP was bound to the cell surface by Con A. This simple method successfully detected Con A binding sites on the cell surface.

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

    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 ...

  14. Mutations in the hemagglutinin receptor-binding site can change the biological properties of an influenza virus.

    Naeve, C W; Hinshaw, V S; Webster, R G

    1984-01-01

    Avian influenza virus reassortants containing human influenza virus hemagglutinins do not replicate in ducks. Two mutations in the receptor-binding site of a human hemagglutinin at residues 226 and 228 allowed replication in ducks. The mutations resulted in a receptor-binding-site sequence identical to the known avian influenza virus sequences.

  15. Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

    Buhrman, Greg; O; #8242; Connor, Casey; Zerbe, Brandon; Kearney, Bradley M.; Napoleon, Raeanne; Kovrigina, Elizaveta A.; Vajda, Sandor; Kozakov, Dima; Kovrigin, Evgenii L.; Mattos, Carla (NCSU); (MCW); (BU)

    2012-09-17

    We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

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

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...... 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...

  17. Target Detection Assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein.

    Thiesen, H J; Bach, C.

    1990-01-01

    We developed a rapid method designated Target Detection Assay (TDA) to determine DNA binding sites for putative DNA binding proteins. A purified, functionally active DNA binding protein and a pool of random double-stranded oligonucleotides harbouring PCR primer sites at each end are included the TDA cycle which consists of four separate steps: a DNA protein incubation step, a protein DNA complex separation step, a DNA elution step and a polymerase chain reaction (PCR) DNA amplification step. ...

  18. Identification of High Affinity Fatty Acid Binding Sites on Human Serum Albumin by MM-PBSA Method

    Fujiwara, Shin-ichi; Amisaki, Takashi

    2007-01-01

    Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the “three-trajectory method” and the “single-trajectory method”. ...

  19. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D’Andrea, Luca Domenico

    2016-08-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  20. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    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.

  1. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Studies of [3H]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 [3H]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

  2. The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

    We have characterized the interaction of the serotonin transporter ligand [3H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [3H]-DASB, a tritiated version of the widely used [11C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K d=0.20±0.04 nM). The serotonin transporter density (B max) obtained for rhesus frontal cortex was found to be 66±8 fmol/mg protein using [3H]-DASB, similar to the B max value obtained using the reference radioligand [3H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83±22 fmol/mg protein). Specific binding sites of both [3H]-DASB and [3H]-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 [3H]-citalopram binding in a competition autoradiographic study, with K i values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [3H]-DASB and [3H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [11C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates

  3. The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

    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.

  4. Binding of dioxygen to non-metal sites in proteins: exploration of the importance of binding site size versus hydrophobicity in the copper amine oxidase from Hansenula polymorpha.

    Goto, Yoshio; Klinman, Judith P

    2002-11-19

    Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O2) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O2) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O2) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O2) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O2) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O2, in relation to the co-gas N2, since substitution of the inert gas N

  5. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase.

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E; Leonidas, Demetres D; Gimisis, Thanasis; Oikonomakos, Nikos G

    2007-08-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 A resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  6. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike (Vanderbilt)

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  7. Sequence-based prediction of protein-peptide binding sites using support vector machine.

    Taherzadeh, Ghazaleh; Yang, Yuedong; Zhang, Tuo; Liew, Alan Wee-Chung; Zhou, Yaoqi

    2016-05-15

    Protein-peptide interactions are essential for all cellular processes including DNA repair, replication, gene-expression, and metabolism. As most protein-peptide interactions are uncharacterized, it is cost effective to investigate them computationally as the first step. All existing approaches for predicting protein-peptide binding sites, however, are based on protein structures despite the fact that the structures for most proteins are not yet solved. This article proposes the first machine-learning method called SPRINT to make Sequence-based prediction of Protein-peptide Residue-level Interactions. SPRINT yields a robust and consistent performance for 10-fold cross validations and independent test. The most important feature is evolution-generated sequence profiles. For the test set (1056 binding and non-binding residues), it yields a Matthews' Correlation Coefficient of 0.326 with a sensitivity of 64% and a specificity of 68%. This sequence-based technique shows comparable or more accurate than structure-based methods for peptide-binding site prediction. SPRINT is available as an online server at: http://sparks-lab.org/. © 2016 Wiley Periodicals, Inc. PMID:26833816

  8. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-11-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  9. Influence of heterogeneity of binding sites of humic acid on its complexation with actinyl ions

    The influence of heterogeneity of binding sites of humic acid was investigated using three different experimental approaches. 1) Complexation with UO22+ was studied by fluorescence spectroscopy. Considering the quenching phenomena, the apparent stability constants of complexation of humic acid with UO22+ were evaluated and compared with the values reported in the literature. 2) Complexation kinetics of NpO2+ with humic acid and fulvic acid was investigated by kinetic spectra. The complexation kinetics was found to be controlled by the heterogeneity of binding sites. 3) The migration of humic acid-NpO2+ complexes was discussed experimentally by column experiments and analytically by a migration model. The concentration profile of NpO2+ in the column was qualitatively simulated. (author)

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

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

    2008-01-01

    promoter sequences. Controlling all aspects of our input data we are able to identify the optimal statistics across multiple threshold values and for sequence sets containing different distributions of transcription factor binding sites. CONCLUSIONS: We show that our implementation is significantly faster...... 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...... than more naïve scanning algorithms when searching with many weight matrices in large sequence sets. When comparing the various statistics, we show that those based on binomial over-representation and Fisher's exact test performs almost equally good and better than the others. An online server is...

  11. Spatial relationships between the amine binding site and the copper in plasma amine oxidase

    Porcine plasma amine oxidase was covalently modified with a series of fluorine containing phenylhydrazine inhibitors. One mole of phenylhydrazine modifies one mole of enzyme at the amine substrate binding site. NMR relaxation measurements on the fluorine nuclei were obtained at two field strengths for each inhibitor-enzyme complex. These measurements were used to calculate the exact distance and spatial orientation between the inhibitor binding site and the copper cofactor. The copper lies in the plane of the aromatic ring of the inhibitor 10.9, 14.3, and 15.5 A from the ortho-, meta-, and para-positions of the ring, respectively. Since the inhibitors react with the active carbonyl cofactor, this defines the spatial relationship between the copper and the active carbonyl cofactor in the enzyme

  12. Genome wide mapping of Foxo1 binding-sites in murine T lymphocytes

    Will Liao

    2014-12-01

    Full Text Available The Forkhead box O (Foxo family of transcription factors has a critical role in controlling the development, differentiation, and function of T cells. However, the direct target genes of Foxo transcription factors in T cells have not been well characterized. In this study, we focused on mapping the genome wide Foxo1-binding sites in naïve CD4+ T cells, CD8+ T cells, and Foxp3+ regulatory T (Treg cells. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq, we identified Foxo1 binding sites that were shared among or specific to the three T cell populations. Here we describe the experiments, quality controls, as well as the deep sequencing data. Part of the data analysis has been published by Ouyang W et al. in Nature 2012 [1] and Kim MV et al. in Immunity 2013 [2], and the associated data set were uploaded to NCBI Gene Expression Omnibus.

  13. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-01

    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. PMID:21797258

  14. Functional and phylogenetic analyses of chromosome 21 promoters and hominid-specific transcription factor binding sites

    Querfurth, Robert

    2011-01-01

    The focus of this work addresses functional studies of human and primate promoters, and the genome-wide localization and validation of human-specific transcription factor binding sites of the essential transcription factor GABPa. In this context, the development of an improved PCR protocol, including the careful adjustment of PCR additives to compose an efficient enhancer mix, was central to the amplification of large GC-rich promoter fragments used as source for the functional studies. Based...

  15. Identification of a chloride ion binding site in Na+/Cl−-dependent transporters

    Forrest, Lucy R.; Tavoulari, Sotiria; Zhang, Yuan-Wei; Rudnick, Gary; Honig, Barry

    2007-01-01

    The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl− independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl− ions. However, the only Cl− ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is...

  16. Glycosaminoglycans that bind cold-insoluble globulin in cell-substratum adhesion sites of murine fibroblasts.

    Laterra, J; Ansbacher, R; Culp, L A

    1980-01-01

    Glycosaminoglycans (GAGs) and glycoprotein-derived glycopeptide from mouse BALB/c3T3 and simian virus 40-transformed 3T3 whole cells or their adhesion sites, which are left bound to the serum-coated tissue culture substratum after detachment of cells mediated by [ethylenebis-(oxyethylenenitrilo]tetraacetic acid (EGTA), were analyzed for specific binding to Sepharose columns derivatized with cold-insoluble globulin (CIg). CIg is the serum-contained form of fibronectin and is required for the a...

  17. SPIC: A novel similarity metric for comparing transcription factor binding site motifs based on information contents

    Zhang, Shaoqiang; Zhou, Xiguo; Du, Chuanbin; Su, Zhengchang

    2013-01-01

    Background Discovering transcription factor binding sites (TFBS) is one of primary challenges to decipher complex gene regulatory networks encrypted in a genome. A set of short DNA sequences identified by a transcription factor (TF) is known as a motif, which can be expressed accurately in matrix form such as a position-specific scoring matrix (PSSM) and a position frequency matrix. Very frequently, we need to query a motif in a database of motifs by seeking its similar motifs, merge similar ...

  18. Flexible protein-peptide docking using CABS-dock with knowledge about the binding site

    Kurcinski, Mateusz; Ciemny, Maciej Pawel; Blaszczyk, Maciej; Kolinski, Andrzej; Kmiecik, Sebastian

    2016-01-01

    Despite considerable efforts, structural prediction of protein-peptide complexes is still a very challenging task, mainly due to two reasons: high flexibility of the peptides and transient character of their interactions with proteins. Recently we have developed an automated web server CABS-dock (http://biocomp.chem.uw.edu.pl/CABSdock), which conducts flexible protein-peptide docking without any knowledge about the binding site. Our method allows for full flexibility of the peptide, whereas t...

  19. Probing the orthosteric binding site of GABAA receptors with heterocyclic GABA carboxylic acid bioisosteres

    Petersen, Jette G; Bergmann, Rikke; Krogsgaard-Larsen, Povl; Balle, Thomas; Frølund, Bente

    2013-01-01

    selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map...... the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available...

  20. Protective action of resveratrol in human skin: possible involvement of specific receptor binding sites.

    Stéphane Bastianetto

    Full Text Available BACKGROUND: Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006 J Pharmacol Exp Ther 318:238-245. The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol. METHODS AND FINDINGS: Using human skin tissue, we report here the presence of specific [(3H]-resveratrol binding sites (K(D  =  180 nM that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3-3 mM resulted in cell death which was reduced by resveratrol (EC(50  =  14.7 µM, and to a much lesser extent by the resveratrol analogue piceatannol (EC(50  =  95 µM and epigallocatechin gallate (EC(50  =  200 µM, a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP. CONCLUSION: Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.

  1. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    Tiraidis, C.; Alexacou, K. M.; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, T.; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where th...

  2. Dual Binding Site and Selective Acetylcholinesterase Inhibitors Derived from Integrated Pharmacophore Models and Sequential Virtual Screening

    2014-01-01

    In this study, we have employed in silico methodology combining double pharmacophore based screening, molecular docking, and ADME/T filtering to identify dual binding site acetylcholinesterase inhibitors that can preferentially inhibit acetylcholinesterase and simultaneously inhibit the butyrylcholinesterase also but in the lesser extent than acetylcholinesterase. 3D-pharmacophore models of AChE and BuChE enzyme inhibitors have been developed from xanthostigmine derivatives through HypoGen an...

  3. Polymorphisms in miRNA binding sites of nucleotide excision repair genes and colorectal cancer risk

    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

  4. Transcriptional Activation of sclA by Mga Requires a Distal Binding Site in Streptococcus pyogenes

    Almengor, Audry C.; McIver, Kevin S.

    2004-01-01

    Streptococcus pyogenes (the group A streptococcus [GAS]) is a medically significant pathogen of humans, causing a range of diseases from pharyngitis to necrotizing fasciitis. Several important GAS virulence genes are under the control of a pleiotropic regulator called Mga, or the multiple gene regulator of GAS, including the gene encoding the streptococcal collagen-like protein, or sclA. Analysis of the genome sequence upstream of sclA revealed two potential Mga-binding sites with homology to...

  5. Genome-wide de novo prediction of cis-regulatory binding sites in prokaryotes

    Zhang, Shaoqiang; Xu, Minli; Li, Shan; Su, Zhengchang

    2009-01-01

    Although cis-regulatory binding sites (CRBSs) are at least as important as the coding sequences in a genome, our general understanding of them in most sequenced genomes is very limited due to the lack of efficient and accurate experimental and computational methods for their characterization, which has largely hindered our understanding of many important biological processes. In this article, we describe a novel algorithm for genome-wide de novo prediction of CRBSs with high accuracy. We desi...

  6. Consistent improvement of cross docking results using binding site ensembles generated with Elastic Network Normal Modes

    Rueda, Manuel; Bottegoni, Giovanni; Abagyan, Ruben

    2009-01-01

    The representation of protein flexibility is still a challenge for the state-of-the-art flexible ligand docking protocols. In this article we use a large and diverse benchmark to prove that is possible to improve consistently the cross docking performance against a single receptor conformation by using an equilibrium ensemble of binding site conformers. The benchmark contained 28 proteins, and the top ranked near native poses for the ligand were found 20% more efficiently than using a single ...

  7. Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites

    Chaloupková, R.; Prudnikova, T.; Řezáčová, Pavlína; Prokop, Z.; Koudeláková, T.; Daniel, L.; Březovský, J.; Ikeda-Ohtsubo, W.; Sato, Y.; Kutý, Michal; Nagata, Y.; Kutá-Smatanová, Ivana; Damborský, J.

    2014-01-01

    Roč. 70, July (2014), s. 1884-1897. ISSN 0907-4449 Grant ostatní: GA ČR(CZ) GAP207/12/0775; GA MŠk(CZ) LO1214; GA MŠk(CZ) LM2010005 Institutional support: RVO:61388963 ; RVO:67179843 Keywords : haloalkane dehalogenase * halide-binding site * catalytic activity * substrate specificity * enzyme stability Subject RIV: CE - Biochemistry Impact factor: 7.232, year: 2013

  8. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG3k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [3H]naloxone. The antibody which did not inhibit the binding of [3H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG3k antibody that blocked the binding of [3H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  9. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor.

    Maillet, Emeline L; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Margolskee, Robert F; Osman, Roman; Max, Marianna

    2015-10-01

    The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. PMID:26377607

  10. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG{sub 3}k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of ({sup 3}H)naloxone. The antibody which did not inhibit the binding of ({sup 3}H)naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG{sub 3}k antibody that blocked the binding of ({sup 3}H)naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form.

  11. Identification of critical residues in loop E in the 5-HT3ASR binding site

    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.

  12. Hydrophobicity of reactive site loop of SCCA1 affects its binding to hepatitis B virus

    Min Chen; Tong Cheng; Chen-Yu Xu; Ting Wu; Shan-Hai Ou; Tao Zhang; Jun Zhang; Ning-Shao Xia

    2005-01-01

    AIM: To investigate the role of SCCA2 and other SCCA1 molecules in the process of hepatitis B virus (HBV) binding to mammalian cells.METHODS: SCCA1 and SCCA2 were isolated from HepG2. Binding protein (BP) genes were obtained through PCR. Recombinant baculoviruses expressing SCCA1, SCCA2, BP, and different mutants were constructed and utilized to infect mammalian cells to investigate the binding ability of infected cells to HBV.RESULTS: A SCCA1 gene (A1) was isolated from HepG2, but it appeared to lack the binding ability of infected cells to HBV. Two mutants, A1-BP and BP-A1, were constructed by interchanging the carboxyl terminal of A1 and BP. Cells expressing A1-BP showed an increased virus bindingcapacity, but not BP-A1. Comparison of A1 sequence with the sequence of BP indicated the presence of only three amino acid changes in the carboxyl terminal, two of them were found in the reactive site loop (RSL) of SCCA1. Primary structure assay revealed that the hydrophobicity of BP and AJ515706 in this domain was strong, but A1 was relatively weak. Changing the aa349 of A1 from low hydrophobic glutamic acid to high hydrophobic valine enhanced HBV binding. In contrast, HBV binding was reduced by changing the aa349 of BP from valine to glutamic acid. CONCLUSION: The reslts suggest that the hydrophobicity of RSL of SCCA1 may play an important role in HBV binding to cells.

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

    Wilkens, Casper; Cockburn, Darrell; Andersen, Susan;

    2015-01-01

    Certain interactions between carbohydrate active enzymes and polysaccharides involve surface binding sites (SBS) situated on catalytic domains outside of the active site. We recently undertook to develop a toolbox for SBS identification and characterization. In affinity gel electrophoresis (AGE......) SBS containing proteins are migrating slower in native polyacrylamide electrophoresis gels cast with polysaccharide versus without polysaccharide. Amylolytic enzymes from GH13 and GH77 and xylanases from GH10 and GH11 are the best studied GH families with respect to SBS, presenting about half of the...

  14. Site-directed mutagenesis of boar proacrosin reveals residues involved in binding of zona pellucida glycoproteins.

    Jansen, S; Jones, R; Jenneckens, I; Marschall, B; Kriegesmann, B; Coadwell, J; Brenig, B

    1998-10-01

    Proacrosin, the zymogen form of the serine protease beta-acrosin, is thought to function as a secondary binding molecule between mammalian gametes during fertilization (Jansen et al., 1995: Int J Dev Biol 39, 501-510). The interaction involves strong ionic bonds between positively charged amino acids on proacrosin and negatively charged polysulphate groups on zona pellucida glycoproteins. In this investigation, we identified the basic residues on proacrosin that are important for this binding. Site-directed mutagenesis shows that two groups of amino acids comprising His47, Arg50, and Arg51 together with Arg250, Lys252, and Arg253 are crucial because their deletion or replacement severely reduces affinity for zona glycoproteins. Molecular models of proacrosin reveal that these residues are located along one face of the protein on two exposed surface loops that project over and around the catalytic site. These findings support the hypothesis that polysulphate binding sites on proacrosin are formed by a restricted number of basic amino acids on the surface of the protein, presenting a specific orientation that is complementary to negatively charged sulphate groups on zona glycoproteins. Identification and elucidation of the stereochemistry of these charged moieties will aid design of new kinds of nonsteroidal antifertility agents. PMID:9740326

  15. LASAGNA-Search: an integrated web tool for transcription factor binding site search and visualization.

    Lee, Chic; Huang, Chun-Hsi

    2013-03-01

    The release of ChIP-seq data from the ENCyclopedia Of DNA Elements (ENCODE) and Model Organism ENCyclopedia Of DNA Elements (modENCODE) projects has significantly increased the amount of transcription factor (TF) binding affinity information available to researchers. However, scientists still routinely use TF binding site (TFBS) search tools to scan unannotated sequences for TFBSs, particularly when searching for lesser-known TFs or TFs in organisms for which ChIP-seq data are unavailable. The sequence analysis often involves multiple steps such as TF model collection, promoter sequence retrieval, and visualization; thus, several different tools are required. We have developed a novel integrated web tool named LASAGNA-Search that allows users to perform TFBS searches without leaving the web site. LASAGNA-Search uses the LASAGNA (Length-Aware Site Alignment Guided by Nucleotide Association) algorithm for TFBS alignment. Important features of LASAGNA-Search include (i) acceptance of unaligned variable-length TFBSs, (ii) a collection of 1726 TF models, (iii) automatic promoter sequence retrieval, (iv) visualization in the UCSC Genome Browser, and (v) gene regulatory network inference and visualization based on binding specificities. LASAGNA-Search is freely available at http://biogrid.engr.uconn.edu/lasagna_search/. PMID:23599922

  16. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    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 [125I]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 [125I]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 [125I]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

  17. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    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.

  18. Improving the prediction of protein binding sites by combining heterogeneous data and Voronoi diagrams

    Fernandez-Fuentes Narcis

    2011-08-01

    Full Text Available Abstract Background Protein binding site prediction by computational means can yield valuable information that complements and guides experimental approaches to determine the structure of protein complexes. Predictions become even more relevant and timely given the current resolution of protein interaction maps, where there is a very large and still expanding gap between the available information on: (i which proteins interact and (ii how proteins interact. Proteins interact through exposed residues that present differential physicochemical properties, and these can be exploited to identify protein interfaces. Results Here we present VORFFIP, a novel method for protein binding site prediction. The method makes use of broad set of heterogeneous data and defined of residue environment, by means of Voronoi Diagrams that are integrated by a two-steps Random Forest ensemble classifier. Four sets of residue features (structural, energy terms, sequence conservation, and crystallographic B-factors used in different combinations together with three definitions of residue environment (Voronoi Diagrams, sequence sliding window, and Euclidian distance have been analyzed in order to maximize the performance of the method. Conclusions The integration of different forms information such as structural features, energy term, evolutionary conservation and crystallographic B-factors, improves the performance of binding site prediction. Including the information of neighbouring residues also improves the prediction of protein interfaces. Among the different approaches that can be used to define the environment of exposed residues, Voronoi Diagrams provide the most accurate description. Finally, VORFFIP compares favourably to other methods reported in the recent literature.

  19. The STAS domain of mammalian SLC26A5 prestin harbours an anion-binding site.

    Lolli, Graziano; Pasqualetto, Elisa; Costanzi, Elisa; Bonetto, Greta; Battistutta, Roberto

    2016-02-15

    Prestin is a unique ATP- and Ca(2+)-independent molecular motor with piezoelectric characteristics responsible for the electromotile properties of mammalian cochlear outer hair cells, i.e. the capacity of these cells to modify their length in response to electric stimuli. This 'electromotility' is at the basis of the exceptional sensitivity and frequency selectivity distinctive of mammals. Prestin belongs to the SLC26 (solute carrier 26) family of anion transporters and needs anions to function properly, particularly Cl(-). In the present study, using X-ray crystallography we reveal that the STAS (sulfate transporter and anti-sigma factor antagonist) domain of mammalian prestin, considered an 'incomplete' transporter, harbours an unanticipated anion-binding site. In parallel, we present the first crystal structure of a prestin STAS domain from a non-mammalian vertebrate prestin (chicken) that behaves as a 'full' transporter. Notably, in chicken STAS, the anion-binding site is lacking because of a local structural rearrangement, indicating that the presence of the STAS anion-binding site is exclusive to mammalian prestin. PMID:26635354

  20. Proteins and Their Interacting Partners: An Introduction to Protein–Ligand Binding Site Prediction Methods

    Daniel Barry Roche

    2015-12-01

    Full Text Available Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP and the Continuous Automated Model EvaluatiOn (CAMEO projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

  1. Antigen-binding site protection during radiolabeling leads to a higher immunoreactive fraction

    It is generally accepted that the immunointegrity of an antibody (Ab) depends on the preservation of its antigen-binding sites. Our goal was to radiolabel an antibody at several iodine:antibody molar ratios under conditions protecting its combining site and to compare its immunoreactive fraction (IRF) and electrophoretic mobility with those of the same antibody radiolabeled without protection. The data indicate that an antibody radiolabeled while its antigen-binding site is occupied by its antigen had the same IRF, regardless of the number of iodine atoms per antibody molecule. On the other hand, even at an I:Ab ratio of 1:1, the IRF of the same antibody radiolabeled without protection was lower than that of a protected one and decreased with increasing I:Ab ratios. In addition, the iodination of these Ab changes their electrophoretic mobility; however, when the Ab is labeled in the protected state, the degree of change is less. The binding of an antibody to its antigen prior to radiolabeling, therefore, enhances its immuno-integrity and prevents major conformational changes as reflected by electrophoresis

  2. Predicting DNA-binding sites of proteins from amino acid sequence

    Wu Feihong

    2006-05-01

    Full Text Available Abstract Background Understanding the molecular details of protein-DNA interactions is critical for deciphering the mechanisms of gene regulation. We present a machine learning approach for the identification of amino acid residues involved in protein-DNA interactions. Results We start with a Naïve Bayes classifier trained to predict whether a given amino acid residue is a DNA-binding residue based on its identity and the identities of its sequence neighbors. The input to the classifier consists of the identities of the target residue and 4 sequence neighbors on each side of the target residue. The classifier is trained and evaluated (using leave-one-out cross-validation on a non-redundant set of 171 proteins. Our results indicate the feasibility of identifying interface residues based on local sequence information. The classifier achieves 71% overall accuracy with a correlation coefficient of 0.24, 35% specificity and 53% sensitivity in identifying interface residues as evaluated by leave-one-out cross-validation. We show that the performance of the classifier is improved by using sequence entropy of the target residue (the entropy of the corresponding column in multiple alignment obtained by aligning the target sequence with its sequence homologs as additional input. The classifier achieves 78% overall accuracy with a correlation coefficient of 0.28, 44% specificity and 41% sensitivity in identifying interface residues. Examination of the predictions in the context of 3-dimensional structures of proteins demonstrates the effectiveness of this method in identifying DNA-binding sites from sequence information. In 33% (56 out of 171 of the proteins, the classifier identifies the interaction sites by correctly recognizing at least half of the interface residues. In 87% (149 out of 171 of the proteins, the classifier correctly identifies at least 20% of the interface residues. This suggests the possibility of using such classifiers to identify

  3. Intracellular binding site kinetics of 201 Tl binding compared to β-adrenergic analog receptors in dog myocardium

    It has been demonstrated with the multiple indicator dilution technique (MID) in an isolated dog heart preparation, that the permeation of thallium ions across the sarcolemma is about ten times larger compared to potassium ions (cellular permeability surface area product PSM 8.90 +- 4.60 vs. 0.65 +- 0.46 ml/min gsup(-1)). Similarly, the intracellular (IC) distribution space of Tlsup(+) is larger compared to that of Ksup(+). These properties may explain in part the rapid and large extraction of Tl in the myocardium. To explain the slow washout rate of Tl from the myocardium (T 1/2>600 sec determined with an on-line residue detection) we proposed a temporary binding of Tl to an IC protein. In experiments the permeation properties of 201 Tl were compared to 125 I-cyanopinodolol (I-CP) and 131 I metabenzylquanidin (I-MBG) by means of MID. The latter two substances act at the β-adrenergic receptor site. Both substances have a lower capillary permeability surface area product PSC of 0.43 +- 0.37 ml/min gsup(-1) compared to that of 201 Tl (1.37 +- 0.49 ml/min gsup(-1)). I-CP and I-MBG are sequestered extracellularly in contrast to Tl, which permeates intracellularly. However, the relation between time and instantaneous extraction during a single bolus passage of 201 Tl is very comparable to that of those receptor substances suggesting also a receptor-type kinetics for Tl with intracellular binding which may elucidate its prolonged washout. (Author)

  4. Computer assisted quantitative densitometric analysis of 125I-apamin binding sites in the central nervous system.

    Janicki, P K; Seibold, G; Siembab, D; Paulo, E A; Szreniawski, Z

    1989-01-01

    The binding sites for 125I-monoiododerivative of apamin in the central nervous system of rat, guinea-pig, chicken and frog were analysed and compared by computer assisted quantitative densitometric autoradiography on X-ray film. The highest level of binding sites in the rat and guinea-pig brain was found in the limbic-olfactory system and in the substantia gelatinosa of the spinal cord. In the chicken brain apamin binds preferentially to the tectum opticum and nuclei isthmi. In the frog brain no specific apamin binding sites were found. The role of presented topography for apamin binding sites is discussed in relation to neurotoxic properties of apamin. PMID:2641421

  5. A functional YY1 binding site is necessary and sufficient to activate Surf-1 promoter activity in response to serum growth factors.

    Cole, E G; Gaston, K

    1997-01-01

    The human Surf-1 and Surf-2 housekeeping genes are divergently transcribed and share a bi-directional, TATA-less promoter. Housekeeping promoters typically contain complex arrays of transcription factor binding sites and several studies have suggested that many of these sites might be functionally redundant. The Surf-1/Surf-2 promoter region contains four factor binding sites; members of the ETS family of transcription factors bind to two of these sites whilst YY1 binds to a third site immedi...

  6. Effect of iodination site on binding of radiolabeled ligand by insulin antibodies and insulin autoantibodies

    Four human insulins and four porcine insulins, each monoiodinated to the same specific activity at one of the four tyrosine residues (A14, A19, B16, B26) and purified by reversed-phase liquid chromatography, were tested in a radiobinding assay against a panel of insulin-antibody (IA)-positive sera from 10 insulin-treated diabetics and insulin-autoantibody-positive (IAA) sera from 10 nondiabetics. Of the 10 IAA-positive sera, five were fully cross reactive with both insulin species, and five were specific for human insulin. The rank order of binding of sera with the four ligands from each species was random for IA (mean rank values of 1.9 for A14, 2.0 for A19, 2.5 for B16, and 3.6 for B26 from a possible ranking range of 1 to 4), but more consistent for non-human-insulin-specific IAA (mean rank values 1.3 for A14, 3.8 for A19, 1.7 for B16, and 3.2 for B26 for labeled human insulins; 1.2 for A14, 4.0 for A19, 1.8 for B16, and 3.0 for B26 for labeled porcine insulins). The rank order of binding was virtually uniform for human-insulin-specific IAA (mean values 1.2 for A14, 3.0 for A19, 1.8 for B16, and 4.0 for B26). The influence of iodination site on the binding of labeled insulin appears to be dependent on the proximity of the labeled tyrosine to the antibody binding site and the clonal diversity, or restriction, of insulin-binding antibodies in the test serum. When IA and IAA are measured, the implications of this study regarding the choice of assay ligand may be important

  7. The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

    Mazin, A V; Kowalczykowski, S C

    1996-01-01

    The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound ...

  8. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

    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 [3H]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 [3H]DM and L-[35S]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

  9. Distinction between high-affinity [3H]phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle

    [3H]Phencyclidine ([3H]PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of [3H]PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the un-ionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific [3H]PCP binding was displaced effectively by several PCP analogs and Ca++ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of [3H]quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities

  10. The distribution of iron between the metal-binding sites of transferrin human serum.

    Williams, J; Moreton, K

    1980-02-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. PMID:7396826

  11. Synthesis of Zn-MOF incorporating titanium-hydrides as active sites binding H2 molecules

    This paper describes the synthetic effort for a Zn-MOF imparting Ti-H as a preferential binding site potentially capturing H2 molecules via Kubas-type interaction. The formation mechanism of Ti-H innate to the final material was potentially demonstrated to follow a radical dissociation rather than a β-hydrogen elimination and a C-H reductive elimination. - Graphical abstract: This study details the synthesis and the formation mechanism of Zn-MOF adsorbent site-isolating TiH3 that can potentially capture H2 molecules via Kubas-binding mechanism. - Highlights: • OH-functionalized Zn-MOF was employed as a reactive template to site-isolate TiH3. • This MOF was post-synthetically modified using a tetracyclohexyl titanium (IV). • This intermediate was hydrogenolyzed to change ligand from cyclohexyl to hydride. • Formation mechanism of TiH3 was investigated via two control GC–MS experiments. • Final Zn-MOF potentially site-isolating TiH3 species was used as a H2 adsorbent

  12. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

    El Sayyed, Hafez; Le Chat, Ludovic; Lebailly, Elise; Vickridge, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco; Espéli, Olivier

    2016-05-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  13. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans.

    Schilling, K M; Bowen, W H

    1992-01-01

    Many researchers have suggested that the role of glucan-mediated interactions in the adherence of Streptococcus mutans is restricted to accumulation of this cariogenic bacterium following its sucrose (i.e., glucan)-independent binding to saliva-coated tooth surfaces. However, the presence of enzymatically active glucosyltransferase in salivary pellicle suggests that glucans could also promote the initial adherence of S. mutans to the teeth. In the present study, the commonly used hydroxyapatite adherence assay was modified to include the incorporation of glucosyltransferase and the synthesis of glucans in situ on saliva-coated hydroxyapatite beads. Several laboratory strains and clinical isolates of S. mutans were examined for their ability to adhere to experimental pellicles, either with or without the prior formation of glucans in situ. Results showed that most strains of S. mutans bound stereospecifically to glucans synthesized in pellicle. Inhibition studies with various polysaccharides and fungal dextranase indicated that alpha 1,6-linked glucose residues were of primary importance in the glucan binding observed. Scanning electron microscopic analysis showed direct binding of S. mutans to hydroxyapatite surface-associated polysaccharide and revealed no evidence of trapping or cell-to-cell binding. S. mutans strains also attached to host-derived structures in experimental pellicles, and the data suggest that the bacterial adhesins which recognize salivary binding sites were distinct from glucan-binding adhesins. Furthermore, glucans formed in experimental pellicles appeared to mask the host-derived components. These results support the concept that glucans synthesized in salivary pellicle can promote the selective adherence of the cariogenic streptococci which colonize human teeth. Images PMID:1530843

  14. Inhibitory mechanisms and binding site location for serotonin selective reuptake inhibitors on nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Bhumireddy, Pankaj; Ortells, Marcelo O

    2010-05-01

    Functional and structural approaches were used to examine the inhibitory mechanisms and binding site location for fluoxetine and paroxetine, two serotonin selective reuptake inhibitors, on nicotinic acetylcholine receptors (AChRs) in different conformational states. The results establish that: (a) fluoxetine and paroxetine inhibit h alpha1beta1 gammadelta AChR-induced Ca(2+) influx with higher potencies than dizocilpine. The potency of fluoxetine is increased approximately 10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs elicited by these antidepressants, (b) fluoxetine and paroxetine inhibit the binding of the phencyclidine analog piperidyl-3,4-(3)H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [(3)H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where neutral dizocilpine and fluoxetine and the conformer of protonated fluoxetine with the highest LUDI score interact with the domain between the valine (position 13') and leucine (position 9') rings. Molecular mechanics calculations also evidence electrostatic interactions of protonated fluoxetine at positions 20', 21', and 24'. Protonated dizocilpine bridges these two binding domains by interacting with the valine and outer (position 20') rings. The high proportion of protonated fluoxetine and dizocilpine calculated at physiological pH suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel between the leucine and valine rings, a domain shared with phencyclidine, finally blocking ion flux and inducing AChR desensitization. PMID:20079457

  15. Influence of 3‧-3‧ inversion of polarity site within d(TGGGGT) on inter quartet cation binding

    Šket, Primož; Korbar, Tjaša; Plavec, Janez

    2014-10-01

    Stability, dynamics and function of nucleic acids are affected by nature of cations that are involved in interaction with specific functionalities. Introduction of inversion of polarity sites represents a very useful and chemically accessible backbone modification, which can alter the binding affinity of cations. NMR study on cation binding between G-quartets in tetramolecular G-quadruplex adopted by d(5‧TGG3‧-3‧GGT5‧) with 3‧-3‧ inversion of polarity sites in the middle of G-tract showed existence of two different G-quadruplex forms with all strands in parallel orientation, where all guanine residues adopt anti conformation around glycosidic bonds in the presence of 15NH4+ ions. In one of the forms all three binding sites are equally populated, while in the second form the binding site next to the inversion of polarity site is not fully populated by 15NH4+ ions.

  16. Automatic generation of 3D motifs for classification of protein binding sites

    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

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

    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)

  18. Binding site on Rhodospirillum rubrum cytochrome c2 for the Rhodospirillum rubrum cytochrome bc1 complex

    Cytochrome c2 and the detergent-solubilized cytochrome bc1 complex, both from Rhodospirillum rubrum, form a tight complex at a low ionic strength that can be isolated by gel permeation chromatography. The dissociation constant of the complex is estimated to be 10-6 M or less. [14C]-labelled and [3H]-labelled acetic anhydride were used in the acetylation experiments, and the 3H/14C ratio was analyzed. The binding site for the cytochrome bc1 complex on cytochrome c2 was analyzed by differential acetylation of lysine residues in free and cytochrome bc1 complex bound cytochrome c2. In bound cytochrome c2, three lysine residues at sequence positions 12, 13, and 97 were less reactive toward acetic anhydride. Lys13, which is located above the exposed heme edge, was the least reactive, i.e., the most shielded by the cytochrome bc1 complex. Correlating this information with the crystal structure of cytochrome c2 indicates that the binding site for the cytochrome bc1 complex on cytochrome c2 involves a surface area above, and probably including, the exposed heme edge. This mode of binding is similar to that observed for horse cytochrome c interacting with the mitochrondrial cytochrome bc1 complex. A simplified version of the method of differential chemical modification is presented

  19. Identification of two potential receptor-binding sites for hGM-CSF

    Eberhardt M.O.

    2003-01-01

    Full Text Available Two receptor-binding sites for hGM-CSF are described. Competitive binding ELISA using four monoclonal antibodies (MAbs showed different epitope recognitions. The antibody combining sites were mapped using sets of overlapping peptides and hexapeptide libraries prepared by the SPOT synthesis technique. We identified the conformationally dependent epitopes A18E21R23R24F119 and R23E21N17W13 bound by MAb CC5B5 and the nonlinear epitope P118F119W13E14 bound by MAb M1B8. The epitopes recognized by these two MAbs are very closely located on the native protein surface. The peptide L61YKQGKLRGSLTK72 was recognized by MAb M7E10 and the peptide A1PAR4, representing the N-terminal sequence of the protein, was bound by the nonneutralizing MAb CC1H7. Inhibition assays of the GM-CSF biological activity demonstrated that MAb M1B8, CC5B5 and M7E10 bind to domains which are responsible for the interaction of the cytokine with the GM-CSF receptor.

  20. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    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.

  1. Wiz binds active promoters and CTCF-binding sites and is required for normal behaviour in the mouse

    Isbel, Luke; Prokopuk, Lexie; Wu, Haoyu; Daxinger, Lucia; Oey, Harald; Spurling, Alex; Lawther, Adam J; Hale, Matthew W; Whitelaw, Emma

    2016-01-01

    We previously identified Wiz in a mouse screen for epigenetic modifiers. Due to its known association with G9a/GLP, Wiz is generally considered a transcriptional repressor. Here, we provide evidence that it may also function as a transcriptional activator. Wiz levels are high in the brain, but its function and direct targets are unknown. ChIP-seq was performed in adult cerebellum and Wiz peaks were found at promoters and transcription factor CTCF binding sites. RNA-seq in Wiz mutant mice identified genes differentially regulated in adult cerebellum and embryonic brain. In embryonic brain most decreased in expression and included clustered protocadherin genes. These also decreased in adult cerebellum and showed strong Wiz ChIP-seq enrichment. Because a precise pattern of protocadherin gene expression is required for neuronal development, behavioural tests were carried out on mutant mice, revealing an anxiety-like phenotype. This is the first evidence of a role for Wiz in neural function. DOI: http://dx.doi.org/10.7554/eLife.15082.001 PMID:27410475

  2. Quinacrine impairs enterovirus 71 RNA replication by preventing binding of polypyrimidine-tract binding protein with internal ribosome entry sites.

    Jianmin Wang

    Full Text Available Since the 1980s, epidemics of enterovirus 71 (EV71 and other enteroviruses have occurred in Asian countries and regions, causing a wide range of human diseases. No effective therapy is available for the treatment of these infections. Internal ribosome entry sites (IRESs are indispensable for the initiation of translation in enteroviruses. Several cellular factors, as well as the ribosome, are recruited to the conserved IRES during this process. Quinacrine intercalates into the RNA architecture and inhibits RNA transcription and protein synthesis, and a recent study showed that quinacrine inhibited encephalomyocarditis virus and poliovirus IRES-mediated translation in vitro without disrupting internal cellular IRES. Here, we report that quinacrine was highly active against EV71, protecting cells from EV71 infection. Replication of viral RNA, expression of viral capsid protein, and production of virus were all strongly inhibited by quinacrine. Interaction of the polypyrimidine tract-binding protein (PTB with the conserved IRES was prevented by quinacrine. Coxsackieviruses and echovirus were also inhibited by quinacrine in cultured cells. These results indicate that quinacrine may serve as a potential protective agent for use in the treatment of patients with chronic enterovirus infection.

  3. MeRNA: a Database of Metal Ion Binding Sites in RNAStructures

    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.

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

    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 seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β......-cyclodextrin (β-CD) albeit with a measurable K D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS...

  5. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. PMID:27016086

  6. Biocompatible medical implant materials with binding sites for a biodegradable drug-delivery system

    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

  7. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    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 Bmax and Kd 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 Bmax 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 Bmax, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in Kd represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing Kd, and dimethylphosphorylation of Ser203 decreasing Kd. 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

  8. (/sup 3/H)nitrobenzylthioinosine binding as a probe for the study of adenosine uptake sites in brain

    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 synaptic region. A study of the tissue distribution of the (/sup 3/H)NBI sites revealed very high densities of binding in erythrocyte, lung, and testis, with much lower binding densities in brain, kidney, liver, muscle, and heart. The binding affinity in the former group was approximately 1.5 nM, whereas that in the latter group was 0.15 nM, suggesting two types of binding sites. The pharmacologic profile of (/sup 3/H)NBI binding was consistent with its function as the adenosine transport site, distinct from the adenosine receptor, since thiopurines were very potent inhibitors of binding whereas adenosine receptor ligands, such as cyclohexyladenosine and 2-chloroadenosine, were three to four orders of magnitude less potent. (/sup 3/H)NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain.

  9. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  10. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, ...

  11. Impact of Alu repeats on the evolution of human p53 binding sites

    Sirotin Michael V

    2011-01-01

    Full Text Available Abstract Background The p53 tumor suppressor protein is involved in a complicated regulatory network, mediating expression of ~1000 human genes. Recent studies have shown that many p53 in vivo binding sites (BSs reside in transposable repeats. The relationship between these BSs and functional p53 response elements (REs remains unknown, however. We sought to understand whether the p53 REs also reside in transposable elements and particularly in the most-abundant Alu repeats. Results We have analyzed ~160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements. In addition, using a position weight matrix approach, we found ~400,000 potential p53 BSs in Alu elements genome-wide. Importantly, these putative BSs are located in the same regions of Alu repeats as the functional p53 REs - namely, in the vicinity of Boxes A/A' and B of the internal RNA polymerase III promoter. Earlier nucleosome-mapping experiments showed that the Boxes A/A' and B have a different chromatin environment, which is critical for the binding of p53 to DNA. Here, we compare the Alu-residing p53 sites with the corresponding Alu consensus sequences and conclude that the p53 sites likely evolved through two different mechanisms - the sites overlapping with the Boxes A/A' were generated by CG → TG mutations; the other sites apparently pre-existed in the progenitors of several Alu subfamilies, such as AluJo and AluSq. The binding affinity of p53 to the Alu-residing sites generally correlates with the age of Alu subfamilies, so that the strongest sites are embedded in the 'relatively young' Alu repeats. Conclusions The primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. One of the selective factors responsible for the frequent occurrence of Alu repeats in introns may be related to the p53-mediated regulation of Alu

  12. Novel mechanisms of intracellular cholesterol transport: oxysterol-binding proteins and membrane contact sites.

    Du, Ximing; Brown, Andrew J; Yang, Hongyuan

    2015-08-01

    Cholesterol is an essential membrane constituent, and also plays a key role in cell signalling. Within a cell, how cholesterol is transported and how its heterogeneous distribution is maintained are poorly understood. Recent advances have identified novel pathways and regulators of cholesterol trafficking. Sterol transfer by lipid-binding proteins, such as OSBP (oxysterol-binding protein), coupled with phosphatidylinositol 4-phosphate exchange at membrane contact sites (MCSs) has emerged as a new theme of cholesterol transport between organellar membranes. Moreover, a previously unappreciated role of peroxisomes in cholesterol trafficking has been revealed recently. These discoveries highlight the crucial role of MCSs, or junctions, in facilitating lipid movement, and provide mechanistic insights into how cholesterol is sorted in cells. PMID:25932595

  13. The use of LeuT as a model in elucidating binding sites for substrates and inhibitors in neurotransmitter transporters

    Løland, Claus Juul

    2015-01-01

    -function relationships on mammalian NSS proteins has so far been unsuccessful. The crystal structure of the bacterial NSS protein, LeuT, has been a turning point in structural investigations. SCOPE OF REVIEW: To provide an update on what is known about the binding sites for substrates and inhibitors in the LeuT. The...... different binding modes and binding sites will be discussed with special emphasis on the possible existence of a second substrate binding site. It is the goal to give an insight into how investigations on ligand binding in LeuT have provided basic knowledge about transporter conformations and translocation......T is a suitable model for the molecular mechanisms behind substrate translocation. GENERAL SIGNIFICANCE: Structure and functional aspects of NSS proteins are central for understanding synaptic transmission. With the purification and crystallization of LeuT as well as the dopamine transporter from...

  14. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis.

    John Gatfield

    Full Text Available The competitive endothelin receptor antagonists (ERA bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min occupancy half-lives at the ET(A receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A receptor-antagonist interaction modes, we performed functional studies using ET(A receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and

  15. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis.

    Gatfield, John; Mueller Grandjean, Celia; Bur, Daniel; Bolli, Martin H; Nayler, Oliver

    2014-01-01

    The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ET(A) receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A) receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A) receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A) receptor-antagonist interaction modes, we performed functional studies using ET(A) receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A) receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A) receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and insurmountable

  16. Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.

    Kiss, András L; Palló, Anna; Náray-Szabó, Gábor; Harmat, Veronika; Polgár, László

    2008-05-01

    It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme. PMID:18325786

  17. [3H]opipramol labels a novel binding site and sigma receptors in rat brain membranes

    Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. [3H]Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). [3H]Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects

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

    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 Ca2+-binding and Na+-binding sites. The Ca2+ 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

  19. Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating

    Szollosi, A; Muallem, D. R.; Csanady, L.; P.; Vergani

    2011-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is tr...

  20. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    Kratochwil Nicole A

    2007-10-01

    Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

  1. NMR investigation of the binding of the anticancer drug actinomycin D to oligodeoxyribonucleotides with isolated 5'd(GC)3' binding sites

    Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G-C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A-T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3 shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A-T and G-C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations. The variety of ActD adducts observed for these relatively simple sequences indicates that ActD binding to natural DNA must be much more complex than previously anticipated

  2. Structural analyses of the Slm1-PH domain demonstrate ligand binding in the non-canonical site.

    Kanchan Anand

    Full Text Available BACKGROUND: Pleckstrin homology (PH domains are common membrane-targeting modules and their best characterized ligands are a set of important signaling lipids that include phosphatidylinositol phosphates (PtdInsPs. PH domains recognize PtdInsPs through two distinct mechanisms that use different binding pockets on opposite sides of the β-strands 1 and 2: i a canonical binding site delimited by the β1-β2 and β3-β4loops and ii a non-canonical binding site bordered by the β1-β2 and β5-β6loops. The PH domain-containing protein Slm1 from budding yeast Saccharomyces cerevisiae is required for actin cytoskeleton polarization and cell growth. We recently reported that this PH domain binds PtdInsPs and phosphorylated sphingolipids in a cooperative manner. PRINCIPAL FINDINGS: To study the structural basis for the Slm1-PH domain (Slm1-PH specificity, we co-crystallized this domain with different soluble compounds that have structures analogous to anionic lipid head groups of reported Slm1 ligands: inositol 4-phosphate, which mimics phosphatidylinositol-4-phosphate (PtdIns(4P, and phosphoserine as a surrogate for dihydrosphingosine 1-phosphate (DHS1-P. We found electron densities for the ligands within the so-called non-canonical binding site. An additional positively charged surface that contacts a phosphate group was identified next to the canonical binding site. CONCLUSIONS: Our results suggest that Slm1-PH utilizes a non-canonical binding site to bind PtdInsPs, similar to that described for the PH domains of β-spectrin, Tiam1 and ArhGAP9. Additionally, Slm1-PH may have retained an active canonical site. We propose that the presence of both a canonical and a non-canonical binding pocket in Slm1-PH may account for the cooperative binding to PtdInsPs and DHS-1P.

  3. Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

    A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, the authors have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung

  4. Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.

    Atsumi, Shogo; Mizuno, Eri; Hara, Hirotaka; Nakanishi, Kazuko; Kitami, Madoka; Miura, Nami; Tabunoki, Hiroko; Watanabe, Ayako; Sato, Ryoichi

    2005-07-01

    We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding. PMID:16000811

  5. Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site

    Kvist, Trine; Steffensen, Thomas Bielefeldt; Greenwood, Jeremy R;

    2013-01-01

    the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual...

  6. DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene

    Esteves, Leda I C V; Javaroni, Afonso C; Nishimoto, Inês N;

    2005-01-01

    Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional...

  7. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site

    S.R. Eberhardy; J. Goncalves; S. Coelho; D.J. Segal; B. Berkhout; C.F. Barbas

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjac

  8. Sulfated polymannuroguluronate inhibits Tat-induced SLK cell adhesion via a novel binding site, a KKR spatial triad

    Yan-lin WU; Jing AI; Jing-ming ZHAO; Bing XIONG; Xiao-jie XIN; Mei-yu GENG; Xian-liang XIN; Han-dong JIANG

    2011-01-01

    Aim: Sulfated polymannuroguluronate (SPMG), a candidate anti-AIDS drug, inhibited HIV replication and interfered with HIV entry into host T lymphocytes. SPMG has high binding affinity for the transactivating factor of the HIV-1 virus (Tat) via its basic domain. However, deletion or substitution of the basic domain affected, but did not completely eliminated Tat-SPMG interactions. Here, we sought to identify other SPMG binding sites in addition to the basic domain.Methods: The potential SPMG binding sites were determined using molecular simulation and a surface plasmon resonance (SPR) based competitive inhibition assay. The effect of SPMG on Tat induced adhesion was evaluated using a cell adhesion assay. Results: The KKR domain, a novel high-affinity heparin binding site, was identified, which consisted of a triad of Lys12, Lys41, and Arg78. The KKR domain, spatially enclosed SPMG binding site on Tat, functions as another binding domain for SPMG. Further func- tional evaluation demonstrated that SPMG inhibits Tat-mediated SLK cell adhesion by directly binding to the KKR region.Conclusion: The KKR domain is a novel high-affinity binding domain for SPMG. Our findings provide important new insights into the molecular mechanisms of SPMG and a potential therapeutic intervention for Tat-induced cell adhesion.

  9. Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

    The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentration. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors

  10. Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

    Medici, N.; Minucci, S.; Nigro, V.; Abbondanza, C.; Armetta, I.; Molinari, A.M.; Puca, G.A. (Istituto di Patologia Generale ed Oncologia, Naples (Italy))

    1989-01-10

    The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Analysis of affinity-labeled ER by ({sup 3}H)tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentration. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.

  11. Probing of possible olanzapine binding site on human serum albumin: Combination of spectroscopic methods and molecular dynamics simulation

    Human serum albumin (HSA)-drug binding affinity is one of the major factors that determine the pharmacokinetics, halftime and bioavailability of drugs in various tissues. In the present study, the interaction of olanzapine (OLZ), a thienobenzodiazepine drug, administered for the treatment of schizophrenia and bipolar disorder, with HSA has been studied using spectroscopic methods such as ultraviolet absorbance, fluorescence and FTIR combined with computational procedures. Analyzing of the Stern–Volmer quenching data showed only one primary binding site on HSA with a binding constant of 4.12×104 M−1 at 298 K. Thermodynamic analyses showed enthalpy change (ΔH°) and entropy change (ΔS°) were 28.03±3.42 kJ mol−1 and −25.52±11.52 J mol−1 K−1, respectively. Molecular docking results suggested the hydrophobic residues such as Val216, Leu327, Ala350 and polar residues such as Glu354 play an important role in the drug binding. Decrement in α-helix content of the protein upon OLZ binding was also confirmed by evidences provided by molecular dynamics simulation as well as FTIR spectroscopy. - Highlights: • Leu327, Ala350 as well as hydrophilic residues of HSA play an important role in the binding reaction. • The drug has only one primary binding site on HSA with a binding constant of 4.12×104 M−1 at 298 K. • The drug binds near to site I

  12. Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations.

    Ratheal, Ian M; Virgin, Gail K; Yu, Haibo; Roux, Benoît; Gatto, Craig; Artigas, Pablo

    2010-10-26

    The Na/K pump is a P-type ATPase that exchanges three intracellular Na(+) ions for two extracellular K(+) ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na(+) or K(+); site III binds only Na(+)) are poorly understood. We studied cation selectivity by outward-facing sites (high K(+) affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium(+), methylguanidinium(+), and aminoguanidinium(+) produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K(+), and (ii) induction of pump-mediated, guanidinium-derivative-carried inward current at negative potentials without Na(+) and K(+). In contrast, formamidinium(+) and acetamidinium(+) induced K(+)-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K(+) congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li(+) induced Na(+)-like VDI, whereas all metals tested except Na(+) induced K(+)-like outward currents. Pump-mediated K(+)-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium(+) derivatives suggest that Na(+) binds to site III in a hydrated form and that the inward current observed without external Na(+) and K(+) represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites. PMID:20937860

  13. Casein kinase II phosphorylation increases the rate of serum response factor-binding site exchange.

    Marais, R M; Hsuan, J J; McGuigan, C.; Wynne, J; Treisman, R

    1992-01-01

    Recombinant baculoviruses were used to express wild-type serum response factor (SRF) and a mutant, SRF.CKIIA, which lacks all four serine residues in the major casein kinase II (CKII) site at residues 77-90. Purified recombinant SRF binds DNA with an affinity and specificity indistinguishable from that of HeLa cell SRF, and activates transcription in vitro. Comparative phosphopeptide analysis of the wild-type and mutant proteins demonstrated that the wild-type protein is phosphorylated at the...

  14. Post-translational Modifications near the Quinone Binding Site of Mammalian Complex I*

    Carroll, Joe; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2013-01-01

    Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicini...

  15. Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function.

    Diamant, S; Eldar-Geva, T.; Atlas, D.

    1992-01-01

    1. An alpha 2-adrenoceptor antagonist, idazoxan, that binds to both alpha 2-adrenoceptors and to imidazoline sites (IR), has been used to characterize human placental IR. Human placenta is shown to be the richest source of IR (1800 +/- 100 fmol mg-1 protein; Kd 38.9 +/- 3.4 nM). 2. Primary cells derived from human placenta and grown in monolayers, also displayed a high density of receptors (3209 +/- 136 fmol mg-1 in cytotrophoblasts and 3642 +/- 144 fmol mg-1 protein in syncytiotrophoblast en...

  16. L-baclofen-sensitive GABAB binding sites in the medial vestibular nucleus localized by immunocytochemistry

    Holstein, G. R.; Martinelli, G. P.; Cohen, B.

    1992-01-01

    L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.

  17. Identification of Inhibitor Binding Site in Human Sirtuin 2 Using Molecular Docking and Dynamics Simulations

    Sugunadevi Sakkiah; Mahreen Arooj; Manian Rajesh Kumar; Soo Hyun Eom; Keun Woo Lee

    2013-01-01

    The ability to identify the site of a protein that can bind with high affinity to small, drug-like compounds has been an important goal in drug design. Sirtuin 2 (SIRT2), histone deacetylase protein family, plays a central role in the regulation of various pathways. Hence, identification of drug for SIRT2 has attracted great interest in the drug discovery community. To elucidate the molecular basis of the small molecules interactions to inhibit the SIRT2 function we employed the molecular doc...

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

    Wilkens, Casper

    with GH62 constitutes clan F of glycoside hydrolases. Sequence alignments and mutational analysis demonstrated the active site catalytic triad as Asp28 (general base), Glu188 (general acid) and Asp136 (pKa-modulator and general acid stabilizer). Barley starch synthase I (HvSSI) has previously been shown...... to contain a SBS, which in the present thesis is shown to be responsible for HvSSIs affinity for β-cyclodextrin (β-CD) and maltooligosaccharides. HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. Using surface plasmon resonance (SPR) it was shown that wild-type HvSSI has...

  19. Evolutionary variation of papillomavirus E2 protein and E2 binding sites

    Angeletti Peter C; Waltke Mackenzie; Rogers Adam

    2011-01-01

    Abstract Background In an effort to identify the evolutionary changes relevant to E2 function, within and between papillomavirus genera, we evaluated the E2 binding sites (E2BS)s inside the long-control-region (LCR), and throughout the genomes. We identified E2BSs in the six largest genera of papillomaviruses: Alpha, Beta, Gamma, Delta, Lambda, and Xi-papillomaviruses (128 genomes), by comparing the sequences with a model consensus we created from known functional E2BSs (HPV16, HPV18, BPV1). ...

  20. Characterization of 5-HT1D receptor binding sites in post-mortem human brain cortex.

    Martial, J; de Montigny, C; Cecyre, D; Quirion, R

    1991-01-01

    The present study provides further evidence for the presence of serotonin1D (5-HT1D) receptors in post-mortem human brain. Receptor binding parameters in temporal cortex homogenates were assessed using [3H]5-HT in the presence of 100 nM 8-OH-DPAT, 1 microM propranolol and 1 microM mesulergine to prevent labelling of the 5-HT1A, 5-HT1B and 5-HT1C sites, respectively. Under these conditions, [3H]5-HT apparently bound to a class of high affinity (Kd = 5.0 +/- 1.0 nM) low capacity (Bmax = 96 +/- ...

  1. A role for the weak DnaA binding sites in bacterial replication origins

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility that...

  2. Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site.

    Wakefield, J K; Rhim, H; Morrow, C D

    1994-01-01

    The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing m...

  3. Signatures of RNA binding proteins globally coupled to effective microRNA target sites

    Jacobsen, Anders; Wen, Jiayu; Marks, Debora S;

    2010-01-01

    in 3' untranslated regions (UTRs) correlate with expression changes following transfection of small RNAs. The most significantly overrepresented motifs in down-regulated mRNAs are two novel U-rich motifs (URMs), UUUUAAA and UUUGUUU, recently discovered as binding sites for the ELAVL4 (also known as...... HuD) RNA-BP. Surprisingly, the most significantly overrepresented motif in up-regulated mRNAs is the heptanucleotide AU-rich element (ARE), UAUUUAU, which is known to affect mRNA stability via at least 20 different RNA-BPs. We show that destabilization mediated by the transfected miRNA is generally...

  4. (+)- and (-)-N-allylnormetazocine binding sites in mouse brain: in vitro and in vivo characterization and regional distribution

    Compton, D.R.; Bagley, R.B.; Katzen, J.S.; Martin, B.R.

    1987-06-01

    In vivo and in vitro binding studies, both in whole brain and in selected areas, indicate that non-identical (+)- and (-)-NANM sites exist in the mouse brain, and each exhibits a different regional distribution. The in vivo binding of (+)-/sup 3/H-NANM was found to be saturable at pharmacologically relevant doses, and represents a relatively small (10 - 22%) portion of total brain (+)-/sup 3/H-NANM concentrations. The in vivo binding of (+)-/sup 3/H-NANM was selectively displaced by (+)-NANM and PCP, and more sensitive to haloperidol and (+)-ketocyclazocine than the (-)-/sup 3/H-NANM site. The in vivo binding of (-)-/sup 3/H-NANM was selectively displaced by (-)-NANM, and more sensitive to naloxone and (-) ketocyclazocine than the (+)-/sup 3/H-NANM site, and insensitive to PCP. This study indicates that the investigation of NANM binding sites is possible using in vivo binding techniques, and that each isomer apparently binds, in the mouse brain, to a single class of distinct sites. 32 references, 4 figures, 2 tables.

  5. Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites

    The present study demonstrates a differential effect of various detergent treatments on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 caused a decrease of about 70% in [3H]Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on [3H]PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in [3H]Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in [3H]Ro 5-4864 and [3H]PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in [3H]Ro 5-4864 binding, while [3H]PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin

  6. Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

    Oikonomakos, N G; Zographos, S E; Tsitsanou, K E; Johnson, L N; Acharya, K R

    1996-12-01

    It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis. PMID:8976550

  7. Characterization of imidazole binding sites labeled by 3H-p-aminoclonidine in the bovine ventrolateral medulla (VLM)

    Since vasodepressor responses to clonidine (CLON) are mediated by neurons in the C1 area of VLM, and CLON, an imidazole (IM), bind to IM as well as alpha-adrenergic receptors in brain, the authors characterized binding sites for CLON in RVL. IM sites were measured in preincubated, washed P2 membranes (0.1 mg protein/ml) of YLM using 3H-p-aminoclonidine (3H-PAC) as a radioligand (40 min., 250C). (-)-Norepinephrine (NE; 10 μM) inhibited only 70% of specific 3H-PAC binding (1.0 nM) as defined by 10 μM phentolamine. The sites displaced by NE resemble alpha2-adrenergic receptors. When 10μM NE was included in the incubation medium, the remaining 3H-PAC sites had a high affinity for CLON (IC50 = 6 +/- 1 nM) and were saturable (K/sub D/ = 1.5 +/- 0.4 nM). Imidazole-4-acetic acid (IC50 = 800 +/- 23 nM) and cimetadine (IC50 = 480 +/- 88 nM) potently inhibited these non-adrenergic sites, but did not affect total 3H-PAC binding to cerebral cortical membranes indicating regional specificity. Histamine was a weak inhibitor (IC50 = 38 +/- 3 μM) in cortex, but was more potent (IC50 = 2 +/- 1 μM) in the RVL. The putative endogenous ligand clonidine displacing substance (CDS), has a 9-fold selectivity of the IM component of 3H-PAC binding in VLM. The authors conclude CLON binds to IM and adrenergic recognition sites in VLM. Binding to one or both sites may mediate the vasodepressor VLM. Binding to one or both sites may mediate the vasodepressor actions of the drug actions of the drug

  8. Correlation between oxytocin neuronal sensitivity and oxytocin-binding sites in the amygdala of the rat: electrophysiological and histoautoradiographic study.

    Condés-Lara, M; Veinante, P; Rabai, M; Freund-Mercier, M J

    1994-02-21

    Central nucleus (Ce), basomedial and medial nuclei of the amygdala (AMG), and some parts of the striato-pallidal system, present high densities of oxytocin (OT)-binding sites. In order to examine whether these OT-binding sites are functional receptors, the OT neuronal sensitivity and the presence of OT-binding sites were investigated using electrophysiological and autoradiographical techniques. To identify the AMG cells, electrical stimulation of the oval subnucleus of the bed nucleus of the stria terminalis (Ov) and of the parabrachial nucleus (Pb) were performed. Somatic and auditory sensory stimulations were also tested. OT was applied by iontophoresis during extracellular single unit recordings of cells which were localized in frontal brain sections subsequently used for histoautoradiographic detection of OT-binding sites. Cells responding to Ov nucleus stimulation were located in the AMG, mainly in the Ce nucleus, whereas those responding to Pb nucleus stimulation were distributed in the Ce nucleus and in the postero lateral part of the caudate putamen. Iontophoretic OT application excited 45% of the recorded cells (43/96) among which OT alone activated spontaneous firing rate of 30 and potentiated the L-Glutamate (GLU)-induced activation on 13. These OT-sensitive neurons were located mainly in the AMG and caudate putamen areas containing OT-binding sites. These results strongly suggest that OT-binding sites found in the AMG are functional receptors upon which OT could act as a neurotransmitter and as a neuromodulator to regulate autonomic functions. PMID:8180808

  9. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  10. Interaction of D-LSD with binding sites in brain: a study in vivo and in vitro

    The localization of [3H]-d-lysergic acid diethylamide ([3H]LSD) binding sites in the mouse brain was compared in vivo and in vitro. Radioautography of brain sections incubated with [3H]LSD in vitro revealed substantial specific [3H]LSD binding in cortical layers III-IV and areas CA1 and dentate gyrus in hippocampus. In contrast, in brain sections from animals that received [3H]LSD in vivo, binding in hippocampus was scant and diffuse, although the pattern of labeling in cortex was similar to that seen in vitro. The low specific binding in hippocampus relative to cortex was confirmed by homogenate filtration studies of brain areas from mice that received injections of [3H]LSD. Time-course studies established that peak specific binding at ten minutes was the same in cortex and hippocampus. At all times, binding in hippocampus was about one-third of that in cortex; in contrast, the concentration of free [3H]LSD did not vary between regions. This finding was unexpected, because binding studies in vitro in membrane preparations indicated that the density and affinity of [3H]LSD binding sites were similar in both brain regions. Saturation binding studies in vivo showed that the lower amount of [3H]LSD binding in hippocampus was attributable to a lower density of sites labeled by [3H]LSD. The pharmacological identify of [3H]LSD binding sites in vivo may be relevant to the hallucinogenic properties of LSD and of other related hallucinogens

  11. Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

    Friedman, H M; Glorioso, J C; Cohen, G H; Hastings, J C; Harris, S L; Eisenberg, R J

    1986-01-01

    The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigen...

  12. Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase.

    Francis, Kevin; Gadda, Giovanni

    2009-10-01

    A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK(is)versus the ionic volume of the anion with slopes of 0.041+/-0.001 mM/A(3) and 0.027+/-0.001 mM/A(3) for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K(d) values of approximately 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme. PMID:19683782

  13. A Novel Alignment-Free Method for Comparing Transcription Factor Binding Site Motifs

    Xu, Minli; Su, Zhengchang

    2010-01-01

    Background Transcription factor binding site (TFBS) motifs can be accurately represented by position frequency matrices (PFM) or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between the two compared motifs. In some applications, alignment-free methods might be preferred; however, few such methods with high accuracy have been described. Methodology/Principal Findings Here we describe a novel alignment-free method for quantifying the similarity of motifs using their PFMs by converting PFMs into k-mer vectors. The motifs could then be compared by measuring the similarity among their corresponding k-mer vectors. Conclusions/Significance We demonstrate that our method in general achieves similar performance or outperforms the existing methods for clustering motifs according to their binding preference and identifying similar motifs of transcription factors of the same family. PMID:20098703

  14. A novel alignment-free method for comparing transcription factor binding site motifs.

    Minli Xu

    Full Text Available BACKGROUND: Transcription factor binding site (TFBS motifs can be accurately represented by position frequency matrices (PFM or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between the two compared motifs. In some applications, alignment-free methods might be preferred; however, few such methods with high accuracy have been described. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel alignment-free method for quantifying the similarity of motifs using their PFMs by converting PFMs into k-mer vectors. The motifs could then be compared by measuring the similarity among their corresponding k-mer vectors. CONCLUSIONS/SIGNIFICANCE: We demonstrate that our method in general achieves similar performance or outperforms the existing methods for clustering motifs according to their binding preference and identifying similar motifs of transcription factors of the same family.

  15. Interleukin 3 activates human blood basophils via high-affinity binding sites

    Pure populations of human basophilic granulocytes were obtained from chronic myeloid leukemia (CML) blood by negative selection using a mixture of monoclonal antibodies and complement. 125I-radiolabeled recombinant human interleukin 3 (rhIL-3) bound to purified basophils in a specific manner. Quantitative binding studies and Scatchard plot analyses performed on samples from two donors revealed the presence of a single class of high-affinity IL-3 binding sites. Purified CML basophils maintained in suspension in the presence of rhIL-3 incorporated up to 12 times more [3H]thymidine than basophils in control cultures. Furthermore, after preincubation in vitro with rhIL-3 for 30 min, normal blood basophils released 2- to 3-fold more histamine than basophils pretreated with control medium when exposed to various concentrations of an anti-IgE antibody. Together, these results show that rhIL-3 binds to a specific receptor on blood basophils and is a regulator of basophil function

  16. An external sodium ion binding site controls allosteric gating in TRPV1 channels.

    Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J

    2016-01-01

    TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. PMID:26882503

  17. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    Kulakovskiy, Ivan V.

    2012-11-21

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  18. Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase

    Li DU; Ya-xue ZHAO; Liu-meng YANG; Yong-tang ZHENG; Yun TANG; Xu SHEN; Hua-liang JIANG

    2008-01-01

    Aim:To characterize the functional and pharmacological features of a symmetrical 1-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide,as a new anti-HIV compound which could competitively inhibit HIV-1 integrase (IN) binding to viral DNA.Methods:A surface plasma resonance (SPR)-based competitive assay was employed to determine the compound's inhibitory activity,and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell assay was used to qualify the antiviral activity.The potential binding sites were predicted by molecular modeling and determined by site-directed mutagenesis and a SPR binding assay.Results:l-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide could competitively inhibit IN binding to viral DNA with a 50% inhibitory concentration (IC50) value of 7.29±0.68 μmol/L as investigated by SPR-based investigation.Another antiretroviral activity assay showed that this compound exhibited inhibition against HⅣ-Ⅰ(ⅢB) replication with a 50% effective concentration (EC50) value of 40.54 μmol/L in C8166 cells,and cytotoxicity with a cytotoxic concentration value of 173.84 μmol/L in mock-infected C8166 cells.Molecular docking predicted 3 potential residues as 1-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene)bis-1-pyrrolidineacetamide binding sites.The importance of 3 key amino acid residues (Lys103,Lys173,and Thr174) involved in the binding was further identified by site-directed mutagenesis and a SPR binding assay.Conclusion:This present work identified a new anti-HIV compound through a new IN-binding site which is expected to supply new potential drug-binding site information for HIV-1 integrase inhibitor discovery and development.

  19. The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter.

    Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H; Ecker, Gerhard F; Freissmuth, Michael; Sucic, Sonja

    2010-12-01

    The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na(+)/Cl(-)-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERT(Y95F) mutation, which greatly reduced the affinity for [(3)H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [(3)H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP(+), paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

  20. Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli.

    Nou, X; Braaten, B; Kaltenbach, L; Low, D A

    1995-01-01

    Pyelonephritis-associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC-I and GATC II, is essential for the transition between active and inactive pap transcriptional states. Here, we identify six Lrp binding sites within the ...

  1. Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography.

    Goudreau, Nathalie; Lemke, Christopher T; Faucher, Anne-Marie; Grand-Maître, Chantal; Goulet, Sylvie; Lacoste, Jean-Eric; Rancourt, Jean; Malenfant, Eric; Mercier, Jean-François; Titolo, Steve; Mason, Stephen W

    2013-05-17

    The HIV-1 capsid (CA) protein, a domain of Gag, which participates in formation of both the mature and immature capsid, represents a potential target for anti-viral drug development. Characterization of hits obtained via high-throughput screening of an in vitro capsid assembly assay led to multiple compounds having this potential. We previously presented the characterization of two inhibitor series that bind the N-terminal domain of the capsid (CA(NTD)), at a site located at the bottom of its helical bundle, often referred to as the CAP-1 binding site. In this work we characterize a novel series of benzimidazole hits. Initial optimization of this series led to compounds with improved in vitro assembly and anti-viral activity. Using NMR spectroscopy we found that this series binds to a unique site on CA(NTD), located at the apex of the helical bundle, well removed from previously characterized binding sites for CA inhibitors. 2D (1)H-(15)N HSQC and (19)F NMR showed that binding of the benzimidazoles to this distinct site does not affect the binding of either cyclophilin A (CypA) to the CypA-binding loop or a benzodiazepine-based CA assembly inhibitor to the CAP-1 site. Unfortunately, while compounds of this series achieved promising in vitro assembly and anti-viral effects, they also were found to be quite sensitive to a number of naturally occurring CA(NTD) polymorphisms observed among clinical isolates. Despite the negative impact of this finding for drug development, the discovery of multiple inhibitor binding sites on CA(NTD) shows that capsid assembly is much more complex than previously realized. PMID:23496828

  2. p53 and TFIIEα share a common binding site on the Tfb1/p62 subunit of TFIIH

    Di Lello, Paola; Miller Jenkins, Lisa M.; Mas, Caroline; Langlois, Chantal; Malitskaya, Elena; Fradet-Turcotte, Amélie; Archambault, Jacques; Legault, Pascale; Omichinski, James G.

    2007-01-01

    The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the α-subunit of the general transcription factor IIE (TFIIEα). We have determined that the acidic carboxyl terminus of TFIIEα (TFIIEα336–439) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEα binding site on p62/Tfb1 is identical to the binding site fo...

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

    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.

  4. HOCOMOCO: expansion and enhancement of the collection of transcription factor binding sites models.

    Kulakovskiy, Ivan V; Vorontsov, Ilya E; Yevshin, Ivan S; Soboleva, Anastasiia V; Kasianov, Artem S; Ashoor, Haitham; Ba-Alawi, Wail; Bajic, Vladimir B; Medvedeva, Yulia A; Kolpakov, Fedor A; Makeev, Vsevolod J

    2016-01-01

    Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences. PMID:26586801

  5. High density of benzodiazepine binding sites in the substantia innominata of the rat

    Sarter, M.; Schneider, H.H.

    1988-07-01

    In order to study the neuronal basis of the pharmacological interactions between benzodiazepine receptor ligands and cortical cholinergic turnover, we examined the regional distribution of specific benzodiazepine binding sites using in vitro autoradiography. In the basal forebrain, the substantia innominata contained a high density of (/sup 3/H)lormetazepam (LMZ) binding sites (Bmax = 277 fmol/mg tissue; Kd = 0.55 nM). The label could be displaced by diazepam (IC50 = 100 nM), the benzodiazepine receptor antagonist beta-carboline ZK 93426 (45 nM) and the partial inverse agonist beta-carboline FG 7142 (540 nM). It is hypothesized that the amnesic effects of benzodiazepine receptor agonists are exerted through benzodiazepine receptors which are situated on cholinergic neurons in the substantia innominata and are involved in a tonic inhibition of cortical acetylcholine release. The benzodiazepine receptor antagonist ZK 93426 may exert its nootropic effects via benzodiazepine receptors in the substantia innominata and, consequently, by disinhibiting cortical acetylcholine release.

  6. Genome-wide de novo prediction of cis-regulatory binding sites in prokaryotes

    Zhang, Shaoqiang; Xu, Minli; Su, Zhengchang

    2009-01-01

    Although cis-regulatory binding sites (CRBSs) are at least as important as the coding sequences in a genome, our general understanding of them in most sequenced genomes is very limited due to the lack of efficient and accurate experimental and computational methods for their characterization, which has largely hindered our understanding of many important biological processes. In this article, we describe a novel algorithm for genome-wide de novo prediction of CRBSs with high accuracy. We designed our algorithm to circumvent three identified difficulties for CRBS prediction using comparative genomics principles based on a new method for the selection of reference genomes, a new metric for measuring the similarity of CRBSs, and a new graph clustering procedure. When operon structures are correctly predicted, our algorithm can predict 81% of known individual binding sites belonging to 94% of known cis-regulatory motifs in the Escherichia coli K12 genome, while achieving high prediction specificity. Our algorithm has also achieved similar prediction accuracy in the Bacillus subtilis genome, suggesting that it is very robust, and thus can be applied to any other sequenced prokaryotic genome. When compared with the prior state-of-the-art algorithms, our algorithm outperforms them in both prediction sensitivity and specificity. PMID:19383880

  7. Somitogenesis clock-wave initiation requires differential decay and multiple binding sites for clock protein.

    Mark Campanelli

    2010-04-01

    Full Text Available Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms.

  8. Design of multiligand inhibitors for the swine flu H1N1 neuraminidase binding site

    Narayanan, Manoj M; Nair, Chandrasekhar B; Sanjeeva, Shilpa K; Rao, PV Subba; Pullela, Phani K; Barrow, Colin J

    2013-01-01

    Viral neuraminidase inhibitors such as oseltamivir and zanamivir prevent early virus multiplication by blocking sialic acid cleavage on host cells. These drugs are effective for the treatment of a variety of influenza subtypes, including swine flu (H1N1). The binding site for these drugs is well established and they were designed based on computational docking studies. We show here that some common natural products have moderate inhibitory activity for H1N1 neuraminidase under docking studies. Significantly, docking studies using AutoDock for biligand and triligand forms of these compounds (camphor, menthol, and methyl salicylate linked via methylene bridges) indicate that they may bind in combination with high affinity to the H1N1 neuraminidase active site. These results also indicate that chemically linked biligands and triligands of these natural products could provide a new class of drug leads for the prevention and treatment of influenza. This study also highlights the need for a multiligand docking algorithm to understand better the mode of action of natural products, wherein multiple active ingredients are present. PMID:23983477

  9. Binding sites for interaction of peroxiredoxin 6 with surfactant protein A.

    Krishnaiah, Saikumari Y; Dodia, Chandra; Sorokina, Elena M; Li, Haitao; Feinstein, Sheldon I; Fisher, Aron B

    2016-04-01

    Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A2 (PLA2) activities. This protein participates in the degradation and remodeling of internalized dipalmitoylphosphatidylcholine (DPPC), the major phospholipid component of lung surfactant. We have shown previously that the PLA2 activity of Prdx6 is inhibited by the lung surfactant-associated protein called surfactant protein A (SP-A) through direct protein-protein interaction. Docking of SPA and Prdx6 was modeled using the ZDOCK (zlab.bu.edu) program in order to predict molecular sites for binding of the two proteins. The predicted peptide sequences were evaluated for binding to the opposite protein using isothermal titration calorimetry and circular dichroism measurement followed by determination of the effect of the SP-A peptide on the PLA2 activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix and 83DEELQTELYEIKHQIL99 in SP-A were identified as the sites for hydrophobic interaction and H(+)-bonding between the 2 proteins. Treatment of mouse endothelial cells with the SP-A peptide inhibited their recovery from lipid peroxidation associated with oxidative stress indicating inhibition of Prdx6 activity by the peptide in the intact cell. PMID:26723227

  10. Igs Expressed by Chronic Lymphocytic Leukemia B Cells Show Limited Binding-Site Structure Variability

    Marcatili, P.

    2013-05-01

    Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated. Copyright © 2013 by The American Association of Immunologists, Inc.

  11. HOCOMOCO: expansion and enhancement of the collection of transcription factor binding sites models

    Kulakovskiy, Ivan V.

    2015-11-19

    Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences.

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

    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.

  13. New binding site conformations of the dengue virus NS3 protease accessed by molecular dynamics simulation.

    Hugo de Almeida

    Full Text Available Dengue fever is caused by four distinct serotypes of the dengue virus (DENV1-4, and is estimated to affect over 500 million people every year. Presently, there are no vaccines or antiviral treatments for this disease. Among the possible targets to fight dengue fever is the viral NS3 protease (NS3PRO, which is in part responsible for viral processing and replication. It is now widely recognized that virtual screening campaigns should consider the flexibility of target protein by using multiple active conformational states. The flexibility of the DENV NS3PRO could explain the relatively low success of previous virtual screening studies. In this first work, we explore the DENV NS3PRO conformational states obtained from molecular dynamics (MD simulations to take into account protease flexibility during the virtual screening/docking process. To do so, we built a full NS3PRO model by multiple template homology modeling. The model comprised the NS2B cofactor (essential to the NS3PRO activation, a glycine flexible link and the proteolytic domain. MD simulations had the purpose to sample, as closely as possible, the ligand binding site conformational landscape prior to inhibitor binding. The obtained conformational MD sample was clustered into four families that, together with principal component analysis of the trajectory, demonstrated protein flexibility. These results allowed the description of multiple binding modes for the Bz-Nle-Lys-Arg-Arg-H inhibitor, as verified by binding plots and pair interaction analysis. This study allowed us to tackle protein flexibility in our virtual screening campaign against the dengue virus NS3 protease.

  14. Novel Vinculin Binding Site of the IpaA Invasin of Shigella

    Park, HaJeung; Valencia-Gallardo, Cesar; Sharff, Andrew; Van Nhieu, Guy Tran; Izard, Tina (Globel Phasing); (Scripps); (CF)

    2012-10-25

    Internalization of Shigella into host epithelial cells, where the bacteria replicates and spreads to neighboring cells, requires a type 3 secretion system (T3SS) effector coined IpaA. IpaA binds directly to and activates the cytoskeletal protein vinculin after injection in the host cell cytosol, and this was previously thought to be directed by two amphipathic {alpha}-helical vinculin-binding sites (VBS) found in the C-terminal tail domain of IpaA. Here, we report a third VBS, IpaA-VBS3, that is located N-terminal to the other two VBSs of IpaA and show that one IpaA molecule can bind up to three vinculin molecules. Biochemical in vitro Shigella invasion assays and the 1.6 {angstrom} crystal structure of the vinculin {center_dot} IpaA-VBS3 complex showed that IpaA-VBS3 is functionally redundant with the other two IpaA-VBSs in cell invasion and in activating the latent F-actin binding functions of vinculin. Multiple VBSs in IpaA are reminiscent of talin, which harbors 11 VBSs. However, most of the talin VBSs have low affinity and are buried in helix bundles, whereas all three of the VBSs of IpaA are high affinity, readily available, and in close proximity to each other in the IpaA structure. Although deletion of IpaA-VBS3 has no detectable effects on Shigella invasion of epithelial cells, deletion of all three VBSs impaired bacterial invasion to levels found in an ipaA null mutant strain. Thus, IpaA-directed mimicry of talin in activating vinculin occurs through three high affinity VBSs that are essential for Shigella pathogenesis.

  15. Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

    Buck, S.H.; Helke, C.J.; Burcher, E.; Shults, C.W.; O' Donohue, T.L.

    1986-11-01

    P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (/sup 125/I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV-VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei.

  16. Theory on thermodynamic coupling of site-specific DNA–protein interactions with fluctuations in DNA-binding domains

    DNA-binding proteins recognize their cognate sites on the template DNA more efficiently when the thermally driven flipping of their DNA-binding domains between the fast- and slow-moving conformations is coupled to the search dynamics. We show that there exists an optimum barrier height (∼kBT ln2) that separates these fast- and slow-moving states of DNA-binding domains, at which the efficiency associated with the thermodynamic coupling of thermally driven flipping and the overall search dynamics is the maximum. Furthermore, the dynamics of DNA-binding domains resembles that of typical downhill folding proteins at their midpoint denaturation temperatures. We further show that the average one-dimensional scanning lengths of slow- and fast-moving states of DNA-binding domains of LacI repressor protein are tuned to minimize the overall search time that is required to locate its cognate sites on DNA. (paper)

  17. In vivo labelling in several rat tissues of 'peripheral type' benzodiazepine binding sites

    Benavides, J.; Guilloux, F.; Rufat, P.; Uzan, A.; Renault, C.; Dubroeucq, M.C.; Gueremy, C.; Le Fur, G. (Pharmuka Laboratoires, 92 - Gennevilliers (France))

    1984-03-16

    'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of (/sup 3/H)PK 11195 and (/sup 3/H)RO5-4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 > PK 11211 > RO5-4864 > diazepam > dipyridamole > clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However, some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make (/sup 3/H)PK 11195 the most suitable ligand for this kind of studies.

  18. Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

    A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca2+,Mg(2+)-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca2+ channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with [3H](+)-PN 200-110, [3H](-)-desmethoxyverapamil [( 3H](-)-D888) and [3H]-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand binding studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca2+ channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle

  19. Theory on thermodynamic coupling of site-specific DNA-protein interactions with fluctuations in DNA-binding domains

    Murugan, R, E-mail: rmurugan@gmail.com [Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India)

    2011-12-16

    DNA-binding proteins recognize their cognate sites on the template DNA more efficiently when the thermally driven flipping of their DNA-binding domains between the fast- and slow-moving conformations is coupled to the search dynamics. We show that there exists an optimum barrier height ({approx}k{sub B}T ln2) that separates these fast- and slow-moving states of DNA-binding domains, at which the efficiency associated with the thermodynamic coupling of thermally driven flipping and the overall search dynamics is the maximum. Furthermore, the dynamics of DNA-binding domains resembles that of typical downhill folding proteins at their midpoint denaturation temperatures. We further show that the average one-dimensional scanning lengths of slow- and fast-moving states of DNA-binding domains of LacI repressor protein are tuned to minimize the overall search time that is required to locate its cognate sites on DNA. (paper)

  20. 2-(/sup 125/I)iodomelatonin binding sites in hamster brain membranes: pharmacological characteristics and regional distribution

    Duncan, M.J.; Takahashi, J.S.; Dubocovich, M.L.

    1988-05-01

    Studies in a variety of seasonally breeding mammals have shown that melatonin mediates photoperiodic effects on reproduction. Relatively little is known, however, about the site(s) or mechanisms of action of this hormone for inducing reproductive effects. Although binding sites for (3H)melatonin have been reported previously in bovine, rat, and hamster brain, the pharmacological selectivity of these sites was never demonstrated. In the present study, we have characterized binding sites for a new radioligand, 2-(125I)iodomelatonin, in brains from a photoperiodic species, the Syrian hamster. 2-(125I)Iodomelatonin labels a high affinity binding site in hamster brain membranes. Specific binding of 2-(125I)iodomelatonin is rapid, stable, saturable, and reversible. Saturation studies demonstrated that 2-(125I)iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 3.3 +/- 0.5 nM and a total binding capacity (Bmax) of 110.2 +/- 13.4 fmol/mg protein (n = 4). The Kd value determined from kinetic analysis (3.1 +/- 0.9 nM; n = 5) was very similar to that obtained from saturation experiments. Competition experiments showed that the relative order of potency of a variety of indoles for inhibition of 2-(125I)iodomelatonin binding site to hamster brain membranes was as follows: 6-chloromelatonin greater than or equal to 2-iodomelatonin greater than N-acetylserotonin greater than or equal to 6-methoxymelatonin greater than or equal to melatonin greater than 6-hydroxymelatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 5-methoxytryptophol greater than 5-methoxytryptamine greater than or equal to 5-methoxy-N,N-dimethyltryptamine greater than N-acetyltryptamine greater than serotonin greater than 5-methoxyindole (inactive).

  1. 2-[125I]iodomelatonin binding sites in hamster brain membranes: pharmacological characteristics and regional distribution

    Studies in a variety of seasonally breeding mammals have shown that melatonin mediates photoperiodic effects on reproduction. Relatively little is known, however, about the site(s) or mechanisms of action of this hormone for inducing reproductive effects. Although binding sites for [3H]melatonin have been reported previously in bovine, rat, and hamster brain, the pharmacological selectivity of these sites was never demonstrated. In the present study, we have characterized binding sites for a new radioligand, 2-[125I]iodomelatonin, in brains from a photoperiodic species, the Syrian hamster. 2-[125I]Iodomelatonin labels a high affinity binding site in hamster brain membranes. Specific binding of 2-[125I]iodomelatonin is rapid, stable, saturable, and reversible. Saturation studies demonstrated that 2-[125I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 3.3 +/- 0.5 nM and a total binding capacity (Bmax) of 110.2 +/- 13.4 fmol/mg protein (n = 4). The Kd value determined from kinetic analysis (3.1 +/- 0.9 nM; n = 5) was very similar to that obtained from saturation experiments. Competition experiments showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding site to hamster brain membranes was as follows: 6-chloromelatonin greater than or equal to 2-iodomelatonin greater than N-acetylserotonin greater than or equal to 6-methoxymelatonin greater than or equal to melatonin greater than 6-hydroxymelatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 5-methoxytryptophol greater than 5-methoxytryptamine greater than or equal to 5-methoxy-N,N-dimethyltryptamine greater than N-acetyltryptamine greater than serotonin greater than 5-methoxyindole (inactive)

  2. The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA-binding site.

    Maes, Michal; Amit, Einav; Danieli, Tsafi; Lebendiker, Mario; Loyter, Abraham; Friedler, Assaf

    2014-11-01

    Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants. PMID:25212215

  3. The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain

    Lorentsen, R H; Graversen, Jonas Heilskov; Caterer, N R;

    2000-01-01

    element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6...

  4. Searching the protein structure database for ligand-binding site similarities using CPASS v.2

    Caprez Adam

    2011-01-01

    Full Text Available Abstract Background A recent analysis of protein sequences deposited in the NCBI RefSeq database indicates that ~8.5 million protein sequences are encoded in prokaryotic and eukaryotic genomes, where ~30% are explicitly annotated as "hypothetical" or "uncharacterized" protein. Our Comparison of Protein Active-Site Structures (CPASS v.2 database and software compares the sequence and structural characteristics of experimentally determined ligand binding sites to infer a functional relationship in the absence of global sequence or structure similarity. CPASS is an important component of our Functional Annotation Screening Technology by NMR (FAST-NMR protocol and has been successfully applied to aid the annotation of a number of proteins of unknown function. Findings We report a major upgrade to our CPASS software and database that significantly improves its broad utility. CPASS v.2 is designed with a layered architecture to increase flexibility and portability that also enables job distribution over the Open Science Grid (OSG to increase speed. Similarly, the CPASS interface was enhanced to provide more user flexibility in submitting a CPASS query. CPASS v.2 now allows for both automatic and manual definition of ligand-binding sites and permits pair-wise, one versus all, one versus list, or list versus list comparisons. Solvent accessible surface area, ligand root-mean square difference, and Cβ distances have been incorporated into the CPASS similarity function to improve the quality of the results. The CPASS database has also been updated. Conclusions CPASS v.2 is more than an order of magnitude faster than the original implementation, and allows for multiple simultaneous job submissions. Similarly, the CPASS database of ligand-defined binding sites has increased in size by ~ 38%, dramatically increasing the likelihood of a positive search result. The modification to the CPASS similarity function is effective in reducing CPASS similarity scores

  5. Radioautographic identification of lactogen binding sites in rat median eminence using 125I-human growth hormone

    The binding characteristics of human growth hormone were exploited to identify radioautographically lactogen binding sites in the rat median eminence following systemic injection 125I-human growth hormone bound preferentially to the lateral palisade zone, a region of median eminence rich in dopamine and LHRH. Coinjection of 125I-human growth hormone with an excess of unlabeled human growth hormone or ovine prolactin, but not bovine growth hormone, competitively blocked 125I-human growth hormone binding to the external median eminence. These observations provide direct evidence of recognition sites for lactogenic hormones in a discrete region of the median eminence associated with hypothalamic regulation of hypophyseal prolactin and luteinizing hormone secretion. Median eminence lactogen binding sites may mediate presumed direct effects of lactogenic hormones on the reproductive functions of the hypophysiotropic hypothalamus. (orig.)

  6. CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site

    Duus, Karen; Thielens, Nicole M; Lacroix, Monique;

    2010-01-01

    and apoptotic material by CD91. Structured digital abstract •  MINT-8040679, MINT-8040706: MBL (uniprotkb: P11226) binds (MI:0407) to CD91 (uniprotkb: Q07954) by enzyme linked immunosorbent assay (MI:0411) •  MINT-8040690: L-ficolin (uniprotkb: Q15485) binds (MI:0407) to CD91 (uniprotkb: Q07954) by...... enzyme linked immunosorbent assay (MI:0411) •  MINT-8040663: C1q B (uniprotkb: P02746), C1q C (uniprotkb: P02747), C1q A (uniprotkb: P02745) and CD91 (uniprotkb: Q07954) physically interact (MI:0915) by enzyme linked immunosorbent assay (MI:0411) •  MINT-8040821, MINT-8040869, MINT-8040928: CD91...... (uniprotkb: Q07954) binds (MI:0407) to MBL (uniprotkb: P11226) by surface plasmon resonance (MI:0107) •  MINT-8040880: CD91 (uniprotkb: Q07954) binds (MI:0407) to L-ficolin (uniprotkb: Q15485) by surface plasmon resonance (MI:0107)....

  7. A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex

    Wiederhold, Katrin; Kloepper, Tobias H; Walter, Alexander M;

    2010-01-01

    coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from...

  8. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter

    Andersen, Jacob; Ringsted, Kristoffer B; Bang-Andersen, Benny; Strømgaard, Kristian; Kristensen, Anders S

    2015-01-01

    in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor...

  9. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).

    Chufan, Eduardo E; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T; Durell, Stewart R; Ambudkar, Suresh V

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each

  10. Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1.

    Eduardo E Chufan

    Full Text Available P-glycoprotein (Pgp, ABCB1 is an ATP-Binding Cassette (ABC transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982 with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available

  11. Characterisation of the effect of ion channel modulators on I1-imidazoline binding sites in bovine adrenal medulla

    Full text: The structure of I1-imidazoline binding sites is still unknown and we have proposed that they represent ion channels (i). In these experiments we characterised the effects of the known ion channel modulators methyltriphenylphosphonium (MTPP), 4-aminopyridine (4-AP) and tetraethyl ammonium (TEA) on [3H] clonidine binding in bovine adrenal medullary membranes as these membranes have a relatively well defined I1-imidazoline binding site (Molderings et al, 1993). Membranes from bovine adrenal medulla's were prepared by a minor modification of the method of Rapier et al. [3H] Clonidine binding was performed by the method of Ernsberger et al (3), with [3H] clonidine (62 Ci/mmol) used at a final concentration of 5 nM. [3H] Clonidine binding was displaced from bovine adrenal medullary membranes by adrenergic drugs with the order of potency being oxymetazoline > clonidine > moxonidine = idazoxan >> yonimbine. This order of potency is consistent with previous studies of I1-imidazoline binding sites (4). Non-linear curve fitting to this data was consistent with a single site model. Both TEA and 4-AP displaced [ H] clonidine with similar potency to its effect on ion channels, TEA having a EC>> of 54 ± 0.3 μM (n=3). The displacement of [3H] clonidine produced by both TEA and 4-AP also fitted to a single site model. Displacement of [3 H] clonidine by MTPP fitted a two site model (p1-imidazoline binding sites defined with [3H] clonidine may represent ion channels. We have used this data to perform molecular modelling and have determined a common conformation of I1-prefering ligands which will aid in the development of I1-selective ligands in the future. Copyright (1998) Australian Neuroscience Society

  12. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

    Wang Pu

    2010-10-01

    Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

  13. U2AF binding selects for the high conservation of the C. elegans 3′ splice site

    HOLLINS, COURTNEY; Zorio, Diego A.R.; MACMORRIS, MARGARET; Blumenthal, Thomas

    2005-01-01

    Caenorhabditis elegans is unusual among animals in having a highly conserved octamer sequence at the 3′ splice site: UUUU CAG/R. This sequence can bind to the essential heterodimeric splicing factor U2AF, with U2AF65 contacting the U tract and U2AF35 contacting the splice site itself (AG/R). Here we demonstrate a strong correspondence between binding to U2AF of RNA oligonucleotides with variant octamer sequences and the frequency with which such variations occur in splice sites. C. elegans U2...

  14. X-Ray Absorption Studies of Zn2+ Binding Sites in Bacterial, Avian, and Bovine Cytochrome bc1 Complexes

    Giachini, Lisa; Francia, Francesco; Veronesi, Giulia; Lee, Dong-Woo; Daldal, Fevzi; Huang, Li-Shar; Edward A. Berry; Cocco, Tiziana; Papa, Sergio; Boscherini, Federico; Venturoli, Giovanni

    2007-01-01

    Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge x-ray absorption fine-structure spectroscopy, we report here on the local structure of Zn2+ bound stoichiome...

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

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

    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

  17. Consistent improvement of cross-docking results using binding site ensembles generated with elastic network normal modes.

    Rueda, Manuel; Bottegoni, Giovanni; Abagyan, Ruben

    2009-03-01

    The representation of protein flexibility is still a challenge for the state-of-the-art flexible ligand docking protocols. In this article, we use a large and diverse benchmark to prove that is possible to improve consistently the cross-docking performance against a single receptor conformation, using an equilibrium ensemble of binding site conformers. The benchmark contained 28 proteins, and our method predicted the top-ranked near native ligand poses 20% more efficiently than using a single receptor. The multiple conformations were derived from the collective variable space defined by all heavy-atom elastic network normal modes, including backbone and side chains. We have found that the binding site displacements for best positioning of the ligand seem rather independent from the global collective motions of the protein. We also found that the number of binding site conformations needed to represent nonredundant flexibility was Web site at http://abagyan.scripps.edu/MRC. PMID:19434904

  18. Iodine 125-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells

    125I-Lysergic acid diethylamide (125I-LSD) binds with high affinity to serotonergic sites on rat choroid plexus. These sites were localized to choroid plexus epithelial cells by use of a novel high resolution stripping film technique for light microscopic autoradiography. In membrane preparations from rat choroid plexus, the serotonergic site density was 3100 fmol/mg of protein, which is 10-fold higher than the density of any other serotonergic site in brain homogenates. The choroid plexus site exhibits a novel pharmacology that does not match the properties of 5-hydroxytryptamine-1a (5-HT1a), 5-HT1b, or 5-HT2 serotonergic sites. 125I-LSD binding to the choroid plexus site is potently inhibited by mianserin, serotonin, and (+)-LSD. Other serotonergic, dopaminergic, and adrenergic agonists and antagonists exhibit moderate to weak affinities for this site. The rat choroid plexus 125I-LSD binding site appears to represent a new type of serotonergic site which is located on non-neuronal cells in this tissue

  19. Genome-scale study of the importance of binding site context for transcription factor binding and gene regulation

    Ronne Hans

    2008-11-01

    Full Text Available Abstract Background The rate of mRNA transcription is controlled by transcription factors that bind to specific DNA motifs in promoter regions upstream of protein coding genes. Recent results indicate that not only the presence of a motif but also motif context (for example the orientation of a motif or its location relative to the coding sequence is important for gene regulation. Results In this study we present ContextFinder, a tool that is specifically aimed at identifying cases where motif context is likely to affect gene regulation. We used ContextFinder to examine the role of motif context in S. cerevisiae both for DNA binding by transcription factors and for effects on gene expression. For DNA binding we found significant patterns of motif location bias, whereas motif orientations did not seem to matter. Motif context appears to affect gene expression even more than it affects DNA binding, as biases in both motif location and orientation were more frequent in promoters of co-expressed genes. We validated our results against data on nucleosome positioning, and found a negative correlation between preferred motif locations and nucleosome occupancy. Conclusion We conclude that the requirement for stable binding of transcription factors to DNA and their subsequent function in gene regulation can impose constraints on motif context.

  20. MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

    Tapp, Hazel; Al-Naggar, Iman M; Yarmola, Elena G; Harrison, Alexis; Shaw, Gerry; Edison, Arthur S; Bubb, Michael R

    2005-03-18

    Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. PMID:15640140