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Sample records for 125i-galanin binding sites

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-11-01

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

  2. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  3. Statistics for Transcription Factor Binding Sites

    OpenAIRE

    2008-01-01

    Transcription factors (TFs) play a key role in gene regulation. They interact with specific binding sites or motifs on the DNA sequence and regulate expression of genes downstream of these binding sites. In silico prediction of potential binding of a TF to a binding site is an important task in computational biology. From a statistical point of view, the DNA sequence is a long text consisting of four different letters ('A','C','G', and 'T'). The binding of a TF to the sequence corresponds to ...

  4. Tissue specificity of endothelin binding sites

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

    1990-09-01

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

  5. Identification of consensus binding sites clarifies FMRP binding determinants.

    Science.gov (United States)

    Anderson, Bart R; Chopra, Pankaj; Suhl, Joshua A; Warren, Stephen T; Bassell, Gary J

    2016-08-19

    Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3' UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Predicted metal binding sites for phytoremediation.

    Science.gov (United States)

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

    2009-09-05

    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 not appear in the essential oil and that some of these species are able to grow in metal contaminated sites. A pattern search against the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases yielded true positives in each case showing the high specificity of the motifs designed for the ions of nickel, lead, molybdenum, manganese, cadmium, zinc, iron, cobalt and xenobiotic compounds. Motifs were also studied against PDB structures. Results of the study suggested the presence of binding sites on the surface of protein molecules involved. PDB structures of proteins were finally predicted for the binding sites functionality in their respective phytoremediation usage. This was further validated through CASTp server to study its physico-chemical properties. Bioinformatics implications would help in designing strategy for developing transgenic plants with increased metal binding capacity. These metal binding factors can be used to restrict metal update by plants. This helps in reducing the possibility of metal movement into the food chain.

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

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

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    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.

  9. Computational identification of uncharacterized cruzain binding sites.

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    Jacob D Durrant

    Full Text Available Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention.

  10. Oxytocin binding sites in bovine mammary tissue

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    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

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

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

  12. STUDY OF ESTROGEN BINDING SITE ON HUMAN EJACULATED SPERMATOZOA

    Institute of Scientific and Technical Information of China (English)

    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. Being a binding site: characterizing residue composition of binding sites on proteins.

    Science.gov (United States)

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-12-30

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

  14. Chloride binding site of neurotransmitter sodium symporters.

    Science.gov (United States)

    Kantcheva, Adriana K; Quick, Matthias; Shi, Lei; Winther, Anne-Marie Lund; Stolzenberg, Sebastian; Weinstein, Harel; Javitch, Jonathan A; Nissen, Poul

    2013-05-21

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

  15. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

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

    2015-12-29

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

  17. Substrate and drug binding sites in LeuT.

    Science.gov (United States)

    Nyola, Ajeeta; Karpowich, Nathan K; Zhen, Juan; Marden, Jennifer; Reith, Maarten E; Wang, Da-Neng

    2010-08-01

    LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine, and dopamine. The original crystal structure of LeuT shows a primary leucine-binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters.

  18. The enigmatic drug binding site for sodium channel inhibitors.

    Science.gov (United States)

    Mike, Arpad; Lukacs, Peter

    2010-11-01

    Local anesthetics have been in clinical use since 1884, and different aspects of the local anesthetic binding site have been studied in enormous detail. In spite of all these efforts, some of the most fundamental questions--such as which exact residues constitute the binding site, how many binding sites exist, do local anesthetics share their binding site(s) with other sodium channel inhibitors, and what are the mechanisms of inhibition--are still largely unanswered. We review accumulated data on the "local anesthetic receptor"and discuss controversial points, such as possible mechanisms of inhibition, the possibility of additional binding sites, the orientation of S6 helices, and the internal vs. external position of the anticonvulsant binding site. We describe the four following specific groups of functionally important residues: i) conserved asparagines six residues below the hinge residues; we propose that they are oriented toward the external surface of S6 helices, and have a critical role in the coupling of voltage sensors to gating, ii) residues lining the inner vestibule and constructing the "orthodox" binding site, iii) residues around the outer vestibule, which have been proposed to constitute an alternative external binding site, and iv) residues determining external access for quaternary amine inhibitors, such as QX314. We conclude that sodium channel inhibitors must be heterogenous in terms of binding sites and inhibition mechanisms, and propose that this heterogeneity should be taken into consideration during drug development.

  19. Defining the bacteroides ribosomal binding site.

    Science.gov (United States)

    Wegmann, Udo; Horn, Nikki; Carding, Simon R

    2013-03-01

    The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

  20. Protein function annotation by local binding site surface similarity.

    Science.gov (United States)

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  1. Predicted metal binding sites for phytoremediation

    OpenAIRE

    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...

  2. Structure and localisation of drug binding sites on neurotransmitter transporters.

    Science.gov (United States)

    Ravna, Aina W; Sylte, Ingebrigt; Dahl, Svein G

    2009-10-01

    The dopamine (DAT), serotontin (SERT) and noradrenalin (NET) transporters are molecular targets for different classes of psychotropic drugs. The crystal structure of Aquifex aeolicus LeuT(Aa) was used as a template for molecular modeling of DAT, SERT and NET, and two putative drug binding sites (pocket 1 and 2) in each transporter were identified. Cocaine was docked into binding pocket 1 of DAT, corresponding to the leucine binding site in LeuT(Aa), which involved transmembrane helices (TMHs) 1, 3, 6 and 8. Clomipramine was docked into binding pocket 2 of DAT, involving TMHs 1, 3, 6, 10 and 11, and extracellular loops 4 and 6, corresponding to the clomipramine binding site in a crystal structure of a LeuT(Aa)-clomipramine complex. The structures of the proposed cocaine- and tricyclic antidepressant-binding sites may be of particular interest for the design of novel DAT interacting ligands.

  3. Frequent gain and loss of functional transcription factor binding sites.

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    Scott W Doniger

    2007-05-01

    Full Text Available Cis-regulatory sequences are not always conserved across species. Divergence within cis-regulatory sequences may result from the evolution of species-specific patterns of gene expression or the flexible nature of the cis-regulatory code. The identification of functional divergence in cis-regulatory sequences is therefore important for both understanding the role of gene regulation in evolution and annotating regulatory elements. We have developed an evolutionary model to detect the loss of constraint on individual transcription factor binding sites (TFBSs. We find that a significant fraction of functionally constrained binding sites have been lost in a lineage-specific manner among three closely related yeast species. Binding site loss has previously been explained by turnover, where the concurrent gain and loss of a binding site maintains gene regulation. We estimate that nearly half of all loss events cannot be explained by binding site turnover. Recreating the mutations that led to binding site loss confirms that these sequence changes affect gene expression in some cases. We also estimate that there is a high rate of binding site gain, as more than half of experimentally identified S. cerevisiae binding sites are not conserved across species. The frequent gain and loss of TFBSs implies that cis-regulatory sequences are labile and, in the absence of turnover, may contribute to species-specific patterns of gene expression.

  4. Identification and characterization of anion binding sites in RNA

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. DBD2BS: connecting a DNA-binding protein with its binding sites

    OpenAIRE

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes c...

  6. Influence of sulfhydryl sites on metal binding by bacteria

    Science.gov (United States)

    Nell, Ryan M.; Fein, Jeremy B.

    2017-02-01

    The role of sulfhydryl sites within bacterial cell envelopes is still unknown, but the sites may control the fate and bioavailability of metals. Organic sulfhydryl compounds are important complexing ligands in aqueous systems and they can influence metal speciation in natural waters. Though representing only approximately 5-10% of the total available binding sites on bacterial surfaces, sulfhydryl sites exhibit high binding affinities for some metals. Due to the potential importance of bacterial sulfhydryl sites in natural systems, metal-bacterial sulfhydryl site binding constants must be determined in order to construct accurate models of the fate and distribution of metals in these systems. To date, only Cd-sulfhydryl binding has been quantified. In this study, the thermodynamic stabilities of Mn-, Co-, Ni-, Zn-, Sr- and Pb-sulfhydryl bacterial cell envelope complexes were determined for the bacterial species Shewanella oneidensis MR-1. Metal adsorption experiments were conducted as a function of both pH, ranging from 5.0 to 7.0, and metal loading, from 0.5 to 40.0 μmol/g (wet weight) bacteria, in batch experiments in order to determine if metal-sulfhydryl binding occurs. Initially, the data were used to calculate the value of the stability constants for the important metal-sulfhydryl bacterial complexes for each metal-loading condition studied, assuming a single binding reaction for the dominant metal-binding site type under the pH conditions of the experiments. For most of the metals that we studied, these calculated stability constant values increased significantly with decreasing metal loading, strongly suggesting that our initial assumption was not valid and that more than one type of binding occurs at the assumed binding site. We then modeled each dataset with two distinct site types with identical acidity constants: one site with a high metal-site stability constant value, which we take to represent metal-sulfhydryl binding and which dominates under low

  7. An additional substrate binding site in a bacterial phenylalanine hydroxylase.

    Science.gov (United States)

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Corn, Isaac R; Wagner, Kyle T; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2013-09-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.

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

    Directory of Open Access Journals (Sweden)

    T Meri

    Full Text Available 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."

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

    Science.gov (United States)

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

    2017-09-01

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

  10. Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

    OpenAIRE

    Hiller, Y; Bayer, E A; Wilchek, M

    1991-01-01

    The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 ...

  11. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

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

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs...

  12. Expression of muscarinic binding sites in primary human brain tumors.

    Science.gov (United States)

    Gurwitz, D; Razon, N; Sokolovsky, M; Soreq, H

    1984-05-01

    The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.

  13. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    Directory of Open Access Journals (Sweden)

    Javier Estrada

    Full Text Available DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  14. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    Science.gov (United States)

    Estrada, Javier; Ruiz-Herrero, Teresa; Scholes, Clarissa; Wunderlich, Zeba; DePace, Angela H

    2016-01-01

    DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  15. Cation binding site of cytochrome c oxidase: progress report.

    Science.gov (United States)

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-07-01

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

  17. Modulation of RNase E activity by alternative RNA binding sites.

    Directory of Open Access Journals (Sweden)

    Daeyoung Kim

    Full Text Available Endoribonuclease E (RNase E affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37 of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne that was UV crosslinked with a 5'-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13 containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433, whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71. Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

  18. Modeling lanthanide series binding sites on humic acid.

    Science.gov (United States)

    Pourret, Olivier; Martinez, Raul E

    2009-02-01

    Lanthanide (Ln) binding to humic acid (HA) has been investigated by combining ultrafiltration and ICP-MS techniques. A Langmuir-sorption-isotherm metal-complexation model was used in conjunction with a linear programming method (LPM) to fit experimental data representing various experimental conditions both in HA/Ln ratio (varying between 5 and 20) and in pH range (from 2 to 10) with an ionic strength of 10(-3) mol L(-1). The LPM approach, not requiring prior knowledge of surface complexation parameters, was used to solve the existing discrepancies in LnHA binding constants and site densities. The application of the LPM to experimental data revealed the presence of two discrete metal binding sites at low humic acid concentrations (5 mg L(-1)), with log metal complexation constants (logK(S,j)) of 2.65+/-0.05 and 7.00 (depending on Ln). The corresponding site densities were 2.71+/-0.57x10(-8) and 0.58+/-0.32x10(-8) mol of Ln(3+)/mg of HA (depending on Ln). Total site densities of 3.28+/-0.28x10(-8), 4.99+/-0.02x10(-8), and 5.01+/-0.01x10(-8) mol mg(-1) were obtained by LPM for humic acid, for humic acid concentrations of 5, 10, and 20 mg L(-1), respectively. These results confirm that lanthanide binding occurs mainly at weak sites (i.e., ca. 80%) and second at strong sites (i.e., ca. 20%). The first group of discrete metal binding sites may be attributed to carboxylic groups (known to be the main binding sites of Ln in HA), and the second metal binding group to phenolic moieties. Moreover, this study evidences heterogeneity in the distribution of the binding sites among Ln. Eventually, the LPM approach produced feasible and reasonable results, but it was less sensitive to error and did not require an a priori assumption of the number and concentration of binding sites.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-01

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

  20. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

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

  1. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

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

  2. CTCF: the protein, the binding partners, the binding sites and their chromatin loops

    NARCIS (Netherlands)

    Holwerda, S.J.; de Laat, W.

    2013-01-01

    CTCF has it all. The transcription factor binds to tens of thousands of genomic sites, some tissue-specific, others ultra-conserved. It can act as a transcriptional activator, repressor and insulator, and it can pause transcription. CTCF binds at chromatin domain boundaries, at enhancers and gene pr

  3. Probing binding hot spots at protein-RNA recognition sites.

    Science.gov (United States)

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

    2016-01-29

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

  4. Probing binding hot spots at protein–RNA recognition sites

    Science.gov (United States)

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

    2016-01-01

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein–RNA interfaces to probe the binding hot spots at protein–RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein–protein and protein–RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein–RNA recognition sites with desired affinity. PMID:26365245

  5. In vitro characterization of cocaine binding sites in human hair.

    Science.gov (United States)

    Joseph, R E; Tsai, W J; Tsao, L I; Su, T P; Cone, E J

    1997-09-01

    In vitro studies were performed to characterize [3H]cocaine binding to dark and light ethnic hair types. In vitro binding to hair was selective, was reversible and increased linearly with increasing hair concentration. Scatchard analyses revealed high-affinity (6-112 nM) and low-affinity (906-4433 nM) binding in hair. Competition studies demonstrated that the potencies of 3beta-(4-bromophenyl)tropane-2beta-carboxylic acid methyl ester, and 5-(4-chlorophenyl)-2,5-dihydro-3H-imidazol[2,1-alpha]isoindole-5-ol and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane were similar to or less than that of (-)-cocaine. The potency of (-)-cocaine was 10-fold greater than that of (+)-cocaine at inhibiting radioligand specific binding to hair. Multivariate analysis indicated that significantly greater nonspecific and specific radioligand binding occurred in dark hair than in light hair. Multivariate analysis also demonstrated a significant ethnicity x sex effect on specific and nonspecific binding to hair. Greater radioligand binding occurred in male Africoid hair than in female Africoid hair and in all Caucasoid hair types. Melanin was considered the most likely binding site for cocaine in hair. Typically, the concentration of melanin is much greater in dark than in light hair. Scatchard analysis indicated that dark hair had a 5- to 43-fold greater binding capacity than light hair. Differences in radioligand binding between hair types appeared to be due to differences in the density of binding sites formed by melanin in hair.

  6. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-27

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

  7. Autoradiographic localization of estrogen binding sites in human mammary lesions

    Energy Technology Data Exchange (ETDEWEB)

    Buell, R.H.

    1984-01-01

    The biochemical assay of human mammary carcinomas for estrogen receptors is of proven clinical utility, but the cellular localization of estrogen binding sites within these lesions is less certain. The author describes the identification of estrogen binding sites as visualized by thaw-mount autoradiography after in vitro incubation in a series of 17 benign and 40 malignant human female mammary lesions. The results on the in vitro incubation method compared favorably with data from in vivo studies in mouse uterus, a well-characterized estrogen target organ. In noncancerous breast biopsies, a variable proportion of epithelial cells contained specific estrogen binding sites. Histologically identifiable myoepithelial and stromal cells were, in general, unlabeled. In human mammary carcinomas, biochemically estrogen receptor-positive, labeled and unlabeled neoplastic epithelial cells were identified by autoradiography. Quantitative results from the autoradiographic method compared favorably with biochemical data.

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

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

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

  9. A novel non-opioid binding site for endomorphin-1.

    Science.gov (United States)

    Lengyel, I; Toth, F; Biyashev, D; Szatmari, I; Monory, K; Tomboly, C; Toth, G; Benyhe, S; Borsodi, A

    2016-08-01

    Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the μ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the μ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the μ-opioid receptor. Our recent data indicate that a radiolabelled [(3)H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...

  11. Binding-site assessment by virtual fragment screening.

    Directory of Open Access Journals (Sweden)

    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.

  12. Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

    Science.gov (United States)

    Gitlin, G; Bayer, E A; Wilchek, M

    1990-01-01

    The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin. PMID:2386489

  13. Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

    Science.gov (United States)

    Gitlin, G; Bayer, E A; Wilchek, M

    1990-07-15

    The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.

  14. Characterization of Heparin-binding Site of Tissue Transglutaminase

    Science.gov (United States)

    Wang, Zhuo; Collighan, Russell J.; Pytel, Kamila; Rathbone, Daniel L.; Li, Xiaoling; Griffin, Martin

    2012-01-01

    Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. PMID:22298777

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-01

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

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

    DEFF Research Database (Denmark)

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

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  17. Objective models for steroid binding sites of human globulins

    Science.gov (United States)

    Schnitker, Jurgen; Gopalaswamy, Ramesh; Crippen, Gordon M.

    1997-01-01

    We report the application of a recently developed alignment-free 3D QSAR method [Crippen,G.M., J. Comput. Chem., 16 (1995) 486] to a benchmark-type problem. The test systeminvolves the binding of 31 steroid compounds to two kinds of human carrier protein. Themethod used not only allows for arbitrary binding modes, but also avoids the problems oftraditional least-squares techniques with regard to the implicit neglect of informative outlyingdata points. It is seen that models of considerable predictive power can be obtained even witha very vague binding site description. Underlining a systematic, but usually ignored, problemof the QSAR approach, there is not one unique type of model but, rather, an entire manifoldof distinctly different models that are all compatible with the experimental information. Fora given model, there is also a considerable variation in the found binding modes, illustratingthe problems that are inherent in the need for 'correct` molecular alignment in conventional3D QSAR methods.

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

    Indian Academy of Sciences (India)

    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

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

    Science.gov (United States)

    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 provides scoring matrices for TFs, nearly 78% of the TFs in the public release do not have matrices available. As work on TFBS alignment algorithms has been limited, it is highly desirable to have an alignment algorithm tailored to TFBSs. Results We designed a novel algorithm named LASAGNA, which is aware of the lengths of input TFBSs and utilizes position dependence. Results on 189 TFs of 5 species in the TRANSFAC database showed that our method significantly outperformed ClustalW2 and MEME. We further compared a PSSM method dependent on LASAGNA to an alignment-free TFBS search method. Results on 89 TFs whose binding sites can be located in genomes showed that our method is significantly more precise at fixed recall rates. Finally, we described LASAGNA-ChIP, a more sophisticated version for ChIP (Chromatin immunoprecipitation) experiments. Under the one-per-sequence model, it showed comparable performance with MEME in discovering motifs in ChIP-seq peak sequences. Conclusions We conclude that the LASAGNA algorithm is simple and effective in aligning variable-length binding sites. It has been integrated into a user-friendly webtool for TFBS search and visualization called LASAGNA-Search. The tool currently stores precomputed PSSM models for 189 TFs and 133 TFs built from TFBSs in the TRANSFAC Public database (release 7.0) and the ORegAnno database (08Nov10 dump), respectively. The webtool is available at http://biogrid.engr.uconn.edu/lasagna_search/. PMID:23522376

  20. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

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

  1. Variable structure motifs for transcription factor binding sites.

    Science.gov (United States)

    Reid, John E; Evans, Kenneth J; Dyer, Nigel; Wernisch, Lorenz; Ott, Sascha

    2010-01-14

    Classically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets. We re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance. We have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable models of motifs of variable

  2. Variable structure motifs for transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Wernisch Lorenz

    2010-01-01

    Full Text Available Abstract Background Classically, models of DNA-transcription factor binding sites (TFBSs have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs. Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets. Results We re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance. Conclusions We have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does

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

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

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

  4. Central melatonin binding sites in rainbow trout (Onchorhynchus mykiss).

    Science.gov (United States)

    Davies, B; Hannah, L T; Randall, C F; Bromage, N; Williams, L M

    1994-10-01

    A combination of in vitro autoradiography and membrane homogenate receptor assays has been used to localize and characterized 2-[125I]iodomelatonin binding sites in the brain of the rainbow trout (Onchorhynchus mykiss). Specific 2-[125I]iodomelatonin binding, defined as that displaced by 1 microM melatonin, increased linearly with increasing protein concentration in membrane homogenates of whole trout brain. Specific binding was both time and temperature dependent and reversible in the presence of 1 microM melatonin. Binding was saturable at between 100-150 pM 2-[125I]iodomelatonin and Scatchard analysis of saturation isotherms revealed a dissociation constant (Kd) of 15.00 +/- 0.95 pM and a maximum receptor number (Bmax) of 42.35 +/- 2.70 fm/mg protein (n = 16). Addition of 10(-4) M GTP gamma S (an analogue of guanosine triphosphate) to saturation isotherms apparently reduced the Bmax by 75% on average with no apparent change in the affinity of the binding. Scatchard analysis of saturation isotherms generated from whole brain membrane homogenates of trout kept on long days (15 hr light:9 hr dark) and killed either during the midlight or middark phase showed no significant differences in either the Kd or the Bmax of 2-[125I]iodomelatonin binding, although a robust rhythm in melatonin concentration was confirmed in these fish. Displacement of 2-[125I]iodomelatonin binding with increasing concentrations of competing ligands gave an order of potency of 2-iodomelatonin > melatonin > 5-HT. Localization of specific central 2-[125I]iodomelatonin binding in the rainbow trout showed high levels of binding associated with neuronal areas involved in the processing of visual signals, particularly the optic tectum and nucleus rotundus.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Bifunctional avidin with covalently modifiable ligand binding site.

    Directory of Open Access Journals (Sweden)

    Jenni Leppiniemi

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

  6. Detection of Binding Site Molecular Interaction Field Similarities.

    Science.gov (United States)

    Chartier, Matthieu; Najmanovich, Rafael

    2015-08-24

    Protein binding-site similarity detection methods can be used to predict protein function and understand molecular recognition, as a tool in drug design for drug repurposing and polypharmacology, and for the prediction of the molecular determinants of drug toxicity. Here, we present IsoMIF, a method able to identify binding site molecular interaction field similarities across protein families. IsoMIF utilizes six chemical probes and the detection of subgraph isomorphisms to identify geometrically and chemically equivalent sections of protein cavity pairs. The method is validated using six distinct data sets, four of those previously used in the validation of other methods. The mean area under the receiver operator curve (AUC) obtained across data sets for IsoMIF is higher than those of other methods. Furthermore, while IsoMIF obtains consistently high AUC values across data sets, other methods perform more erratically across data sets. IsoMIF can be used to predict function from structure, to detect potential cross-reactivity or polypharmacology targets, and to help suggest bioisosteric replacements to known binding molecules. Given that IsoMIF detects spatial patterns of molecular interaction field similarities, its predictions are directly related to pharmacophores and may be readily translated into modeling decisions in structure-based drug design. IsoMIF may in principle detect similar binding sites with distinct amino acid arrangements that lead to equivalent interactions within the cavity. The source code to calculate and visualize MIFs and MIF similarities are freely available.

  7. Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.

    Science.gov (United States)

    Bakhautdin, Bakytzhan; Goksoy Bakhautdin, Esen; Fox, Paul L

    2014-10-31

    Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.

  8. Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

    Science.gov (United States)

    Hiller, Y; Bayer, E A; Wilchek, M

    1991-09-01

    The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

  9. (/sup 3/H)desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons

    Energy Technology Data Exchange (ETDEWEB)

    Baeckstroem, I.T.Ro.; Ross, S.B.; Marcusson, J.O.

    1989-04-01

    The pharmacological and biochemical characteristics of (3H)desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one (3H)desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The (3H)desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 microM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of approximately 1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). In contrast, when 10 microM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with KD values of 0.5 and greater than 100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (Bmax) for binding defined by 0.1 microM nisoxetine varied between brain regions, with very low density in the striatum (Bmax not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of (3H)noradrenaline uptake but not with 5-(3H)hydroxytryptamine uptake.

  10. The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase.

    Science.gov (United States)

    Francia, Francesco; Giachini, Lisa; Boscherini, Federico; Venturoli, Giovanni; Capitanio, Giuseppe; Martino, Pietro Luca; Papa, Sergio

    2007-02-20

    EXAFS analysis of Zn binding site(s) in bovine-heart cytochrome c oxidase and characterization of the inhibitory effect of internal zinc on respiratory activity and proton pumping of the liposome reconstituted oxidase are presented. EXAFS identifies tetrahedral coordination site(s) for Zn(2+) with two N-histidine imidazoles, one N-histidine imidazol or N-lysine and one O-COOH (glutamate or aspartate), possibly located at the entry site of the proton conducting D pathway in the oxidase and involved in inhibition of the oxygen reduction catalysis and proton pumping by internally trapped zinc.

  11. Minimal Zn2+ Binding Site of Amyloid-β

    Science.gov (United States)

    Tsvetkov, Philipp O.; Kulikova, Alexandra A.; Golovin, Andrey V.; Tkachev, Yaroslav V.; Archakov, Alexander I.; Kozin, Sergey A.; Makarov, Alexander A.

    2010-01-01

    Zinc-induced aggregation of amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aβ region 6–14 as the minimal Zn2+ binding site wherein the ion is coordinated by His6, Glu11, His13, and His14. With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11–14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn2+-induced aggregation of Aβ. PMID:21081056

  12. Minimal Zn(2+) binding site of amyloid-β.

    Science.gov (United States)

    Tsvetkov, Philipp O; Kulikova, Alexandra A; Golovin, Andrey V; Tkachev, Yaroslav V; Archakov, Alexander I; Kozin, Sergey A; Makarov, Alexander A

    2010-11-17

    Zinc-induced aggregation of amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aβ region 6-14 as the minimal Zn(2+) binding site wherein the ion is coordinated by His(6), Glu(11), His(13), and His(14). With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11-14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn(2+)-induced aggregation of Aβ.

  13. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh (Michigan)

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

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

    Directory of Open Access Journals (Sweden)

    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

  15. Creating BHb-imprinted magnetic nanoparticles with multiple binding sites.

    Science.gov (United States)

    Li, Yanxia; Chen, Yiting; Huang, Lu; Lou, BenYong; Chen, Guonan

    2017-01-16

    A kind of protein imprinted over magnetic Fe3O4@Au multifunctional nanoparticles (NPs) with multiple binding sites was synthesized and investigated. Magnetic Fe3O4@Au NPs as carrier materials were modified with 4-mercaptophenylboronic acid (MPBA) and mercaptopropionic acid (MPA) to introduce boronic acids and carboxyl groups. Using Bovine Hemoglobin (BHb) as a template, a polydopamine(PDA)-based molecular imprinted film was fabricated to produce a kind of magnetic molecularly imprinted nanoparticle (MMIP), possessing multiple binding sites with benzene-diol, amino groups, boronic acids and carboxyl groups. The MMIPs exhibited an excellent imprinting effect and adsorption capacity (89.65± 0.38 mg g(-1)) toward the template protein. The results show that the MMIPs reached saturated adsorption at 0.5 mg mL(-1) within 90 min. The synthesized MMIPs are suitable for the removal and enrichment of the template protein in proteomics. The strategy of multiple binding sites paves the way for the preparation of functional nanomaterials in molecular imprinting techniques.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-07-02

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

  17. Cloud Computing for Protein-Ligand Binding Site Comparison

    Science.gov (United States)

    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

  18. Cloud Computing for Protein-Ligand Binding Site Comparison

    Directory of Open Access Journals (Sweden)

    Che-Lun Hung

    2013-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

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

  20. Steered molecular dynamics study of inhibitor binding in the internal binding site in dehaloperoxidase-hemoglobin.

    Science.gov (United States)

    Zhang, Zhisen; Santos, Andrew P; Zhou, Qing; Liang, Lijun; Wang, Qi; Wu, Tao; Franzen, Stefan

    2016-04-01

    The binding free energy of 4-bromophenol (4-BP), an inhibitor that binds in the internal binding site in dehaloperoxidase-hemoglobin (DHP) was calculated using Molecular Dynamics (MD) methods combined with pulling or umbrella sampling. The effects of systematic changes in the pulling speed, pulling force constant and restraint force constant on the calculated potential of mean force (PMF) are presented in this study. The PMFs calculated using steered molecular dynamics (SMD) were validated by umbrella sampling (US) in the strongly restrained regime. A series of restraint force constants ranging from 1000 down to 5 kJ/(mol nm(2)) were used in SMD simulations. This range was validated using US, however noting that weaker restraints give rise to a broader sampling of configurations. This comparison was further tested by a pulling simulation conducted without any restraints, which was observed to have a value closest to the experimentally measured free energy for binding of 4-BP to DHP based on ultraviolet-visible (UV-vis) and resonance Raman spectroscopies. The protein-inhibitor system is well suited for fundamental study of free energy calculations because the DHP protein is relatively small and the inhibitor is quite rigid. Simulation configuration structures are compared to the X-ray crystallography structures of the binding site of 4-BP in the distal pocket above the heme.

  1. Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid-tubulin binding site.

    Science.gov (United States)

    Naik, Pradeep K; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C

    2011-06-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ΔΔG(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (ΔΔG(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.

  2. Characterization of Binding Sites of Eukaryotic Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    Jiang Qian; Jimmy Lin; Donald J. Zack

    2006-01-01

    To explore the nature of eukaryotic transcription factor (TF) binding sites and determine how they differ from surrounding DNA sequences, we examined four features associated with DNA binding sites: G+C content, pattern complexity,palindromic structure, and Markov sequence ordering. Our analysis of the regulatory motifs obtained from the TRANSFAC database, using yeast intergenic sequences as background, revealed that these four features show variable enrichment in motif sequences. For example, motif sequences were more likely to have palindromic structure than were background sequences. In addition, these features were tightly localized to the regulatory motifs, indicating that they are a property of the motif sequences themselves and are not shared by the general promoter "environment" in which the regulatory motifs reside. By breaking down the motif sequences according to the TF classes to which they bind, more specific associations were identified. Finally, we found that some correlations, such as G+C content enrichment, were species-specific, while others, such as complexity enrichment, were universal across the species examined. The quantitative analysis provided here should increase our understanding of protein-DNA interactions and also help facilitate the discovery of regulatory motifs through bioinformatics.

  3. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  4. Molecular modeling and competition binding study of Br-noscapine and colchicine provides insight into noscapinoid-tubulin binding site

    OpenAIRE

    Naik, Pradeep K.; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C.

    2011-01-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: 1) In silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; 2) the molecular mechanics-generalized Born/surface area (MM-GB/SA)...

  5. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  7. A Unitary Anesthetic-Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-01-01

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

  9. Leveraging cross-species transcription factor binding site patterns

    DEFF Research Database (Denmark)

    Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward

    2014-01-01

    to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2...... diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele...... that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms....

  10. CBS domains: Ligand binding sites and conformational variability.

    Science.gov (United States)

    Ereño-Orbea, June; Oyenarte, Iker; Martínez-Cruz, Luis Alfonso

    2013-12-01

    Cystathionine β-synthase (CBS) domains or CBS motifs are conserved structural domains that are present in thousands of non functionally-related proteins from all kingdoms of life. Their importance is underlined by the range of hereditary diseases associated with mutations in their amino acid sequence. CBS motifs associate in pairs referred to as Bateman modules. In contrast with initial assumptions, it is now well documented that CBS motifs and/or Bateman modules may suffer conformational changes upon binding of adenosine derivatives, metal ions or nucleic acids. The degree and direction of these structural changes depend on the type of ligand, the intrinsic features of the binding sites and the association manner of the Bateman modules. This review aims to provide a summary of the current knowledge on the structural basis of ligand recognition and on the structural effects caused by these ligands in CBS domain containing proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    CERN Document Server

    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...

  13. Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

    Science.gov (United States)

    Meshalkina, L; Nilsson, U; Wikner, C; Kostikowa, T; Schneider, G

    1997-03-01

    The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

  14. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-10-28

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

  16. Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

    Science.gov (United States)

    Gitlin, G; Bayer, E A; Wilchek, M

    1987-01-01

    Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site. PMID:3109401

  17. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    Science.gov (United States)

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-01-01

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin. PMID:3355517

  18. Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

    OpenAIRE

    Gitlin, G; Bayer, E A; Wilchek, M

    1987-01-01

    Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

  19. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    Science.gov (United States)

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-02-15

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

  20. DBD2BS: connecting a DNA-binding protein with its binding sites.

    Science.gov (United States)

    Chien, Ting-Ying; Lin, Chih-Kang; Lin, Chih-Wei; Weng, Yi-Zhong; Chen, Chien-Yu; Chang, Darby Tien-Hao

    2012-07-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein-DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD-DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein-DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

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

  3. Monoclonal Anti—CD4 Antibody MT310 Binds HIV-1 gp120 Binding Site on CD4

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Tests show the monoclonal anti—CD4 antibody (mAb) MT310 recognizes the gp120-binding site on CD4 as part of its mechanism for strongly inhibiting human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T cells. In competition tests, mAb MT310 and mAb Leu3a (an anti-CD4 mAb recognizing the gp120-binding site) all inhibited gp120-binding to CD4+ T lymphocytes, while mAb MT405 did not. This result suggests that MT310, like Leu3a, recognizes the gp120-binding site on CD4. To further confirm whether MT310 recognizes the gp120-binding site on CD4, we prepared rabbit anti-idiotypic antisera (Ab2) against MT310 (Ab1). The anti-idiotypic antisera against MT310 inhibited binding of MT310 and Leu3a to human CD4+ T lymphocytes, but did not block binding of MT151 with the second domain of CD4, while rabbit anti-idiotypic antisera to MT151 could block binding of itself to these cells, but could not inhibit the binding of MT310 and Leu3a, further indicating that MT310 recognized the gp120-binding site on CD4.

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

    KAUST Repository

    Gao, Xin

    2016-05-06

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

  5. Distribution of intercalative dye binding sites in chromatin.

    Science.gov (United States)

    Lurquin, P F; Seligy, V L

    1976-04-01

    Actinomycin D (AMD) and ethidium bromide (EB) were found to bind to chromatin isolated from a variety of gander tissues according to a strong and weak process analogous to that found for deproteinized DNA. Distribution of the dye intercalation sites in chromatin and DNA were evaluated at low r-values (dye bound per nucleotide) by following the appearance of free dye released from chromatin and DNA during thermal denaturation. The AMD dissociation profiles closely resembled the DNA or chromatin-DNA denaturation profiles; whereas the EB derivative dissociation profiles, indicated 3 major transitions for transcriptionally active chromatin with the main component corresponding to the single component which characterizes DNA. The DNA-like component was greatly reduced for mature erythrocyte chromatin but could be generated by removal of histone I and V. Removal of residual non acid-soluble proteins from dehistonized chromatin, urea treatment or dissociation and reconstitution of chromatin favoured conversion to the DNA-like component with loss of the other two. This study indicates that more than one type of binding exists generally in chromatin.

  6. ncDNA and drift drive binding site accumulation

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Leeder J Steven

    2003-09-01

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

  8. Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1.

    Science.gov (United States)

    Pham, Thu-Hang; Minderjahn, Julia; Schmidl, Christian; Hoffmeister, Helen; Schmidhofer, Sandra; Chen, Wei; Längst, Gernot; Benner, Christopher; Rehli, Michael

    2013-07-01

    The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-08-12

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

  11. Target-mediated drug disposition model for drugs with two binding sites that bind to a target with one binding site.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2017-07-19

    The paper extended the TMDD model to drugs with two identical binding sites (2-1 TMDD). The quasi-steady-state (2-1 QSS), quasi-equilibrium (2-1 QE), irreversible binding (2-1 IB), and Michaelis-Menten (2-1 MM) approximations of the model were derived. Using simulations, the 2-1 QSS approximation was compared with the full 2-1 TMDD model. As expected and similarly to the standard TMDD for monoclonal antibodies (mAb), 2-1 QSS predictions were nearly identical to 2-1 TMDD predictions, except for times of fast changes following initiation of dosing, when equilibrium has not yet been reached. To illustrate properties of new equations and approximations, several variations of population PK data for mAbs with soluble (slow elimination of the complex) or membrane-bound (fast elimination of the complex) targets were simulated from a full 2-1 TMDD model and fitted to 2-1 TMDD models, to its approximations, and to the standard (1-1) QSS model. For a mAb with a soluble target, it was demonstrated that the 2-1 QSS model provided nearly identical description of the observed (simulated) free drug and total target concentrations, although there was some minor bias in predictions of unobserved free target concentrations. The standard QSS approximation also provided a good description of the observed data, but was not able to distinguish between free drug concentrations (with no target attached and both binding site free) and partially bound drug concentrations (with one of the binding sites occupied by the target). For a mAb with a membrane-bound target, the 2-1 MM approximation adequately described the data. The 2-1 QSS approximation converged 10 times faster than the full 2-1 TMDD, and its run time was comparable with the standard QSS model.

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

    Science.gov (United States)

    Clifford, Jacob; Adami, Christoph

    2015-09-02

    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 about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

  13. Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site

    Directory of Open Access Journals (Sweden)

    Fortin Sébastien

    2010-04-01

    Full Text Available Abstract Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide (EBI to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

  14. Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site

    Directory of Open Access Journals (Sweden)

    Moreau Emmanuel

    2010-01-01

    Full Text Available Abstract Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide (EBI to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

  15. Prediction of nucleosome positioning based on transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Xianfu Yi

    Full Text Available BACKGROUND: The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs have been suggested to play a role in nucleosome positioning in vivo. PRINCIPAL FINDINGS: Here, the minimum redundancy maximum relevance (mRMR feature selection algorithm, the nearest neighbor algorithm (NNA, and the incremental feature selection (IFS method were used to identify the most important TFs that either favor or inhibit nucleosome positioning by analyzing the numbers of transcription factor binding sites (TFBSs in 53,021 nucleosomal DNA sequences and 50,299 linker DNA sequences. A total of nine important families of TFs were extracted from 35 families, and the overall prediction accuracy was 87.4% as evaluated by the jackknife cross-validation test. CONCLUSIONS: Our results are consistent with the notion that TFs are more likely to bind linker DNA sequences than the sequences in the nucleosomes. In addition, our results imply that there may be some TFs that are important for nucleosome positioning but that play an insignificant role in discriminating nucleosome-forming DNA sequences from nucleosome-inhibiting DNA sequences. The hypothesis that TFs play a role in nucleosome positioning is, thus, confirmed by the results of this study.

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

    Science.gov (United States)

    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.

  17. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    Science.gov (United States)

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  18. Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

    Science.gov (United States)

    Suji, George; Khedkar, Santosh A; Singh, Sreelekha K; Kishore, Nand; Coutinho, Evans C; Bhor, Vikrant M; Sivakami, S

    2008-06-01

    The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    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.......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...

  20. Peripheral benzodiazepine binding sites on striated muscles of the rat: Properties and effect of denervation

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, W.E.; Ickstadt, A. (Mainz Univ. (Germany, F.R.). Pharmakologisches Inst.); Hopf, H.Ch. (Mainz Univ. (Germany, F.R.))

    1985-01-01

    In order to test the hypothesis that peripheral benzodiazepine binding sites mediate some direct effects of benzodiazepines on striated muscles, the properties of specific /sup 3/H-Ro 5-4864 binding to rat biceps and rat diaphragm homogenates were investigated. In both tissues a single population of sites was found with a Ksub(D) value of 3 nmol/l. The density of these sites in both muscles was higher than the density in rat brain, but was considerably lower than in rat kidney. Competition experiments indicate a substrate specificity of specific /sup 3/H-Ro 5-4864 binding similar to the properties already demonstrated for the specific binding of this ligand to peripheral benzodiazepine binding sites in many other tissues. The properties of these sites in the rat diaphragm are not changed after motoric denervation by phrenicectomy. It is concluded that peripheral benzodiazepine binding sites are not involved in direct effects of benzodiazepines on striated muscles.

  1. In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.

    Directory of Open Access Journals (Sweden)

    Nima Najand

    Full Text Available We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.

  2. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

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

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

    Indian Academy of Sciences (India)

    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.

  4. Determination of energies and sites of binding of PFOA and PFOS to human serum albumin.

    Science.gov (United States)

    Salvalaglio, Matteo; Muscionico, Isabella; Cavallotti, Carlo

    2010-11-25

    Structure and energies of the binding sites of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) to human serum albumin (HSA) were determined through molecular modeling. The calculations consisted of a compound approach based on docking, followed by molecular dynamics simulations and by the estimation of the free binding energies adopting WHAM-umbrella sampling and semiempirical methodologies. The binding sites so determined are common either to known HSA fatty acids sites or to other HSA sites known to bind to pharmaceutical compounds such as warfarin, thyroxine, indole, and benzodiazepin. Among the PFOA binding sites, five have interaction energies in excess of -6 kcal/mol, which become nine for PFOS. The calculated binding free energy of PFOA to the Trp 214 binding site is the highest among the PFOA complexes, -8.0 kcal/mol, in good agreement with literature experimental data. The PFOS binding site with the highest energy, -8.8 kcal/mol, is located near the Trp 214 binding site, thus partially affecting its activity. The maximum number of ligands that can be bound to HSA is 9 for PFOA and 11 for PFOS. The calculated data were adopted to predict the level of complexation of HSA as a function of the concentration of PFOA and PFOS found in human blood for different levels of exposition. The analysis of the factors contributing to the complex binding energy permitted to outline a set of guidelines for the rational design of alternative fluorinated surfactants with a lower bioaccumulation potential.

  5. Prediction of calcium-binding sites by combining loop-modeling with machine learning

    Directory of Open Access Journals (Sweden)

    Altman Russ B

    2009-12-01

    Full Text Available Abstract Background Protein ligand-binding sites in the apo state exhibit structural flexibility. This flexibility often frustrates methods for structure-based recognition of these sites because it leads to the absence of electron density for these critical regions, particularly when they are in surface loops. Methods for recognizing functional sites in these missing loops would be useful for recovering additional functional information. Results We report a hybrid approach for recognizing calcium-binding sites in disordered regions. Our approach combines loop modeling with a machine learning method (FEATURE for structure-based site recognition. For validation, we compared the performance of our method on known calcium-binding sites for which there are both holo and apo structures. When loops in the apo structures are rebuilt using modeling methods, FEATURE identifies 14 out of 20 crystallographically proven calcium-binding sites. It only recognizes 7 out of 20 calcium-binding sites in the initial apo crystal structures. We applied our method to unstructured loops in proteins from SCOP families known to bind calcium in order to discover potential cryptic calcium binding sites. We built 2745 missing loops and evaluated them for potential calcium binding. We made 102 predictions of calcium-binding sites. Ten predictions are consistent with independent experimental verifications. We found indirect experimental evidence for 14 other predictions. The remaining 78 predictions are novel predictions, some with intriguing potential biological significance. In particular, we see an enrichment of beta-sheet folds with predicted calcium binding sites in the connecting loops on the surface that may be important for calcium-mediated function switches. Conclusion Protein crystal structures are a potentially rich source of functional information. When loops are missing in these structures, we may be losing important information about binding sites and active

  6. Basis for half-site ligand binding in yeast NAD(+)-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Lin, An-Ping; McAlister-Henn, Lee

    2011-09-27

    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 can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2(C150S) octameric enzymes were conducted in the presence of dithiothreitol. These assays demonstrated the presence of eight isocitrate and four AMP binding sites for the wild-type enzyme in the presence of dithiothreitol and for the IDH1/IDH2(C150S) enzyme in the absence or presence of this reagent, suggesting that interactions between sulfhydryl side chains of IDH2 Cys-150 residues limit access to these sites. However, only two NAD(+) sites could be measured for either enzyme. A tetrameric form of IDH (an IDH1(G15D)/IDH2 mutant enzyme) demonstrated half-site binding for isocitrate (two sites) in the absence of dithiothreitol and full-site binding (four sites) in the presence of dithiothreitol. Only one NAD(+) site could be measured for the tetramer under both conditions. In the context of the structure of the enzyme, these results suggest that an observed asymmetry between heterotetramers in the holoenzyme contributes to interactions between IDH2 Cys-150 residues and to half-site binding of isocitrate, but that a form of negative cooperativity may limit access to apparently equivalent NAD(+) binding sites.

  7. Structural Perspectives on the Evolutionary Expansion of Unique Protein-Protein Binding Sites.

    Science.gov (United States)

    Goncearenco, Alexander; Shaytan, Alexey K; Shoemaker, Benjamin A; Panchenko, Anna R

    2015-09-15

    Structures of protein complexes provide atomistic insights into protein interactions. Human proteins represent a quarter of all structures in the Protein Data Bank; however, available protein complexes cover less than 10% of the human proteome. Although it is theoretically possible to infer interactions in human proteins based on structures of homologous protein complexes, it is still unclear to what extent protein interactions and binding sites are conserved, and whether protein complexes from remotely related species can be used to infer interactions and binding sites. We considered biological units of protein complexes and clustered protein-protein binding sites into similarity groups based on their structure and sequence, which allowed us to identify unique binding sites. We showed that the growth rate of the number of unique binding sites in the Protein Data Bank was much slower than the growth rate of the number of structural complexes. Next, we investigated the evolutionary roots of unique binding sites and identified the major phyletic branches with the largest expansion in the number of novel binding sites. We found that many binding sites could be traced to the universal common ancestor of all cellular organisms, whereas relatively few binding sites emerged at the major evolutionary branching points. We analyzed the physicochemical properties of unique binding sites and found that the most ancient sites were the largest in size, involved many salt bridges, and were the most compact and least planar. In contrast, binding sites that appeared more recently in the evolution of eukaryotes were characterized by a larger fraction of polar and aromatic residues, and were less compact and more planar, possibly due to their more transient nature and roles in signaling processes.

  8. Oestradiol and testosterone binding sites in mice tibiae and their relationship with bone growth.

    Science.gov (United States)

    Lopez, A; Ventanas, J; Burgos, J

    1986-11-01

    High affinity oestradiol and testosterone binding sites were found in tibiae cytosol from entire male and female of different ages. Scatchard assay allowed to estimate a Kd of 2.7 X 10(-9) M for oestradiol binding sites indicating that the 3H-oestradiol binding was of high affinity. Oestradiol and testosterone binding sites abundance in mice tibiae are subject to change with age. It is not easy to establish a direct correlation between these changes and the values reported here on bone growth in weight and length, however seems possible to point a negative relationship between bone lengthening and oestradiol binding site levels in female, as well a positive relationship with testosterone in both sexes. The presence of oestradiol and testosterone binding sites in epiphyses and not in the diaphyses reinforces the hypothesis that both are playing some role in bone growth.

  9. Identification of a second substrate-binding site in solute-sodium symporters.

    Science.gov (United States)

    Li, Zheng; Lee, Ashley S E; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P; Abramson, Jeff; Quick, Matthias; Shi, Lei

    2015-01-02

    The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ~1. In addition, the related and more experimentally tractable SSS member PutP (the Na(+)/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.

  10. Alignment-free ultra-high-throughput comparison of druggable protein-ligand binding sites.

    Science.gov (United States)

    Weill, Nathanaël; Rognan, Didier

    2010-01-01

    Inferring the biological function of a protein from its three-dimensional structure as well as explaining why a drug may bind to various targets is of crucial importance to modern drug discovery. Here we present a generic 4833-integer vector describing druggable protein-ligand binding sites that can be applied to any protein and any binding cavity. The fingerprint registers counts of pharmacophoric triplets from the Calpha atomic coordinates of binding-site-lining residues. Starting from a customized data set of diverse protein-ligand binding site pairs, the most appropriate metric and a similarity threshold could be defined for similar binding sites. The method (FuzCav) has been used in various scenarios: (i) screening a collection of 6000 binding sites for similarity to different queries; (ii) classifying protein families (serine endopeptidases, protein kinases) by binding site diversity; (iii) discriminating adenine-binding cavities from decoys. The fingerprint generation and comparison supports ultra-high throughput (ca. 1000 measures/s), does not require prior alignment of protein binding sites, and is able to detect local similarity among subpockets. It is thus particularly well suited to the functional annotation of novel genomic structures with low sequence identity to known X-ray templates.

  11. CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site.

    Science.gov (United States)

    Duus, Karen; Thielens, Nicole M; Lacroix, Monique; Tacnet, Pascale; Frachet, Philippe; Holmskov, Uffe; Houen, Gunnar

    2010-12-01

    CD91 plays an important role in the scavenging of apoptotic material, possibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found to bind CD91. The MBL-CD91 interaction was time- and concentration-dependent and could be inhibited by known ligands of CD91. MBL-associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP-MBL interaction site. This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic material by CD91.

  12. CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site

    DEFF Research Database (Denmark)

    Duus, Karen; Thielens, Nicole M; Lacroix, Monique;

    2010-01-01

    CD91 plays an important role in the scavenging of apoptotic material, possibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found...... to bind CD91. The MBL-CD91 interaction was time- and concentration-dependent and could be inhibited by known ligands of CD91. MBL-associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP-MBL interaction site....... This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic...

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

    OpenAIRE

    Amaral, L; Lee, Y.; Schwarz, U.; Lorian, V

    1986-01-01

    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. At low pH, PBPs 1b, 1c, 2, and 3 demonstrated the greatest amount of binding. At high pH, these PBPs bound the least amount of penicillin. PBPs 1a and 5/6 exhibited the greatest amount of binding at pH 10 and the least amount at pH 4. With the exception of PBP 5/6, the effect of pH on the...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1).

    Science.gov (United States)

    Günther, S; Huchzermeyer, B

    1998-12-01

    The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-03-15

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

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

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.; Goberna, R.; Guerrero, J.M. (Univ. of Seville School of Medicine, Sevilla (Spain))

    1991-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

    OpenAIRE

    Borgeson, Claudia D.; Samson, Marie-Laure

    2005-01-01

    The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3′-untranslated region (3′-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3′-UTR. This site resembles ELAV-binding sites identified prev...

  2. Does transcription play a role in creating a condensin binding site?

    Science.gov (United States)

    Bernard, Pascal; Vanoosthuyse, Vincent

    2015-01-01

    The highly conserved condensin complex is essential for the condensation and integrity of chromosomes through cell division. Published data argue that high levels of transcription contribute to specify some condensin-binding sites on chromosomes but the exact role of transcription in this process remains elusive. Here we discuss our recent data addressing the role of transcription in establishing a condensin-binding site.

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

    DEFF Research Database (Denmark)

    Kjeldsen, K; Richter, Erik; Galbo, H

    1986-01-01

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

  4. Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

    Science.gov (United States)

    Luo, G; Lewis, R A

    1992-12-01

    RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

  5. There is communication between all four Ca(2+)-bindings sites of calcineurin B.

    Science.gov (United States)

    Gallagher, S C; Gao, Z H; Li, S; Dyer, R B; Trewhella, J; Klee, C B

    2001-10-09

    We have used site-directed mutagenesis, flow dialysis, and Fourier transform infrared (FTIR) spectroscopy to study Ca(2+)-binding to the regulatory component of calcineurin. Single Glu-Gln(E --> Q) mutations were used to inactivate each of the four Ca(2+)-binding sites of CnB in turn, generating mutants Q1, Q2, Q3, and Q4, with the number indicating which Ca(2+) site is inactivated. The binding data derived from flow dialysis reveal two pairs of sites in the wild-type protein, one pair with very high affinity and the other with lower affinity Ca(2+)-binding sites. Also, only three sites are titratable in the wild-type protein because one site cannot be decalcified. Mutation of site 2 leaves the protein with only two titratable sites, while mutation of sites 1, 3, or 4 leave three titratable sites that are mostly filled with 3 Ca(2+) equiv added. The binding data further show that each of the single-site mutations Q2, Q3, and Q4 affects the affinities of at least one of the remaining sites. Mutation in either of sites 3 or 4 results in a protein with no high-affinity sites, indicating communication between the two high-affinity sites, most likely sites 3 and 4. Mutation in site 2 decreases the affinity of all three remaining sites, though still leaving two relatively high-affinity sites. The FTIR data support the conclusions from the binding data with respect to the number of titratable sites as well as the impact of each mutation on the affinities of the remaining sites. We conclude therefore that there is communication between all four Ca(2+)-binding sites. In addition, the Ca(2+) induced changes in the FTIR spectra for the wild-type and Q4 mutant are most similar, suggesting that the same three Ca(2+)-binding sites are being titrated, i.e., site 4 is the very high-affinity site under the conditions of the FTIR experiments.

  6. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs

    OpenAIRE

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and avail...

  7. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    OpenAIRE

    Zhichao Miao; Eric Westhof

    2015-01-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and avail...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.

    Science.gov (United States)

    Seidel, Susanne A I; Wienken, Christoph J; Geissler, Sandra; Jerabek-Willemsen, Moran; Duhr, Stefan; Reiter, Alwin; Trauner, Dirk; Braun, Dieter; Baaske, Philipp

    2012-10-15

    Look, no label! Microscale thermophoresis makes use of the intrinsic fluorescence of proteins to quantify the binding affinities of ligands and discriminate between binding sites. This method is suitable for studying binding interactions of very small amounts of protein in solution. The binding of ligands to iGluR membrane receptors, small-molecule inhibitorss to kinase p38, aptamers to thrombin, and Ca(2+) ions to synaptotagmin was quantified.

  10. Spatial distribution of predicted transcription factor binding sites in Drosophila ChIP peaks.

    Science.gov (United States)

    Pettie, Kade P; Dresch, Jacqueline M; Drewell, Robert A

    2016-08-01

    In the development of the Drosophila embryo, gene expression is directed by the sequence-specific interactions of a large network of protein transcription factors (TFs) and DNA cis-regulatory binding sites. Once the identity of the typically 8-10bp binding sites for any given TF has been determined by one of several experimental procedures, the sequences can be represented in a position weight matrix (PWM) and used to predict the location of additional TF binding sites elsewhere in the genome. Often, alignments of large (>200bp) genomic fragments that have been experimentally determined to bind the TF of interest in Chromatin Immunoprecipitation (ChIP) studies are trimmed under the assumption that the majority of the binding sites are located near the center of all the aligned fragments. In this study, ChIP/chip datasets are analyzed using the corresponding PWMs for the well-studied TFs; CAUDAL, HUNCHBACK, KNIRPS and KRUPPEL, to determine the distribution of predicted binding sites. All four TFs are critical regulators of gene expression along the anterio-posterior axis in early Drosophila development. For all four TFs, the ChIP peaks contain multiple binding sites that are broadly distributed across the genomic region represented by the peak, regardless of the prediction stringency criteria used. This result suggests that ChIP peak trimming may exclude functional binding sites from subsequent analyses.

  11. Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase.

    Science.gov (United States)

    Zhang, Xiaohua; Caner, Sami; Kwan, Emily; Li, Chunmin; Brayer, Gary D; Withers, Stephen G

    2016-11-01

    Starch provides the major source of caloric intake in many diets. Cleavage of starch into malto-oligosaccharides in the gut is catalyzed by pancreatic α-amylase. These oligosaccharides are then further cleaved by gut wall α-glucosidases to release glucose, which is absorbed into the bloodstream. Potential surface binding sites for starch on the pancreatic amylase, distinct from the active site of the amylase, have been identified through X-ray crystallographic analyses. The role of these sites in the degradation of both starch granules and soluble starch was probed by the generation of a series of surface variants modified at each site to disrupt binding. Kinetic analysis of the binding and/or cleavage of substrates ranging from simple maltotriosides to soluble starch and insoluble starch granules has allowed evaluation of the potential role of each such surface site. In this way, two key surface binding sites, on the same face as the active site, are identified. One site, containing a pair of aromatic residues, is responsible for attachment to starch granules, while a second site featuring a tryptophan residue around which a malto-oligosaccharide wraps is shown to heavily influence soluble starch binding and hydrolysis. These studies provide insights into the mechanisms by which enzymes tackle the degradation of largely insoluble polymers and also present some new approaches to the interrogation of the binding sites involved.

  12. Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels.

    Science.gov (United States)

    Lyman, Kyle A; Han, Ye; Heuermann, Robert J; Cheng, Xiangying; Kurz, Jonathan E; Lyman, Reagan E; Van Veldhoven, Paul P; Chetkovich, Dane M

    2017-09-08

    Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein-protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal (PTS1) motifs. A homolog of PEX5, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide-binding domain (CNBD) and the C-terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN CNBD through a 37-residue domain and the HCN C-terminus through the TPR domains. Using a combination of fluorescence polarization and co-immunoprecipitation based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to PTS1 substrates. These results raise the possibility that other TPR domains may similarly be influenced by allosteric mechanisms as a general feature of protein-protein interactions. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

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

    Science.gov (United States)

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ionas Erb

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

  15. Characterization of the binding sites for dicarboxylic acids on bovine serum albumin.

    Science.gov (United States)

    Tonsgard, J H; Meredith, S C

    1991-06-15

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic.

  16. Functional identification and characterization of sodium binding sites in Na symporters.

    Science.gov (United States)

    Loo, Donald D F; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A; Wright, Ernest M

    2013-11-19

    Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na(+) in binding sites is beyond the resolution of the structures, two Na(+) binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na(+) binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na(+) and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na(+) binding, and that Na(+) binding to Na2 promotes binding to Na1 and also sugar binding.

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

    Science.gov (United States)

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

    2015-12-21

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

  18. [Type-I and -II estradiol binding sites in the endometrium during blastocyst implantation].

    Science.gov (United States)

    Bernal, A; Calzada, L; Hicks, J J; Velázquez, A

    1989-04-01

    The properties of type I and occupied and unoccupied type II cytosolic estrogen binding sites in the rat endometrium were analyzed on day five of pregnancy; the samples studied correspond to blastocyst receptive endometrium (implantation sites), nonreceptive endometrium and ovariectomized uterine horn endometrium, from the same pregnancy rats. The occupied binding site type II was analyzed by exchange assays. Dissociation constant obtained from experiments carried out at 4 or 25 degrees C are similar for each one of the binding site at the three different endometrium samples; the binding capacity (femtomoles/mg protein) from the sites type I and type II and the ratio between occupied (by endogenous estradiol) and unoccupied site type II, seems to be characteristic for each one of the three analyzed endometrium.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-08-28

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

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

    NARCIS (Netherlands)

    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),

  1. Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species.

    Directory of Open Access Journals (Sweden)

    Robert K Bradley

    2010-03-01

    Full Text Available Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances.

  2. DO-RIP-seq to quantify RNA binding sites transcriptome-wide.

    Science.gov (United States)

    Nicholson, Cindo O; Friedersdorf, Matthew B; Bisogno, Laura S; Keene, Jack D

    2016-11-10

    Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al., 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al., 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information. Therefore, DO-RIP-seq is useful for quantifying RBP binding events that are regulated during dynamic biological processes.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Lin, An-Ping; McAlister-Henn, Lee

    2002-06-21

    Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer containing two types of homologous subunits. Ligand-binding analyses were conducted to examine effects of residue changes in putative catalytic and regulatory isocitrate-binding sites respectively contained in IDH2 and IDH1 subunits. Replacement of homologous serine residues in either subunit site, S98A in IDH2 or S92A in IDH1, was found to reduce by half the total number of holoenzyme isocitrate-binding sites, confirming a correlation between detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP. Replacement of both serine residues eliminates isocitrate binding and measurable catalytic activity. The putative isocitrate-binding sites of IDH1 and IDH2 contain five identical and four nonidentical residues. Reciprocal replacement of the four nonidentical residues in either or both subunits (A108R, F136Y, T241D, and N245D in IDH1 and/or R114A, Y142F, D248T, and D252N in IDH2) was found to be permissive for isocitrate binding. This provides further evidence for two types of binding sites in IDH, although the authentic residues have been shown to be necessary for normal kinetic contributions. Finally, the mutant enzymes with residue replacements in the IDH1 site were found to be unable to bind AMP, suggesting that allosteric activation is dependent both upon binding of isocitrate at the IDH1 site and upon the changes in the enzyme normally elicited by this binding.

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

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Oscar Harari

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  10. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models

    Science.gov (United States)

    Mehta, Pankaj; Schwab, David J.; Sengupta, Anirvan M.

    2011-04-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where the density of binding sites is low. We then use techniques from statistical mechanics to derive a scaling principle relating the specificity (binding energy) of a TF to the minimum amount of training data necessary to learn it.

  11. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models.

    Science.gov (United States)

    Mehta, Pankaj; Schwab, David J; Sengupta, Anirvan M

    2011-04-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where the density of binding sites is low. We then use techniques from statistical mechanics to derive a scaling principle relating the specificity (binding energy) of a TF to the minimum amount of training data necessary to learn it.

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

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

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

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...... for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon...... sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites...

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

    Science.gov (United States)

    Colas, B; Bodegas, G; Sanz, M; Prieto, J C; Arilla, E

    1988-05-01

    1. 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. 2. Five weeks after surgery both the somatostatin concentration and the number of somatostatin-binding sites returned towards control values. 3. These results suggest that an increase in bowel somatostatin content could lead to down-regulation of somatostatin-binding sites in the intestinal mucosa.

  16. Brominated lipids identify lipid binding sites on the surface of the reaction center from Rhodobacter sphaeroides.

    Science.gov (United States)

    Roszak, Aleksander W; Gardiner, Alastair T; Isaacs, Neil W; Cogdell, Richard J

    2007-03-20

    This study describes the use of brominated phospholipids to distinguish between lipid and detergent binding sites on the surface of a typical alpha-helical membrane protein. Reaction centers isolated from Rhodobacter sphaeroides were cocrystallized with added brominated phospholipids. X-ray structural analysis of these crystals has revealed the presence of two lipid binding sites from the characteristic strong X-ray scattering from the bromine atoms. These results demonstrate the usefulness of this approach to mapping lipid binding sites at the surface of membrane proteins.

  17. Identification of ligands that target the HCV-E2 binding site on CD81

    Science.gov (United States)

    Olaby, Reem Al; Azzazy, Hassan M.; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

  18. An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

    Science.gov (United States)

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D.

    2010-01-01

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro. PMID:20018884

  19. Identification of ligands that target the HCV-E2 binding site on CD81.

    Science.gov (United States)

    Olaby, Reem Al; Azzazy, Hassan M; Harris, Rodney; Chromy, Brett; Vielmetter, Jost; Balhorn, Rod

    2013-04-01

    Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-26

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    DEFF Research Database (Denmark)

    Dupont, Daniel M; 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...... 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-12

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

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Predicting DNA-binding sites of proteins based on sequential and 3D structural information.

    Science.gov (United States)

    Li, Bi-Qing; Feng, Kai-Yan; Ding, Juan; Cai, Yu-Dong

    2014-06-01

    Protein-DNA interactions play important roles in many biological processes. To understand the molecular mechanisms of protein-DNA interaction, it is necessary to identify the DNA-binding sites in DNA-binding proteins. In the last decade, computational approaches have been developed to predict protein-DNA-binding sites based solely on protein sequences. In this study, we developed a novel predictor based on support vector machine algorithm coupled with the maximum relevance minimum redundancy method followed by incremental feature selection. We incorporated not only features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, solvent accessibility, but also five three-dimensional (3D) structural features calculated from PDB data to predict the protein-DNA interaction sites. Feature analysis showed that 3D structural features indeed contributed to the prediction of DNA-binding site and it was demonstrated that the prediction performance was better with 3D structural features than without them. It was also shown via analysis of features from each site that the features of DNA-binding site itself contribute the most to the prediction. Our prediction method may become a useful tool for identifying the DNA-binding sites and the feature analysis described in this paper may provide useful insights for in-depth investigations into the mechanisms of protein-DNA interaction.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit...... that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley alpha-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends...

  7. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site...

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

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

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

  9. Identification of the binding sites of regulatory proteins in bacterial genomes

    OpenAIRE

    Li, Hao; Rhodius, Virgil; Gross, Carol; Siggia, Eric D.

    2002-01-01

    We present an algorithm that extracts the binding sites (represented by position-specific weight matrices) for many different transcription factors from the regulatory regions of a genome, without the need for delineating groups of coregulated genes. The algorithm uses the fact that many DNA-binding proteins in bacteria bind to a bipartite motif with two short segments more conserved than the intervening region. It identifies all statistically significant patterns of the form W1NxW2, where W1...

  10. Three-dimensional binding sites volume assessment during cardiac pacing lead extraction

    Directory of Open Access Journals (Sweden)

    Bich Lien Nguyen

    2015-07-01

    Conclusions: Real-time 3D binding sites assessment is feasible and improves transvenous lead extraction outcomes. Its role as a complementary information requires extensive validation, and might be beneficial for a tailored strategy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Duman, Memed, E-mail: memi@hacettepe.edu.tr

    2014-10-01

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

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  13. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  15. Identifying ligand binding sites and poses using GPU-accelerated Hamiltonian replica exchange molecular dynamics.

    Science.gov (United States)

    Wang, Kai; Chodera, John D; Yang, Yanzhi; Shirts, Michael R

    2013-12-01

    We present a method to identify small molecule ligand binding sites and poses within a given protein crystal structure using GPU-accelerated Hamiltonian replica exchange molecular dynamics simulations. The Hamiltonians used vary from the physical end state of protein interacting with the ligand to an unphysical end state where the ligand does not interact with the protein. As replicas explore the space of Hamiltonians interpolating between these states, the ligand can rapidly escape local minima and explore potential binding sites. Geometric restraints keep the ligands from leaving the vicinity of the protein and an alchemical pathway designed to increase phase space overlap between intermediates ensures good mixing. Because of the rigorous statistical mechanical nature of the Hamiltonian exchange framework, we can also extract binding free energy estimates for all putative binding sites. We present results of this methodology applied to the T4 lysozyme L99A model system for three known ligands and one non-binder as a control, using an implicit solvent. We find that our methodology identifies known crystallographic binding sites consistently and accurately for the small number of ligands considered here and gives free energies consistent with experiment. We are also able to analyze the contribution of individual binding sites to the overall binding affinity. Our methodology points to near term potential applications in early-stage structure-guided drug discovery.

  16. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Diniz, Carlos Roberto; Nascimento, Marta Cordeiro [FUNED, Belo Horizonte, MG (Brazil); Lima, Maria Elena de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia

    1996-07-01

    The neurotoxin Tx1, isolated from the venom of the South American spider Phoneutria nigriventer produces tail elevation and spastic paralysis of posterior limbs after intracerebral ventricular injection in mice. Tx1 also produces ileum contraction in bioassay. We have investigated the binding of radioiodinated-Tx1 ({sup 125} I-Tx1) on the preparation of myenteric plexus-longitudinal muscle membrane from guinea pig ileum (MPLM) as a tool to characterize the interaction of this neurotoxin with its site. The neurotoxin Tx1 was radioiodinated with Na{sup 125} I by the lactoperoxidase method. {sup 125} I-Tx1 specifically binds to a single class of noninteracting binding sites of high affinity (Kd= 3.5 x 10{sup -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 {sup 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)

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

    Directory of Open Access Journals (Sweden)

    Quader Saad

    2012-07-01

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

  19. Quantitative determination of angiotensin II binding sites in rat brain and pituitary gland by autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Israel, A.; Correa, F.M.A.; Niwa, M.; Saavedra, J.M. (National Inst. of Mental Health, Bethesda, MD (USA))

    1984-11-26

    Rat brain and pituitary angiotensin II (AII) binding sites were quantitated by incubation of tissue sections with /sup 125/I-(Sar/sup 1/) AII, Ultrofilm radioautography, computerized densitometry, and comparison with /sup 125/I-standards at appropriate film exposure times. The highest number of AII binding sites was found in anterior pituitary and the circumventricular organs, organon subfornicalis and organon vasculosum laminae terminalis.

  20. Guanine Nucleotides Modulate Cell Surface cAMP-Binding Sites in Membranes from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1984-01-01

    D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, t½ = 15 s). A second class is fast dissociating (t½ about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low a

  1. Cycloxaprid insecticide: nicotinic acetylcholine receptor binding site and metabolism.

    Science.gov (United States)

    Shao, Xusheng; Swenson, Tami L; Casida, John E

    2013-08-21

    Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs.

  2. In Silico Docking, Molecular Dynamics and Binding Energy Insights into the Bolinaquinone-Clathrin Terminal Domain Binding Site

    Directory of Open Access Journals (Sweden)

    Mohammed K. Abdel-Hamid

    2014-05-01

    Full Text Available Clathrin-mediated endocytosis (CME is a process that regulates selective internalization of important cellular cargo using clathrin-coated vesicles. Perturbation of this process has been linked to many diseases including cancer and neurodegenerative conditions. Chemical proteomics identified the marine metabolite, 2-hydroxy-5-methoxy-3-(((1S,4aS,8aS-1,4a,5-trimethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-2-ylmethylcyclohexa- 2,5-diene-1,4-dione (bolinaquinone as a clathrin inhibitor. While being an attractive medicinal chemistry target, the lack of data about bolinaquinone’s mode of binding to the clathrin enzyme represents a major limitation for its structural optimization. We have used a molecular modeling approach to rationalize the observed activity of bolinaquinone and to predict its mode of binding with the clathrin terminal domain (CTD. The applied protocol started by global rigid-protein docking followed by flexible docking, molecular dynamics and linear interaction energy calculations. The results revealed the potential of bolinaquinone to interact with various pockets within the CTD, including the clathrin-box binding site. The results also highlight the importance of electrostatic contacts over van der Waals interactions for proper binding between bolinaquinone and its possible binding sites. This study provides a novel model that has the potential to allow rapid elaboration of bolinaquinone analogues as a new class of clathrin inhibitors.

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

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

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

  4. CTCF genomic binding sites in Drosophila and the organisation of the bithorax complex.

    Directory of Open Access Journals (Sweden)

    Eimear E Holohan

    2007-07-01

    Full Text Available Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C. We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; "Fab-2," "Fab-3," and "Fab-4." With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation.

  5. MBSTAR: multiple instance learning for predicting specific functional binding sites in microRNA targets

    Science.gov (United States)

    Bandyopadhyay, Sanghamitra; Ghosh, Dip; Mitra, Ramkrishna; Zhao, Zhongming

    2015-01-01

    MicroRNA (miRNA) regulates gene expression by binding to specific sites in the 3'untranslated regions of its target genes. Machine learning based miRNA target prediction algorithms first extract a set of features from potential binding sites (PBSs) in the mRNA and then train a classifier to distinguish targets from non-targets. However, they do not consider whether the PBSs are functional or not, and consequently result in high false positive rates. This substantially affects the follow up functional validation by experiments. We present a novel machine learning based approach, MBSTAR (Multiple instance learning of Binding Sites of miRNA TARgets), for accurate prediction of true or functional miRNA binding sites. Multiple instance learning framework is adopted to handle the lack of information about the actual binding sites in the target mRNAs. Biologically validated 9531 interacting and 973 non-interacting miRNA-mRNA pairs are identified from Tarbase 6.0 and confirmed with PAR-CLIP dataset. It is found that MBSTAR achieves the highest number of binding sites overlapping with PAR-CLIP with maximum F-Score of 0.337. Compared to the other methods, MBSTAR also predicts target mRNAs with highest accuracy. The tool and genome wide predictions are available at http://www.isical.ac.in/~bioinfo_miu/MBStar30.htm.

  6. Propofol Shares the Binding Site with Isoflurane and Sevoflurane on Leukocyte Function-Associated Antigen-1

    Science.gov (United States)

    Yuki, Koichi; Bu, Weiming; Xi, Jin; Shimaoka, Motomu; Eckenhoff, Roderic

    2013-01-01

    Background We previously demonstrated that propofol interacted with the leukocyte adhesion molecule leukocyte function–associated antigen-1 (LFA-1) and inhibited the production of interleukin-2 via LFA-1 in a dependent manner. However, the binding site(s) of propofol on LFA-1 remains unknown. Methods First, the inhibition of LFA-1's ligand binding by propofol was confirmed in an ELISA-type assay. The binding site of propofol on LFA-1 was probed with a photolabeling experiment using a photoactivatable propofol analog called azi-propofol-m. The adducted residues of LFA-1 by this compound were determined using liquid chromatography–mass spectrometry. In addition, the binding of propofol to the ligand-binding domain of LFA-1 was examined using 1-aminoanthracene (1-AMA) displacement assay. Furthermore, the binding site(s) of 1-AMA and propofol on LFA-1 was studied using the docking program GLIDE. Results We demonstrated that propofol impaired the binding of LFA-1 to its ligand intercellular adhesion molecule-1. The photolabeling experiment demonstrated that the adducted residues were localized in the allosteric cavity of the ligand-binding domain of LFA-1 called “lovastatin site. ” The shift of fluorescence spectra was observed when 1-AMA was coincubated with the low-affinity conformer of LFA-1 ligand-binding domain (wild-type [WT] αL I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT αL I domain. In the 1-AMA displacement assay, propofol decreased 1-AMA fluorescence signal (at 520 nm), suggesting that propofol competed with 1-AMA and bound to the WT αL I domain. The docking simulation demonstrated that both 1-AMA and propofol bound to the lovastatin site, which agreed with the photolabeling experiment. Conclusions We demonstrated that propofol bound to the lovastatin site in LFA-1. Previously we showed that the volatile anesthetics isoflurane and sevoflurane bound to this site. Taken together, the lovastatin site is an

  7. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  8. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    Energy Technology Data Exchange (ETDEWEB)

    Gil, D.W.; Wolfe, B.B.

    1986-05-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands (/sup 3/H)quinuclidinyl benzilate or (/sup 3/H)PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of (/sup 3/H)quinuclidinyl benzilate in a biphasic manner.

  9. Phylogenetic distribution of [3H]kainic acid receptor binding sites in neuronal tissue.

    Science.gov (United States)

    London, E D; Klemm, N; Coyle, J T

    1980-06-23

    The phylogenetic distribution of specific binding sites for kainic acid was determined in 14 species including invertebrates and vertebrates. The highest level of binding was observed in brains of the frog (Xenopus laevis), followed by the spiny dogfish (Heterodontus francisci), the goldfish (Carasius auratus) and the chick (Gallus domesticus). Although significant specific binding was noted in some of the lowest forms tested (e.g. Hydra littoralis), this was not a consistent observation in the invertebrates. In most cases, specific binding to both high and low affinity sites was detected; notable exceptions were the cockroach brain (Periplaneta americana), which had negligible high affinity binding, and the crayfish brain (Procambarus) which had negligible low affinity binding. In the spiny dogfish, the smooth dogfish and the chick, the highest level of binding occurred in cerebellum with less in the forebrain and the least in the medulla; in the mammalian species, the highest level of binding occurred in the forebrain structures with less in the cerebellum and least in the medulla. Eadie plots of the saturation isotherms for [3H]kainic acid revealed similar kinetics of binding for frog whole brain, rat forebrain and human parietal cortex with two apparent populations of binding sites: KD1 = 25--50 nM and KD2 = 3--14 nM. While binding in the spiny dogfish forebrain and human caudate nucleus occurred exclusively at a high affinity component, the cerebella of chick, rat and man exhibited only a low affinity binding site. In the 3 species studied most extensively, frog, rat and man, unlabeled kainic acid was the most potent inhibitor of the specific binding of [3H]-kainic acid. L-Glutamic acid was 20--20-fold less potent than kainic acid, and D-glutamic acid was 4--2500-fold less potent than its L-isomer. Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid decreased the affinity of the derivative 115--30,000-fold. Hill coefficients

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

    Directory of Open Access Journals (Sweden)

    Amy Sebeson

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

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

    Science.gov (United States)

    Sebeson, Amy; Xi, Liqun; Zhang, Quanwei; Sigmund, Audrey; Wang, Ji-Ping; Widom, Jonathan; Wang, Xiaozhong

    2015-01-01

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

  12. In vitro and in vivo characterisation of [3H]ANSTO-14 binding to the sigma 1 binding sites.

    Science.gov (United States)

    Nguyen, V H; Mardon, K; Kassiou, M; Christie, M D

    1999-02-01

    N-(4-phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3, 10).0(5,9) .0(8,11)]dodecane (ANSTO-14) showed the highest activity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity. The present study showed that [3H]ANSTO-14 binds to a single high-affinity site in guinea pig brain membranes with an equilibrium Ki of 8.0 +/- 0.3 nM, in good agreement with the kinetic studies (Kd = 13.3 +/- 5.4 nM, n = 4), and a Bmax of 3.199 +/- 105 fmol/mg protein (n = 4). The in vivo biodistribution of [3H]ANSTO-14 showed a high uptake in the diencephalon. Pretreatment of rats with sigma ligands including (+)-pentazocine (sigma 1), ANSTO-14 (sigma 1), and DTG (sigma 1 and sigma 2) did not significantly reduce radiotracer uptake in the brain, but did in the spleen. A labelled metabolite was found in the liver and brain. Due to its insensitivity to sigma ligands, the accumulation of [3H]ANSTO-14 in the brain indicates high nonspecific binding. Therefore, [3H]ANSTO-14 is a suitable ligand for labelling sigma 1 sites in vitro but is not suitable for brain imaging of sigma binding sites in vivo.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... inhibition of dopamine transport by cocaine....

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert...

  15. Putative cholesterol-binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5.

    Science.gov (United States)

    Zhukovsky, Mikhail A; Lee, Po-Hsien; Ott, Albrecht; Helms, Volkhard

    2013-04-01

    Using molecular docking, we identified a cholesterol-binding site in the groove between transmembrane helices 1 and 7 near the inner membrane-water interface of the G protein-coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol-binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol-binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol-binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins.

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

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

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

  17. A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site.

    Science.gov (United States)

    Duffy, Caroline M; Hilbert, Brendan J; Kelch, Brian A

    2016-03-27

    The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

  18. Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers.

    Science.gov (United States)

    Farley, Emma K; Olson, Katrina M; Zhang, Wei; Rokhsar, Daniel S; Levine, Michael S

    2016-06-07

    Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.

  19. Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.; Mantyh, P.W. (Wadsworth VA Medical Center, Los Angeles, CA (USA))

    1988-09-01

    To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for {sup 125}I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between {sup 125}I-rat ANF and brown fat. Specific binding of {sup 125}I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.

  20. Characterization of gonadotropin binding sites in the intracellular organelles of bovine corpora lutea and comparison with plasma membrane sites.

    Science.gov (United States)

    Rao, C V; Mitra, S; Carman, F R

    1981-03-25

    The specific binding of 125I-human choriogonadotropin (hCG) to plasma membranes, nuclear membranes, lysosomes, rough endoplasmic reticulum, heavy golgi, and medium and light golgi of bovine corpora lutea was dependent on the amount of protein, 125I-hCG concentration and incubation time. The bound hormone in all the organelles was able to rebind to fresh corresponding organelles. Scatchard analysis revealed a homogenous population of gonadotropin binding sites in plasma membrane, rough endoplasmic reticulum, heavy golgi, and medium and light golgi, whose binding affinities (Kd = 8.6-11.0 X 10(-11) M) were similar but whose number of available gonadotropin binding sites varied. Scatchard analyses of nuclear membranes and lysosome binding, on the other hand, were heterogenous (Nuclear membranes, 11 and 23 X 10(-11) M lysosomes, 3.4 and 130 X 10(-11) M). The rate constants for association (5.9 to 12.1 X 10(6) M-1 S-1) and dissociation (7.4 to 9.0 X 10(-4) S-1) were similar among different subcellular organelles except for nuclear membranes and lysosomes, where rate constants for association were significantly lower. The ligand binding specificity, lower effectiveness of human luteinizing hormone as compared to hCG in competition, the optimal pH, the lack of ionic requirements for binding, and the molecular size of 125I-hCG-gonadotropin binding site complexes solubilized from various intracellular organelles were similar to those observed for plasma membranes. Numerous differences were also observed between intracellular organelles and plasma membranes as well as among intracellular organelles themselves with respect to binding losses due to exposure to low and high pH values, di- and monovalent cations, increasing preincubation temperatures, and a variety of enzymes and protein reagents. The possible reasons for these similarities as well as differences observed are discussed. The differences are viewed as an additional indication that contamination cannot solely

  1. Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

    Science.gov (United States)

    Chovanec, M; Smetana, K; Purkrábková, T; Holíková, Z; Dvoránková, B; André, S; Pytlík, R; Hozák, P; Plzák, J; Sedo, A; Vacík, J; Gabius, H

    2004-01-01

    The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

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

    Science.gov (United States)

    Gonzalez-Guijarro, L; Lopez-Ruiz, M P; Bodegas, G; Prieto, J C; Arilla, E

    1987-04-01

    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.

  3. A propofol binding site on mammalian GABAA receptors identified by photolabeling

    Science.gov (United States)

    Yip, Grace M S; Chen, Zi-Wei; Edge, Christopher J; Smith, Edward H; Dickinson, Robert; Hohenester, Erhard; Townsend, R Reid; Fuchs, Karoline; Sieghart, Werner; Evers, Alex S; Franks, Nicholas P

    2014-01-01

    Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2. PMID:24056400

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

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

  7. A quantitative model for the in vivo assessment of drug binding sites with positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Mintun, M.A.; Raichle, M.E.; Kilbourn, M.R.; Wooten, G.F.; Welch, M.J.

    1984-03-01

    We propose an in vivo method for use with positron emission tomography (PET) that results in a quantitative characterization of neuroleptic binding sites using radiolabeled spiperone. The data are analyzed using a mathematical model that describes transport, nonspecific binding, and specific binding in the brain. The model demonstrates that the receptor quantities Bmax (i.e., the number of binding sites) and KD-1 (i.e., the binding affinity) are not separably ascertainable with tracer methodology in human subjects. We have, therefore, introduced a new term, the binding potential, equivalent to the product BmaxKD-1, which reflects the capacity of a given tissue, or region of a tissue, for ligand-binding site interaction. The procedure for obtaining these measurements is illustrated with data from sequential PET scans of baboons after intravenous injection of carrier-added (18F)spiperone. From these data we estimate the brain tissue nonspecific binding of spiperone to be in the range of 94.2 to 95.3%, and the regional brain spiperone permeability (measured as the permeability-surface area product) to be in the range of 0.025 to 0.036 cm3/(s X ml). The binding potential of the striatum ranged from 17.4 to 21.6; these in vivo estimates compare favorably to in vitro values in the literature. To our knowledge this represents the first direct evidence that PET can be used to characterize quantitatively, locally and in vivo, drug binding sites in brain. The ability to make such measurements with PET should permit the detailed investigation of diseases thought to result from disorders of receptor function.

  8. Cooperativity between calmodulin-binding sites in Kv7.2 channels.

    Science.gov (United States)

    Alaimo, Alessandro; Alberdi, Araitz; Gomis-Perez, Carolina; Fernández-Orth, Juncal; Gómez-Posada, Juan Camilo; Areso, Pilar; Villarroel, Alvaro

    2013-01-01

    Among the multiple roles assigned to calmodulin (CaM), controlling the surface expression of Kv7.2 channels by binding to two discontinuous sites is a unique property of this Ca(2+) binding protein. Mutations that interfere with CaM binding or the sequestering of CaM prevent this M-channel component from exiting the endoplasmic reticulum (ER), which reduces M-current density in hippocampal neurons, enhancing excitability and offering a rational mechanism to explain some forms of benign familial neonatal convulsions (BFNC). Previously, we identified a mutation (S511D) that impedes CaM binding while allowing the channel to exit the ER, hinting that CaM binding may not be strictly required for Kv7.2 channel trafficking to the plasma membrane. Alternatively, this interaction with CaM might escape detection and, indeed, we now show that the S511D mutant contains functional CaM-binding sites that are not detected by classical biochemical techniques. Surface expression and function is rescued by CaM, suggesting that free CaM in HEK293 cells is limiting and reinforcing the hypothesis that CaM binding is required for ER exit. Within the CaM-binding domain formed by two sites (helix A and helix B), we show that CaM binds to helix B with higher apparent affinity than helix A, both in the presence and absence of Ca(2+), and that the two sites cooperate. Hence, CaM can bridge two binding domains, anchoring helix A of one subunit to helix B of another subunit, in this way influencing the function of Kv7.2 channels.

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

    Directory of Open Access Journals (Sweden)

    Tomasz Brzoska

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

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

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2012-01-01

    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 mo...... for a wide variety of human neurological disorders....

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-20

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

  12. Discriminating between HuR and TTP binding sites using the k-spectrum kernel method

    Science.gov (United States)

    Goldberg, Debra S.; Dowell, Robin

    2017-01-01

    Background The RNA binding proteins (RBPs) human antigen R (HuR) and Tristetraprolin (TTP) are known to exhibit competitive binding but have opposing effects on the bound messenger RNA (mRNA). How cells discriminate between the two proteins is an interesting problem. Machine learning approaches, such as support vector machines (SVMs), may be useful in the identification of discriminative features. However, this method has yet to be applied to studies of RNA binding protein motifs. Results Applying the k-spectrum kernel to a support vector machine (SVM), we first verified the published binding sites of both HuR and TTP. Additional feature engineering highlighted the U-rich binding preference of HuR and AU-rich binding preference for TTP. Domain adaptation along with multi-task learning was used to predict the common binding sites. Conclusion The distinction between HuR and TTP binding appears to be subtle content features. HuR prefers strongly U-rich sequences whereas TTP prefers AU-rich as with increasing A content, the sequences are more likely to be bound only by TTP. Our model is consistent with competitive binding of the two proteins, particularly at intermediate AU-balanced sequences. This suggests that fine changes in the A/U balance within a untranslated region (UTR) can alter the binding and subsequent stability of the message. Both feature engineering and domain adaptation emphasized the extent to which these proteins recognize similar general sequence features. This work suggests that the k-spectrum kernel method could be useful when studying RNA binding proteins and domain adaptation techniques such as feature augmentation could be employed particularly when examining RBPs with similar binding preferences. PMID:28333956

  13. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    Science.gov (United States)

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

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

    Directory of Open Access Journals (Sweden)

    Manuela Maurer

    2016-04-01

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

  15. Characterization of two different melatonin binding sites in peripheral tissues of the teleost Tinca tinca.

    Science.gov (United States)

    López Patiño, M A; Guijarro, A I; Alonso-Gómez, A L; Delgado, M J

    2012-01-01

    The aim of the present study was to localize and characterize 2-iodo-melatonin ([(125)I]Mel) binding sites in peripheral tissues of the teleost Tinca tinca. A wide distribution of [(125)I]Mel binding sites in peripheral locations of the tench is found, with highest densities being measured in the heart, gills and kidney, and low density of [(125)I]Mel binding sites in gastrointestinal tract, spleen, liver and gonads. Saturation, kinetics, and pharmacological approaches revealed the presence of, at least, two different [(125)I]Mel binding sites in the tench peripheral tissues. The unique characterized subtype in the heart fulfils all the criteria for a canonical melatonin receptor belonging to MT(1) family (the binding is saturable, reversible, and inhibited by GTP analogs), and gives support for the presence of a functional melatonin receptor in the heart of the tench. In contrast, kinetic and pharmacological studies in the kidney revealed the preponderance of a melatonin binding site belonging to the MT(3)-like receptor subtype. Moreover, the decrease of specific binding in both, heart and kidney membranes, and the decrease of affinity in the kidney, produced by the addition of a non-hydrolysable GTP analog, and sodium cations suggest the presence of G(i/o)-proteins (that mediate inhibition of cAMP formation) coupled to such melatonin binding sites. Our results also point to different G(i/o)-proteins involved in the underlying mechanism of melatonin binding sites activation in the kidney. Additionally, the kinetics of [(125)I]Mel binding in kidney membrane preparations is a highly thermosensitive process, being necessary to perform the assays at 4 °C since the equilibrium was not reached at 25 °C assay temperature. The time needed to complete association of [(125)I]Mel at such low temperature is only 15s, whereas 100s is required to displace [(125)I]Mel specific binding by the unlabeled melatonin in kidney membranes. Present results support previous reports on

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

    Science.gov (United States)

    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. © 2016

  17. PhyloScan: identification of transcription factor binding sites using cross-species evidence

    Directory of Open Access Journals (Sweden)

    Newberg Lee A

    2007-01-01

    Full Text Available Abstract Background When transcription factor binding sites are known for a particular transcription factor, it is possible to construct a motif model that can be used to scan sequences for additional sites. However, few statistically significant sites are revealed when a transcription factor binding site motif model is used to scan a genome-scale database. Methods We have developed a scanning algorithm, PhyloScan, which combines evidence from matching sites found in orthologous data from several related species with evidence from multiple sites within an intergenic region, to better detect regulons. The orthologous sequence data may be multiply aligned, unaligned, or a combination of aligned and unaligned. In aligned data, PhyloScan statistically accounts for the phylogenetic dependence of the species contributing data to the alignment and, in unaligned data, the evidence for sites is combined assuming phylogenetic independence of the species. The statistical significance of the gene predictions is calculated directly, without employing training sets. Results In a test of our methodology on synthetic data modeled on seven Enterobacteriales, four Vibrionales, and three Pasteurellales species, PhyloScan produces better sensitivity and specificity than MONKEY, an advanced scanning approach that also searches a genome for transcription factor binding sites using phylogenetic information. The application of the algorithm to real sequence data from seven Enterobacteriales species identifies novel Crp and PurR transcription factor binding sites, thus providing several new potential sites for these transcription factors. These sites enable targeted experimental validation and thus further delineation of the Crp and PurR regulons in E. coli. Conclusion Better sensitivity and specificity can be achieved through a combination of (1 using mixed alignable and non-alignable sequence data and (2 combining evidence from multiple sites within an intergenic

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

    Science.gov (United States)

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

    2008-08-26

    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 by direct cross-species comparison between four yeast species. We find ubiquitous compensatory mutations within functional sites, such that the energy phenotype and the function of a site evolve in a significantly more constrained way than does its sequence. We also find evidence for substantial evolution of regulatory function involving point mutations as well as sequence insertions and deletions within binding sites. Genes lose their regulatory link to a given transcription factor at a rate similar to the neutral point mutation rate, from which we infer a moderate average fitness advantage of functional over nonfunctional sites. In a wider context, this study provides an example of inference of selection acting on a quantitative molecular trait.

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

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    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, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-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 /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nye, J.S.

    1988-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-08-01

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

  2. Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

    Science.gov (United States)

    Husu, Ivan; Magyar, Melinda; Szabó, Tibor; Fiser, Béla; Gómez-Bengoa, Enrique; Nagy, László

    2015-04-01

    Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

  3. Putative hAPN receptor binding sites in SARS_CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    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. Membrane androgen binding sites are preferentially expressed in human prostate carcinoma cells

    Directory of Open Access Journals (Sweden)

    Delakas Dimitrios

    2003-01-01

    Full Text Available Abstract Background Prostate cancer is one of the most frequent malignancies in males. Nevertheless, to this moment, there is no specific routine diagnostic marker to be used in clinical practice. Recently, the identification of a membrane testosterone binding site involved in the remodeling of actin cytoskeleton structures and PSA secretion, on LNCaP human prostate cancer cells has been reported. We have investigated whether this membrane testosterone binding component could be of value for the identification of prostate cancer. Methods Using a non-internalizable testosterone-BSA-FITC analog, proven to bind on membrane sites only in LNCaP cells, we have investigated the expression of membrane testosterone binding sites in a series of prostate carcinomas (n = 14, morphologically normal epithelia, taken from areas of the surgical specimens far from the location of the carcinomas (n = 8 and benign prostate hyperplasia epithelia (n = 10. Isolated epithelial cells were studied by flow cytometry, and touching preparations, after 10-min incubation. In addition, routine histological slides were assayed by confocal laser microscopy. Results We show that membrane testosterone binding sites are preferentially expressed in prostate carcinoma cells, while BPH and non-malignant epithelial cells show a low or absent binding. Conclusions Our results indicate that membrane testosterone receptors might be of use for the rapid routine identification of prostate cancer, representing a new diagnostic marker of the disease.

  5. The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.

    Science.gov (United States)

    Tseng, C C; Miyamoto, M; Ramalingam, K; Hemavathy, K C; Levine, M J; Ramasubbu, N

    1999-02-01

    Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type

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

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

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

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

    Science.gov (United States)

    Sunayama, Hirobumi; Takeuchi, Toshifumi

    2014-11-26

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

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

    Directory of Open Access Journals (Sweden)

    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. Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli*

    Science.gov (United States)

    Opačić, Milena; Vos, Erwin P. P.; Hesp, Ben H.; Broos, Jaap

    2010-01-01

    The mannitol transporter from Escherichia coli, EIImtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EIImtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IICmtl). To localize this binding site, 19 single Trp-containing mutants of EIImtl were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IICmtl. Combined with the available two-dimensional projection maps of IICmtl, it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EIImtl becomes phosphorylated at Cys384 in the cytoplasmic B domain. Stably phosphorylated EIImtl mutants were constructed, and FRET experiments showed that the position of mannitol in IICmtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol. PMID:20522557

  10. Localization of the substrate-binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli.

    Science.gov (United States)

    Opacić, Milena; Vos, Erwin P P; Hesp, Ben H; Broos, Jaap

    2010-08-13

    The mannitol transporter from Escherichia coli, EII(mtl), belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII(mtl) is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC(mtl)). To localize this binding site, 19 single Trp-containing mutants of EII(mtl) were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC(mtl). Combined with the available two-dimensional projection maps of IIC(mtl), it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII(mtl) becomes phosphorylated at Cys(384) in the cytoplasmic B domain. Stably phosphorylated EII(mtl) mutants were constructed, and FRET experiments showed that the position of mannitol in IIC(mtl) remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.

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

    Science.gov (United States)

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

    2010-03-22

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

  12. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    Directory of Open Access Journals (Sweden)

    John A Capra

    2009-12-01

    Full Text Available Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/.

  13. Predicting protein ligand binding sites by combining evolutionary sequence conservation and 3D structure.

    Science.gov (United States)

    Capra, John A; Laskowski, Roman A; Thornton, Janet M; Singh, Mona; Funkhouser, Thomas A

    2009-12-01

    Identifying a protein's functional sites is an important step towards characterizing its molecular function. Numerous structure- and sequence-based methods have been developed for this problem. Here we introduce ConCavity, a small molecule binding site prediction algorithm that integrates evolutionary sequence conservation estimates with structure-based methods for identifying protein surface cavities. In large-scale testing on a diverse set of single- and multi-chain protein structures, we show that ConCavity substantially outperforms existing methods for identifying both 3D ligand binding pockets and individual ligand binding residues. As part of our testing, we perform one of the first direct comparisons of conservation-based and structure-based methods. We find that the two approaches provide largely complementary information, which can be combined to improve upon either approach alone. We also demonstrate that ConCavity has state-of-the-art performance in predicting catalytic sites and drug binding pockets. Overall, the algorithms and analysis presented here significantly improve our ability to identify ligand binding sites and further advance our understanding of the relationship between evolutionary sequence conservation and structural and functional attributes of proteins. Data, source code, and prediction visualizations are available on the ConCavity web site (http://compbio.cs.princeton.edu/concavity/).

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

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

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

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

    Directory of Open Access Journals (Sweden)

    Oliver Weth

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

  16. Binding Sites of miR-1273 Family on the mRNA of Target Genes

    Directory of Open Access Journals (Sweden)

    Anatoly Ivashchenko

    2014-01-01

    Full Text Available This study examined binding sites of 2,578 miRNAs in the mRNAs of 12,175 human genes using the MirTarget program. It found that the miRNAs of miR-1273 family have between 33 and 1,074 mRNA target genes, with a free hybridization energy of 90% or more of its maximum value. The miR-1273 family consists of miR-1273a, miR-1273c, miR-1273d, miR-1273e, miR-1273f, miR-1273g-3p, miR-1273g-5p, miR-1273h-3p, and miR-1273h-5p. Unique miRNAs (miR-1273e, miR-1273f, and miR-1273g-3p have more than 400 target genes. We established 99 mRNA nucleotide sequences that contain arranged binding sites for the miR-1273 family. High conservation of each miRNA binding site in the mRNA of the target genes was found. The arranged binding sites of the miR-1273 family are located in the 5′UTR, CDS, or 3′UTR of many mRNAs. Five repeating sites containing some of the miR-1273 family’s binding sites were found in the 3′UTR of several target genes. The oligonucleotide sequences of miR-1273 binding sites located in CDSs code for homologous amino acid sequences in the proteins of target genes. The biological role of unique miRNAs was also discussed.

  17. Prenatal exposure to methylmercury alters development of adrenergic receptor binding sites in peripheral sympathetic target tissues

    Energy Technology Data Exchange (ETDEWEB)

    Slotkin, T.A.; Orband, L.; Cowdery, T.; Kavlock, R.J.; Bartolome, J.

    1987-01-01

    In order to assess the impact of prenatal exposure to methylmercury on sympathetic neurotransmission, effects on development of adrenergic receptor binding sites in peripheral tissues was evaluated. In the liver, methylmercury produced a dose-dependent increase in alpha/sub 1/, alpha/sub 2/, and beta-receptor binding of radioliganda throughout the first 5 weeks of postnatal life. Similarly, renal alpha-receptor subtypes showed increased binding capabilities, but binding to alpha-receptor sites was reduced. At least some of the changes in receptors appear to be of functional significance, as physiological reactivity to adrenergic stimulation is altered in the same directions in these two tissues. The actions of methylmercury displayed tissue specificity in that the same receptor populations were largely unaffected in other tissues (lung, heart). These results suggest that methylmercury exposure in utero alters adrenergic responses through targeted effects on postsynaptic receptor populations in specific tissues.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  20. Novel approach for selecting the best predictor for identifying the binding sites in DNA binding proteins.

    Science.gov (United States)

    Nagarajan, R; Ahmad, Shandar; Gromiha, M Michael

    2013-09-01

    Protein-DNA complexes play vital roles in many cellular processes by the interactions of amino acids with DNA. Several computational methods have been developed for predicting the interacting residues in DNA-binding proteins using sequence and/or structural information. These methods showed different levels of accuracies, which may depend on the choice of data sets used in training, the feature sets selected for developing a predictive model, the ability of the models to capture information useful for prediction or a combination of these factors. In many cases, different methods are likely to produce similar results, whereas in others, the predictors may return contradictory predictions. In this situation, a priori estimates of prediction performance applicable to the system being investigated would be helpful for biologists to choose the best method for designing their experiments. In this work, we have constructed unbiased, stringent and diverse data sets for DNA-binding proteins based on various biologically relevant considerations: (i) seven structural classes, (ii) 86 folds, (iii) 106 superfamilies, (iv) 194 families, (v) 15 binding motifs, (vi) single/double-stranded DNA, (vii) DNA conformation (A, B, Z, etc.), (viii) three functions and (ix) disordered regions. These data sets were culled as non-redundant with sequence identities of 25 and 40% and used to evaluate the performance of 11 different methods in which online services or standalone programs are available. We observed that the best performing methods for each of the data sets showed significant biases toward the data sets selected for their benchmark. Our analysis revealed important data set features, which could be used to estimate these context-specific biases and hence suggest the best method to be used for a given problem. We have developed a web server, which considers these features on demand and displays the best method that the investigator should use. The web server is freely available at

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions...... 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 based...... on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion....

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

    Directory of Open Access Journals (Sweden)

    E. Gout

    2010-01-01

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

  3. Statistical Mechanics of Transcription-Factor Binding Site Discovery Using Hidden Markov Models

    OpenAIRE

    Mehta, Pankaj; Schwab, David J.; Sengupta, Anirvan M.

    2011-01-01

    Hidden Markov Models (HMMs) are a commonly used tool for inference of transcription factor (TF) binding sites from DNA sequence data. We exploit the mathematical equivalence between HMMs for TF binding and the "inverse" statistical mechanics of hard rods in a one-dimensional disordered potential to investigate learning in HMMs. We derive analytic expressions for the Fisher information, a commonly employed measure of confidence in learned parameters, in the biologically relevant limit where th...

  4. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    Science.gov (United States)

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  5. Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

    Science.gov (United States)

    Hanner, M; Moebius, F F; Flandorfer, A; Knaus, H G; Striessnig, J; Kempner, E; Glossmann, H

    1996-07-23

    Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

  6. Deep sequencing of MYC DNA-binding sites in Burkitt lymphoma.

    Directory of Open Access Journals (Sweden)

    Volkhard Seitz

    Full Text Available BACKGROUND: MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP followed by next generation sequencing (ChIP-Seq has been conducted in BL so far. METHODOLOGY/PRINCIPAL FINDINGS: ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing. CONCLUSION/SIGNIFICANCE: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i many B-cell relevant genes are targeted by MYC and (ii that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology.

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

    Science.gov (United States)

    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.

  8. Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase.

    Science.gov (United States)

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2016-01-01

    The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.

  9. Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo.

    Science.gov (United States)

    Schuch, Ronaldo; Agelopoulos, Konstantin; Neumann, Anna; Brandt, Burkhard; Bürger, Horst; Korsching, Eberhard

    2012-02-20

    Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs. The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue. The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme

  10. Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo

    Directory of Open Access Journals (Sweden)

    Schuch Ronaldo

    2012-02-01

    Full Text Available Abstract Background Transcription factors (TFs and their binding sites (TFBSs play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP. However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs. Findings The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase, through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue. Conclusions The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially

  11. An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

    Science.gov (United States)

    Schanke, J T; Marcuzzi, A; Podzorski, R P; Van Ness, B

    1994-01-01

    Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells. Images PMID:7816634

  12. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C

    1998-01-01

    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...... resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-10-01

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

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

    Science.gov (United States)

    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.

  15. Ligand docking and binding site analysis with PyMOL and Autodock/Vina.

    Science.gov (United States)

    Seeliger, Daniel; de Groot, Bert L

    2010-05-01

    Docking of small molecule compounds into the binding site of a receptor and estimating the binding affinity of the complex is an important part of the structure-based drug design process. For a thorough understanding of the structural principles that determine the strength of a protein/ligand complex both, an accurate and fast docking protocol and the ability to visualize binding geometries and interactions are mandatory. Here we present an interface between the popular molecular graphics system PyMOL and the molecular docking suites Autodock and Vina and demonstrate how the combination of docking and visualization can aid structure-based drug design efforts.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  17. Functional diversification of paralogous transcription factors via divergence in DNA binding site motif and in expression.

    Directory of Open Access Journals (Sweden)

    Larry N Singh

    Full Text Available BACKGROUND: Gene duplication is a major driver of evolutionary innovation as it allows for an organism to elaborate its existing biological functions via specialization or diversification of initially redundant gene paralogs. Gene function can diversify in several ways. Transcription factor gene paralogs in particular, can diversify either by changes in their tissue-specific expression pattern or by changes in the DNA binding site motif recognized by their protein product, which in turn alters their gene targets. The relationship between these two modes of functional diversification of transcription factor paralogs has not been previously investigated, and is essential for understanding adaptive evolution of transcription factor gene families. FINDINGS: Based on a large set of human paralogous transcription factor pairs, we show that when the DNA binding site motifs of transcription factor paralogs are similar, the expressions of the genes that encode the paralogs have diverged, so in general, at most one of the paralogs is highly expressed in a tissue. Moreover, paralogs with diverged DNA binding site motifs tend to be diverged in their function. Conversely, two paralogs that are highly expressed in a tissue tend to have dissimilar DNA binding site motifs. We have also found that in general, within a paralogous family, tissue-specific decrease in gene expression is more frequent than what is expected by chance. CONCLUSIONS: While previous investigations of paralogous gene diversification have only considered coding sequence divergence, by explicitly quantifying divergence in DNA binding site motif, our work presents a new paradigm for investigating functional diversification. Consistent with evolutionary expectation, our quantitative analysis suggests that paralogous transcription factors have survived extinction in part, either through diversification of their DNA binding site motifs or through alterations in their tissue-specific expression

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    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 degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-03-01

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

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

    DEFF Research Database (Denmark)

    Møller, Charlotte; Sprenger, Richard R; Stürup, Stefan;

    2011-01-01

    and 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...

  4. The nucleotide-binding site of Aquifex aeolicus LpxC

    OpenAIRE

    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 ...

  5. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites

    OpenAIRE

    Zahid, Henna; Miah, Layeque; Lau, Andy; Brochard, Lea; Hati, Debolina; Bui, T. T.; Drake, A. F.; Gor, Jayesh; Perkins, Stephen J.; McDermott, Lindsay C.

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigate...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  7. Does distant homology with Evf reveal a lipid binding site in Bacillus thuringiensis cytolytic toxins?

    Science.gov (United States)

    Rigden, Daniel J

    2009-05-19

    The Cry and Cyt classes of insecticidal toxins derived from the sporulating bacterium Bacillus thuringiensis are valuable substitutes for synthetic pesticides in agricultural contexts. Crystal structures and many biochemical data have provided insights into their molecular mechanisms, generally thought to involve oligomerization and pore formation, but have not localised the site on Cyt toxins responsible for selective binding of phospholipids containing unsaturated fatty acids. Here, distant homology between the structure of Cyt toxins and Erwinia virulence factor (Evf) is demonstrated which, along with sequence conservation analysis, allows a putative lipid binding site to be localised in the toxins.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

    2016-05-31

    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.

  10. Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.

    OpenAIRE

    Oliphant, A R; Brandl, C J; Struhl, K

    1989-01-01

    We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C...

  11. Evolution of allosteric citrate binding sites on 6-phosphofructo-1-kinase.

    Directory of Open Access Journals (Sweden)

    Aleksandra Usenik

    Full Text Available As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1 level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.

  12. Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin.

    Science.gov (United States)

    Wang, C L; Leavis, P C; Gergely, J

    1984-12-18

    The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.

  13. Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

    OpenAIRE

    Gitlin, G; Bayer, E A; Wilchek, M

    1988-01-01

    Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is beli...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-10-17

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

  15. PIP(2)-binding site in Kir channels: definition by multiscale biomolecular simulations.

    Science.gov (United States)

    Stansfeld, Phillip J; Hopkinson, Richard; Ashcroft, Frances M; Sansom, Mark S P

    2009-11-24

    Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains.

  16. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

    OpenAIRE

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-01-01

    Experiments are presented which suggest that the binding of the primer tRNA to the primer binding site of the HIV-1 5′ UTR is involved in the dimerization of the genome, as part of the packaging process.

  17. Identification of Calcium binding sites on calsequestrin 1 and its implications to polymerization

    Science.gov (United States)

    Kumar, Amit; Chakravarty, Harapriya; Bal, Naresh C.; Balaraju, Tuniki; Jena, Nivedita; Misra, Gauri; Bal, Chandralata; Pieroni, Enrico; Periasamy, Muthu; Sharon, Ashoke

    2013-01-01

    Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70–80 Ca2+ ions. However, the nature of Ca2+-binding sites has not yet been fully characterized. In this study, we employed in-silico approaches to identify the Ca2+ binding sites and to understand the molecular basis of CASQ1-Ca2+ recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: 3UOM) and adding the missing C-terminal residues (aa350–364). Using this model we performed extensive 30 ns molecular dynamics simulations exposed to wide range of Ca2+ concentrations ([Ca2+]). Our results show that the Ca2+-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca2+-binding sites share a similar geometry and interestingly, majority of them were found to be induced by increased [Ca2+]. We also found that the system undergoes maximal Ca2+-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data shows that the CASQ1 back-to-back stacking is progressively stabilized by emergence of an increasing number of hydrophobic interactions with increasing [Ca2+]. Further, this study shows that the CAS domain assumes a compact structure with increase in Ca2+ binding, which suggests that the CAS domain might function as a Ca2+-sensor that may be a novel structural motif to sense metal. We propose the term “Dn-motif” for the CAS domain. PMID:23629537

  18. Study of V2 vasopressin receptor hormone binding site using in silico methods.

    Science.gov (United States)

    Sebti, Yeganeh; Sardari, Soroush; Sadeghi, Hamid Mir Mohammad; Ghahremani, Mohammad Hossein; Innamorati, Giulio

    2015-01-01

    The antidiuretic effect of arginine vasopressin (AVP) is mediated by the vasopressin V2 receptor. The docking study of AVP as a ligand to V2 receptor helps in identifying important amino acid residues that might be involved in AVP binding for predicting the lowest free energy state of the protein complex. Whereas previous researchers were not able to detect the exact site of the ligand-receptor binding, we designed the current study to identify the vasopressin V2 receptor hormone binding site using bioinformatic methods. The 3D structure of nonapeptide hormone vasopressin was extracted from Protein Data Bank. Since no suitable template resembling V2 receptor was found, an ab initio approach was chosen to model the protein receptor. Using protein docking methods such as Hex protein-protein docking, the model of V2 receptor was docked to the peptide ligand AVP to identify possible binding sites. The residues that involved in binding site are W293, W296, D297, A300, and P301. The lowest free energy state of the protein complex was predicted after mutation in the above residues. The amount of gained energies permits us to compare the mutant forms with native forms and help to asses critical changes such as positive and negative mutations followed by ranking the best mutations. Based on the mutation/docking predictions, we found some mutants such as W293D and A300E possess positively inducing effect in ligand binding and some of them such as A300R present negatively inducing effect in ligand binding.

  19. A specific binding site recognizing a fragment of angiotensin II in bovine adrenal cortex membranes.

    Science.gov (United States)

    Bernier, S G; Fournier, A; Guillemette, G

    1994-12-12

    We have characterized a specific binding site for angiotensin IV in bovine adrenal cortex membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 0.24 +/- 0.03 nM). The binding site is saturable and relatively abundant (maximal binding capacity around 0.5 pmol/mg protein). Non-equilibrium kinetic analyses at 37 degrees C revealed a calculated kinetic Kd of 47 pM. The binding site is pharmacologically distinct from the classic angiotensin receptors AT1 or AT2. Competitive binding studies with bovine adrenal cortex membranes demonstrated the following rank order of effectiveness: angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) = angiotensin II-(3-7) (Val-Tyr-Ile-His-Pro) > angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) > or = angiotensin II-(4-7) (Tyr-Ile-His-Pro) > angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > angiotensin II-(1-6) (Asp-Arg-Val-Tyr-Ile-His) > angiotensin II-(4-8) (Tyr-Ile-His-Pro-Phe) > > > angiotensin II-(3-6) (Val-Tyr-Ile-His), angiotensin II-(4-6) (Tyr-Ile-His), L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'(1-H-tetrazol-5-yl)[1,1'-biphenyl]-4-y l) methyl]-3-H-imidazo[4,5-beta]pyridine H2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)4,5,6 ,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid). The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensioffn IV to bovine adrenal cortex membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Identification and characterization of a glycosaminoglycan binding site on interleukin-10 via molecular simulation methods.

    Science.gov (United States)

    Gehrcke, Jan-Philip; Pisabarro, M Teresa

    2015-11-01

    The biological function of the pleiotropic cytokine interleukin-10 (IL-10), which has an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are essential constituents of the extracellular matrix with an important role in modulating the biological function of many proteins. The molecular mechanisms governing the IL-10-GAG interaction, though, are unclear so far. In particular, detailed knowledge about GAG binding sites and recognition mode on IL-10 is lacking, despite of its imminent importance for understanding the functional consequences of IL-10-GAG interaction. In the present work, we report a GAG binding site on IL-10 identified by applying computational methods based on Coulomb potential calculations and specialized molecular dynamics simulations. The identified GAG binding site is constituted of several positively charged residues, which are conserved among species. Exhaustive conformational space sampling of a series of GAG ligands binding to IL-10 led to the observation of two GAG binding modes in the predicted binding site, and to the identification of IL-10 residues R104, R106, R107, and K119 as being most important for molecular GAG recognition. In silico mutation as well as single-residue energy decomposition and detailed analysis of hydrogen-bonding behavior led to the conclusion that R107 is most essential and assumes a unique role in IL-10-GAG interaction. This structural and dynamic characterization of GAG-binding to IL-10 represents an important step for further understanding the modulation of the biological function of IL-10.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  3. Recognition of oxidatively modified bases within the biotin-binding site of avidin.

    Science.gov (United States)

    Conners, Rebecca; Hooley, Elizabeth; Clarke, Anthony R; Thomas, Siân; Brady, R Leo

    2006-03-17

    Oxidative damage of DNA results in the formation of many products, including 8-oxodeoxyguanosine, which has been used as a marker to quantify DNA damage. Earlier studies have demonstrated that avidin, a protein prevalent in egg-white and which has high affinity for the vitamin biotin, binds to 8-oxodeoxyguanosine and related bases. In this study, we have determined crystal structures of avidin in complex with 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. In each case, the base is observed to bind within the biotin-binding site of avidin. However, the mode of association between the bases and the protein varies and, unlike in the avidin:biotin complex, complete ordering of the protein in this region does not accompany binding. Fluorescence studies indicate that in solution the individual bases, and a range of oligonucleotides, bind to avidin with micromolar affinity. Only one of the modes of binding observed is consistent with recognition of oxidised purines when incorporated within a DNA oligomer, and from this structure a model is proposed for the selective binding of avidin to DNA containing oxidatively damaged deoxyguanosine. These studies illustrate the molecular basis by which avidin might act as a marker of DNA damage, although the low levels of binding observed are inconsistent with the recognition of oxidised purines forming a major physiological role for avidin.

  4. Binding isotope effects as a tool for distinguishing hydrophobic and hydrophilic binding sites of HIV-1 RT.

    Science.gov (United States)

    Krzemińska, Agnieszka; Paneth, Piotr; Moliner, Vicent; Świderek, Katarzyna

    2015-01-22

    The current treatment for HIV-1 infected patients consists of a cocktail of inhibitors, in an attempt to improve the potency of the drugs by adding the possible effects of each supplied compound. In this contribution, nine different inhibitors of HIV-1 RT, one of the three key proteins responsible for the virus replication, have been selected to develop and test a computational protocol that allows getting a deep insight into the inhibitors' binding mechanism. The interaction between the inhibitors and the protein have been quantified by computing binding free energies through FEP calculations, while a more detailed characterization of the kind of inhibitor-protein interactions is based on frequency analysis of the ligands in the initial and final state, i.e. in solution and binding the protein. QM/MM calculation of heavy atoms ((13)C, (15)N, and (18)O) binding isotope effects (BIE) have been used to identify the binding sites of the different inhibitors. Specific interactions between the isotopically labeled atoms of the inhibitors and polar residues and magnesium cations on the hydrophilic pocket of the protein are responsible for the frequencies shifting that can be detected when comparing the IR spectra of the compounds in solution and in the protein. On the contrary, it seems that changes in vdW interactions from solution to the final state when the ligand is interacting with residues of the hydrophobic cavity, does not influence frequency modes and then no BIE are observed. Our results suggest that a proper computational protocol can be a valuable tool which in turn can be used to increase the efficiency of anti AIDS drugs.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Erill, Ivan; O'Neill, Michael C

    2009-01-01

    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. PMID:19210776

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

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

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

  9. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites

    OpenAIRE

    Sena, J.A.D. [UNESP; Hernández Rodríguez, Carmen Sara; Ferré Manzanero, Juan

    2009-01-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  10. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites.

    Science.gov (United States)

    Sena, Janete A D; Hernández-Rodríguez, Carmen Sara; Ferré, Juan

    2009-04-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  11. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  12. Mapping the heparin-binding site on the 13-14F3 fragment of fibronectin.

    Science.gov (United States)

    Sachchidanand; Lequin, Olivier; Staunton, David; Mulloy, Barbara; Forster, Mark J; Yoshida, Keiichi; Campbell, Iain D

    2002-12-27

    Fibronectin, a multifunctional glycoprotein of the extracellular matrix, plays a major role in cell adhesion. Various studies have revealed that the human 13th and 14th fibronectin type III domains (labeled (13)F3 and (14)F3 here) contain a heparin-binding site. Mapping of the heparin-binding sites of (13-14)F3, (13)F3, and (14)F3 by NMR chemical shift perturbation, isothermal titration calorimetry, and molecular modeling show that (13)F3 provides the dominant heparin-binding site and that the residues involved are within the first 29 amino acids of (13)F3. Predictions from earlier biochemical and modeling studies as well as the x-ray structure of (12-14)F3 were tested. It was shown that the positively charged residues that project into the solvent from the ABE face of the triple-stranded beta sheet on (13)F3 are involved in binding, but (14)F3 does not appear to contribute significantly to heparin binding.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-22

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

  14. Mapping cocaine binding sites in human and baboon brain in vivo.

    Science.gov (United States)

    Fowler, J S; Volkow, N D; Wolf, A P; Dewey, S L; Schlyer, D J; Macgregor, R R; Hitzemann, R; Logan, J; Bendriem, B; Gatley, S J

    1989-01-01

    The first direct measurements of cocaine binding in the brain of normal human volunteers and baboons have been made by using positron emission tomography (PET) and tracer doses of [N-11C-methyl]-(-)-cocaine ([11C]cocaine). Cocaine's binding and release from brain are rapid with the highest regional uptake of carbon-11 occurring in the corpus striatum at 4-10 minutes after intravenous injection of labeled cocaine. This was followed by a clearance to half the peak value at about 25 minutes with the overall time course paralleling the previously documented time course of the euphoria experienced after intravenous cocaine administration. Blockade of the dopamine reuptake sites with nomifensine reduced the striatal but not the cerebellar uptake of [11C]cocaine in baboons indicating that cocaine binding is associated with the dopamine reuptake site in the corpus striatum. A comparison of labeled metabolites of cocaine in human and baboon plasma showed that while cocaine is rapidly metabolized in both species, the profile of labeled metabolites is different, with baboon plasma containing significant amounts of labeled carbon dioxide, and human plasma containing no significant labeled carbon dioxide. These studies demonstrate the feasibility of using [11C]cocaine and PET to map binding sites for cocaine in human brain, to monitor its kinetics, and to characterize its binding mechanism by using appropriate pharmacological challenges.

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

    Directory of Open Access Journals (Sweden)

    Previato Jose O

    2008-05-01

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  17. Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Saavedra, J.M.; Israel, A.; Plunkett, L.M.; Kurihara, M.; Shigematsu, K.; Correa, F.M.

    1986-07-01

    Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM /sup 125/I-(Sar1)-angiotensin II, (/sup 3/H)-Ultrofilm autoradiography, computerized microdensitometry and comparison with /sup 125/I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with /sup 125/I-(Sar1)-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.

  18. An information transmission model for transcription factor binding at regulatory DNA sites.

    Science.gov (United States)

    Tan, Mingfeng; Yu, Dong; Jin, Yuan; Dou, Lei; Li, Beiping; Wang, Yuelan; Yue, Junjie; Liang, Long

    2012-06-06

    Computational identification of transcription factor binding sites (TFBSs) is a rapid, cost-efficient way to locate unknown regulatory elements. With increased potential for high-throughput genome sequencing, the availability of accurate computational methods for TFBS prediction has never been as important as it currently is. To date, identifying TFBSs with high sensitivity and specificity is still an open challenge, necessitating the development of novel models for predicting transcription factor-binding regulatory DNA elements. Based on the information theory, we propose a model for transcription factor binding of regulatory DNA sites. Our model incorporates position interdependencies in effective ways. The model computes the information transferred (TI) between the transcription factor and the TFBS during the binding process and uses TI as the criterion to determine whether the sequence motif is a possible TFBS. Based on this model, we developed a computational method to identify TFBSs. By theoretically proving and testing our model using both real and artificial data, we found that our model provides highly accurate predictive results. In this study, we present a novel model for transcription factor binding regulatory DNA sites. The model can provide an increased ability to detect TFBSs.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    Directory of Open Access Journals (Sweden)

    Zhichao Miao

    2015-12-01

    Full Text Available Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i the definition of nucleic acid binding sites; ii the training and test datasets; iii the algorithmic methods for the prediction strategies; iv the performance measures and v the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy… are applied. We found that i the tools have been greatly improved over the years; ii some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii RNA binding and DNA binding appear to follow similar driving forces and iv dataset bias may exist in some methods.

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

    Directory of Open Access Journals (Sweden)

    Simon Oakley

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

  2. Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

    Directory of Open Access Journals (Sweden)

    Jose L S Lopes

    Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.

    Science.gov (United States)

    Armitage, Ian M; Drakenberg, Torbjörn; Reilly, Brian

    2013-01-01

    Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca(2+) binding proteins, and metallothioneins.In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, (113)Cd NMR, in conjunction with (13)C and (31)P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd

  5. Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

    OpenAIRE

    Rebelo, Tânia S. C. R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M. H.; Noronha, João P. C.; Sales, M. Goreti F.

    2014-01-01

    This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice...

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

    Directory of Open Access Journals (Sweden)

    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

  7. Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

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    Yoshiaki Okada

    Full Text Available In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4 is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

  8. Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine

    OpenAIRE

    Grigoryan, Hasmik; Li, Bin; Anderson, Erica K.; Xue, Weihua; Nachon, Florian; Lockridge, Oksana; Schopfer, Lawrence M.

    2009-01-01

    Organophosphorus esters (OP) are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-b...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    CERN Document Server

    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...

  11. Signatures of RNA binding proteins globally coupled to effective microRNA target sites

    DEFF Research Database (Denmark)

    Jacobsen, Anders; Wen, Jiayu; Marks, Debora S

    2010-01-01

    may be modulated by other mRNA sequence elements such as binding sites for the hundreds of RNA binding proteins (RNA-BPs) expressed in any cell, and this aspect has not been systematically explored. Across a panel of published experiments, we systematically investigated to what extent sequence motifs...... proteins. This is the first systematic investigation of 3' UTR motifs that globally couple to regulation by miRNAs and may potentially antagonize or cooperate with miRNA/siRNA regulation. Our results suggest that binding sites of miRNAs and RNA-BPs should be considered in combination when interpreting......MicroRNAs (miRNAs) and small interfering RNAs (siRNAs), bound to Argonaute proteins (RISC), destabilize mRNAs through base-pairing with the mRNA. However, the gene expression changes after perturbations of these small RNAs are only partially explained by predicted miRNA/siRNA targeting. Targeting...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

    Nielsen, J R; Pedersen, K E; Klitgaard, N A;

    1989-01-01

    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...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    and [(3)H]-escitalopram binding in transiently transfected human embryonic kidney cells; HEK-293-MSR. Residues responsible for altered affinities inhibitors were pinpointed by generating cross-species chimeras and subsequent point mutations by site directed mutagenesis. drSERT has a higher affinity...

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

    Science.gov (United States)

    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.

  16. Characterization of H2 binding sites in prototypical metal-organic frameworks by inelastic neutron scattering.

    Science.gov (United States)

    Rowsell, Jesse L C; Eckert, Juergen; Yaghi, Omar M

    2005-10-26

    The hindered rotor transitions of H(2) adsorbed in the chemically related and prototypical porous metal-organic frameworks IRMOF-1, IRMOF-8, IRMOF-11, and MOF-177 were studied by inelastic neutron scattering to gain information on the specifics of H(2) binding in this class of adsorbents. Remarkably sharp and complex spectra of these materials signify a diversity of well-defined binding sites. Similarities in the spectral features as a function of H(2) loading and correlations with recent crystallographic studies were used to assign transitions ranging in rotational barrier from adsorption sites on the organic and inorganic components of these frameworks. We find that binding of H(2) at the inorganic cluster sites is affected by the nature of the organic link and is strongest in IRMOF-11 in accord with our adsorption isotherm data. The sites on the organic link have lower binding energies, but a much greater capacity for increases in H(2) loading, which demonstrates their importance for hydrogen uptake by these materials.

  17. Localization of the substrate binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli

    NARCIS (Netherlands)

    Opacic, Milena; Vos, Erwin P. P.; Hesp, Ben H.; Broos, Jaap

    2010-01-01

    The mannitol transporter from Escherichia coli, EIImtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EIImtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IICmtl

  18. Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms.

    Science.gov (United States)

    Noskov, Sergei Y; Roux, Benoît

    2008-03-28

    The x-ray structure of LeuT, a bacterial homologue of Na(+)/Cl(-)-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na(+). Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na(+) over K(+) or Li(+), the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na(+) over K(+) arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na(+) is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a "snug-fit" mechanism.

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

    Directory of Open Access Journals (Sweden)

    A.M. Kettlun

    2000-07-01

    Full Text Available Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10 and Desirée (ATPase/ADPase = 1 isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

  20. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  1. Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy

    2008-01-01

    A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed...

  2. Sulindac-derived RXRα modulators inhibit cancer cell growth by binding to a novel site.

    Science.gov (United States)

    Chen, Liqun; Wang, Zhi-Gang; Aleshin, Alexander E; Chen, Fan; Chen, Jiebo; Jiang, Fuquan; Alitongbieke, Gulimiran; Zeng, Zhiping; Ma, Yue; Huang, Mingfeng; Zhou, Hu; Cadwell, Gregory; Zheng, Jian-Feng; Huang, Pei-Qiang; Liddington, Robert C; Zhang, Xiao-kun; Su, Ying

    2014-05-22

    Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.

  3. Transcription factor binding sites are highly enriched within microRNA precursor sequences

    Directory of Open Access Journals (Sweden)

    Piriyapongsa Jittima

    2011-12-01

    Full Text Available Abstract Background Transcription factors are thought to regulate the transcription of microRNA genes in a manner similar to that of protein-coding genes; that is, by binding to conventional transcription factor binding site DNA sequences located in or near promoter regions that lie upstream of the microRNA genes. However, in the course of analyzing the genomics of human microRNA genes, we noticed that annotated transcription factor binding sites commonly lie within 70- to 110-nt long microRNA small hairpin precursor sequences. Results We report that about 45% of all human small hairpin microRNA (pre-miR sequences contain at least one predicted transcription factor binding site motif that is conserved across human, mouse and rat, and this rises to over 75% if one excludes primate-specific pre-miRs. The association is robust and has extremely strong statistical significance; it affects both intergenic and intronic pre-miRs and both isolated and clustered microRNA genes. We also confirmed and extended this finding using a separate analysis that examined all human pre-miR sequences regardless of conservation across species. Conclusions The transcription factor binding sites localized within small hairpin microRNA precursor sequences may possibly regulate their transcription. Transcription factors may also possibly bind directly to nascent primary microRNA gene transcripts or small hairpin microRNA precursors and regulate their processing. Reviewers This article was reviewed by Guillaume Bourque (nominated by Jerzy Jurka, Dmitri Pervouchine (nominated by Mikhail Gelfand, and Yuriy Gusev.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy.

    Science.gov (United States)

    Künze, Georg; Köhling, Sebastian; Vogel, Alexander; Rademann, Jörg; Huster, Daniel

    2016-02-05

    The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-24

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

    2012-02-05

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    McCann, D.J.; Su, T.P. (National Institute on Drug Abuse, Baltimore, MD (USA))

    1991-05-01

    The zwitterionic detergent 3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-({sup 3}H)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 (+)-({sup 3}H)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 (+)-({sup 3}H)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 ({sup 3}H)progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-({sup 3}H)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 (+)-({sup 3}H)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.

  9. Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3ζ dimer.

    Science.gov (United States)

    Molzan, Manuela; Ottmann, Christian

    2012-11-02

    C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single-site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. The presence of gonadotropin binding sites in the intracellular organelles of human ovaries.

    Science.gov (United States)

    Rao, C V; Mitra, S; Sanfilippo, J; Carman, F R

    1981-03-15

    The nuclei (N), plasma membranes (PM), mitochondria-lysosomes, rough endoplasmic reticulum, and combined (light, medium, and heavy) Golgi (G) fractions were isolated from human ovaries. The purities of these fractions were evaluated by assays of appropriate marker enzymes, which revealed that some fractions were very pure but that others had minor contamination. When tested, all of the fractions exhibited 125I-labeled human chorionic gonadotropin (125I-hCG)-specific binding. This intracellular 125I-hCG binding was not due to PM contamination because: (1) N, which had no detectable 5'-nucleotidase (5'-NE) activity, a marker for PM, exhibited 125I-hCG-specific binding; (2) the G, which had only a fraction of the 5'-NE activity of PM, exhibited as much binding as PM; and (3) the ratios between specific 125I-hCG binding and 5'-NE activity in other fractions were not the same as for PM. They should have been the same if PM contamination was responsible for the 125I-hCG binding observed in other organelles. In conclusion, our results demonstrate that gonadotropin-binding sites are present in various intracellular organelles as well as in PM of human ovaries.

  11. The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

    Science.gov (United States)

    Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

    2000-01-01

    Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

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

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    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

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

    Science.gov (United States)

    Castellanos, Raquel; Xie, Qing; Zheng, Deyou; Cvekl, Ales; Morrow, Bernice E

    2014-01-01

    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 molecular

  14. Prediction of protein binding sites in protein structures using hidden Markov support vector machine

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    Lin Lei

    2009-11-01

    Full Text Available Abstract Background Predicting the binding sites between two interacting proteins provides important clues to the function of a protein. Recent research on protein binding site prediction has been mainly based on widely known machine learning techniques, such as artificial neural networks, support vector machines, conditional random field, etc. However, the prediction performance is still too low to be used in practice. It is necessary to explore new algorithms, theories and features to further improve the performance. Results In this study, we introduce a novel machine learning model hidden Markov support vector machine for protein binding site prediction. The model treats the protein binding site prediction as a sequential labelling task based on the maximum margin criterion. Common features derived from protein sequences and structures, including protein sequence profile and residue accessible surface area, are used to train hidden Markov support vector machine. When tested on six data sets, the method based on hidden Markov support vector machine shows better performance than some state-of-the-art methods, including artificial neural networks, support vector machines and conditional random field. Furthermore, its running time is several orders of magnitude shorter than that of the compared methods. Conclusion The improved prediction performance and computational efficiency of the method based on hidden Markov support vector machine can be attributed to the following three factors. Firstly, the relation between labels of neighbouring residues is useful for protein binding site prediction. Secondly, the kernel trick is very advantageous to this field. Thirdly, the complexity of the training step for hidden Markov support vector machine is linear with the number of training samples by using the cutting-plane algorithm.

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

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    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

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

    Science.gov (United States)

    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%.

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

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    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.

  18. Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore.

    Science.gov (United States)

    Klement, Göran; Eisele, Lina; Malinowsky, David; Nolting, Andreas; Svensson, Mats; Terp, Gitte; Weigelt, Dirk; Dabrowski, Michael

    2013-02-19

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

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

    Science.gov (United States)

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

    2007-07-31

    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 unclear. Here, we use calculations of pK(A)s and homology modeling to predict the location of a functionally important Cl(-) binding site in serotonin transporter and other Cl(-)-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl(-) ion and through the observation of sequence conservation patterns in other Cl(-)-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl(-) and Na(+) ions during transport. Other implications of the model are also discussed.

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

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    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.

  1. Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.

    Science.gov (United States)

    Yang, S A; Klee, C B

    2000-12-26

    Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee

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

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    Deng W-M

    2009-02-01

    Full Text Available Abstract Background Dystroglycan (Dg is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

  3. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-01

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rosenbaum, J.S.; Holford, N.H.; Sadee, W.

    1985-06-01

    The in vivo receptor binding of a series of opioid drugs was investigated in intact rats after s.c. administration of (/sup 3/H)etorphine tracer, which selectively binds to mu sites in vivo. Receptor binding was determined by a membrane filtration assay immediately after sacrifice of the animals and brain homogenization. Coadministration of unlabeled opioid drugs together with tracer led to a dose-dependent decrease of in vivo tracer binding. Estimates of the doses required to occupy 50% of the mu sites in vivo established the following potency rank order: diprenorphine, naloxone, buprenorphine, etorphine, levallorphan, cyclazocine, sufentanil, nalorphine, ethylketocyclazocine, ketocyclazocine, pentazocine, morphine. In vivo-in vitro differences among the relative receptor binding potencies were only partially accounted for by differences in their access to the brain and the regulatory effects of Na+ and GTP, which are expected to reduce agonist affinities in vivo. The relationship among mu receptor occupancy in vivo and pharmacological effects of the opioid drugs is described.

  5. The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists

    Science.gov (United States)

    Kalani, M. Yashar S.; Vaidehi, Nagarajan; Hall, Spencer E.; Trabanino, Rene J.; Freddolino, Peter L.; Kalani, Maziyar A.; Floriano, Wely B.; Tak Kam, Victor Wai; Goddard, William A., III

    2004-03-01

    Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

  6. A short update on the structure of drug binding sites on neurotransmitter transporters

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    Gabrielsen Mari

    2011-12-01

    Full Text Available Abstract Background The dopamine (DAT, noradrenalin (NET and serotonin (SERT transporters are molecular targets for different classes of psychotropic drugs. Cocaine and the SSRI (S-citalopram block neurotransmitter reuptake competitively, but while cocaine is a non-selective reuptake inhibitor, (S-citalopram is a selective SERT inhibitor. Findings Here we present comparisons of the binding sites and the electrostatic potential surfaces (EPS of DAT, NET and SERT homology models based on two different LeuTAa templates; with a substrate (leucine in an occluded conformation (PDB id 2a65, and with an inhibitor (tryptophan in an open-to-out conformation (PDB id 3f3a. In the occluded homology models, two conserved aromatic amino acids (tyrosine and phenylalanine formed a gate between the putative binding pockets, and this contact was interrupted in the open to out conformation. The EPS of DAT and NET were generally negative in the vestibular area, whereas the EPS of the vestibular area of SERT was more neutral. Conclusions The findings presented here contribute as an update on the structure of the binding sites of DAT, NET and SERT. The updated models, which have larger ligand binding site areas than models based on other templates, may serve as improved tools for virtual ligand screening.

  7. A model-based approach to identify binding sites in CLIP-Seq data.

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    Tao Wang

    Full Text Available Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq has made it possible to identify the targeting sites of RNA-binding proteins in various cell culture systems and tissue types on a genome-wide scale. Here we present a novel model-based approach (MiClip to identify high-confidence protein-RNA binding sites from CLIP-seq datasets. This approach assigns a probability score for each potential binding site to help prioritize subsequent validation experiments. The MiClip algorithm has been tested in both HITS-CLIP and PAR-CLIP datasets. In the HITS-CLIP dataset, the signal/noise ratios of miRNA seed motif enrichment produced by the MiClip approach are between 17% and 301% higher than those by the ad hoc method for the top 10 most enriched miRNAs. In the PAR-CLIP dataset, the MiClip approach can identify ∼50% more validated binding targets than the original ad hoc method and two recently published methods. To facilitate the application of the algorithm, we have released an R package, MiClip (http://cran.r-project.org/web/packages/MiClip/index.html, and a public web-based graphical user interface software (http://galaxy.qbrc.org/tool_runner?tool_id=mi_clip for customized analysis.

  8. Identification of Two Secondary Ligand Binding Sites in 14-3-3 Proteins Using Fragment Screening.

    Science.gov (United States)

    Sijbesma, Eline; Skora, Lukasz; Leysen, Seppe; Brunsveld, Luc; Koch, Uwe; Nussbaumer, Peter; Jahnke, Wolfgang; Ottmann, Christian

    2017-08-01

    Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein-protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation.

  9. STarMir Tools for Prediction of microRNA binding sites

    Science.gov (United States)

    Kanoria, Shaveta; Rennie, William; Liu, Chaochun; Carmack, C. Steven; Lu, Jun; Ding, Ye

    2017-01-01

    MicroRNAs (miRNAs) are a class of endogenous short non-coding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs), which results in translational repression and/or mRNA degradation. As regulatory molecules, miRNAs are involved in many mammalian biological processes and also in the manifestation of certain human diseases. As miRNAs play central role in the regulation of gene expression, understanding miRNA-binding patterns is essential to gain an insight of miRNA mediated gene regulation and also holds promise for therapeutic applications. Computational prediction of miRNA binding sites on target mRNAs facilitates experimental investigation of miRNA functions. This chapter provides protocols for using the STarMir web server for improved predictions of miRNA binding sites on a target mRNA. As an application module of the Sfold RNA package, the current version of STarMir is an implementation of logistic prediction models developed with high throughput miRNA binding data from crosslinking immuno-precipitation (CLIP) studies. The models incorporated comprehensive thermodynamic, structural and sequence features, and were found to make improved predictions of both seed and seedless sites, in comparison to the established algorithms [1]. Their broad applicability was indicated by their good performance in cross-species validation. STarMir is freely available at http://sfold.wadsworth.org/starmir.html PMID:27665594

  10. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    Science.gov (United States)

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  11. Spectroscopic Signature of a Ubiquitous Metal Binding Site in the Metallo-beta-lactamase Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    V Campos-Bermudez; J Gonzalez; D Tierney; A Vila

    2011-12-31

    The metallo-{beta}-lactamase (M{beta}L) superfamily is a functionally diverse group of metalloproteins sharing a distinctive {alpha}{beta}/{alpha}{beta} fold and a characteristic metal binding motif. A large number of open reading frames identified in genomic sequencing efforts have been annotated as members of this superfamily through sequence comparisons. However, structural and functional studies performed on purified proteins are normally needed to unequivocally include a newly discovered protein in the M{beta}L superfamily. Here we report the spectroscopic characterization of recombinant YcbL, a gene product annotated as a member of the M{beta}L superfamily whose function in vivo remains unknown. By taking advantage of the structural features characterizing the M{beta}L superfamily metal binding motif, we performed spectroscopic studies on Zn(II)- and Co(II)-substituted YcbL to structurally interrogate the metal binding site. The dinuclear center in Co(II)-YcbL was shown to display characteristic electronic absorption features in the visible region, which were also observed in an engineered M{beta}L aimed at mimicking this metal site. Thus, the spectroscopic features reported herein can be employed as a signature to readily identify and characterize the presence of these ubiquitous metal binding sites.

  12. Nature of the Hydrogen Binding in Metal Organic Frameworks with Exposed Transition Metal Sites

    Science.gov (United States)

    Zhou, Wei; Yildirim, Taner

    2008-03-01

    MOFs with exposed transition metal (TM) sites were recently found to exhibit significantly larger experimental heat of H2 adsorption than classical MOFs, thus attracted great attention. [1, 2] Understanding the nature of the H2 interaction with the exposed metal sites is of critical importance for the further development of these materials. Using Mn4Cl-MOF as an example, here we show that the H2 binding with the exposed TM site is not of the expected Kubas type, in strong contrast to ``similar'' systems investigated previously (e.g., ref. [3] and [4]). In Mn4Cl-MOF, there are a) no charge transfer from TM to H2, b) no significant H-H bond elongation, and c) no evidence of any H2-σ^* Mn-d orbital hybridization. We also study the H2 binding as a function of Mn4-magnetic spin configurations, and find no significant effect of the magnetic state on the binding energy. We further reveal that the major contribution to the overall binding is classical Coulomb interaction arising from the small charge overlap of H2-σ and Mn-d orbitals. This coulomb interaction is very anisotropic, and when the quantum nature of H2-orientation is taken into account, the actual binding energy is significantly reduced from the calculated classical binding energy. [1] J. Am. Chem. Soc. 128, 16876 (2006). [2] Angew. Chem. Int. Ed. 46, 1419 (2007). [3] Phys. Rev. Lett. 94, 087205 (2005). [4] Phys. Rev. B 76, 085434 (2007).

  13. Vif proteins from diverse primate lentiviral lineages use the same binding site in APOBEC3G.

    Science.gov (United States)

    Letko, Michael; Silvestri, Guido; Hahn, Beatrice H; Bibollet-Ruche, Frederick; Gokcumen, Omer; Simon, Viviana; Ooms, Marcel

    2013-11-01

    APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) and other lentiviruses. Most of these viruses encode a Vif protein that directly binds A3G and leads to its proteasomal degradation. Both Vif proteins of HIV-1 and African green monkey simian immunodeficiency virus (SIVagm) bind residue 128 of A3G. However, this position does not control the A3G degradation by Vif variants derived from HIV-2 and SIVmac, which both originated from SIV of sooty mangabey monkeys (SIVsmm), suggesting that the A3G binding site for Vif proteins of the SIVsmm/HIV-2 lineage differs from that of HIV-1. To map the SIVsmm Vif binding site of A3G, we performed immunoprecipitations of individual A3G domains, Vif/A3G degradation assays and a detailed mutational analysis of human A3G. We show that A3G residue 129, but not the adjacent position 128, confers susceptibility to degradation by SIVsmm Vif. An artificial A3G mutant, the P129D mutant, was resistant to degradation by diverse Vifs from HIV-1, HIV-2, SIVagm, and chimpanzee SIV (SIVcpz), suggesting a conserved lentiviral Vif binding site. Gorilla A3G naturally contains a glutamine (Q) at position 129, which makes its A3G resistant to Vifs from diverse lineages. We speculate that gorilla A3G serves as a barrier against SIVcpz strains. In summary, we show that Vif proteins from distinct lineages bind to the same A3G loop, which includes positions 128 and 129. The multiple adaptations within this loop among diverse primates underscore the importance of counteracting A3G in lentiviral evolution.

  14. Enhanced Binding of a Non-hydrogen Bond Ligand to DNA by Introducing an Apurine/Apyrimidine Site

    Institute of Scientific and Technical Information of China (English)

    SHAO Yong; NIU Zhenjiang; CHEN Jianrong; ZHANG Liangke

    2009-01-01

    Intercalators are well known for their DNA binding specificity by inserting between base pairs, whereas the binding event occurring to apurine/apyrimidine site (AP site)-containing DNA for this type of noncovalent interac-tion is still not highlighted although AP site is frequently in vivo produced in living cells. Here proflavine (PF) as an example is used to investigate the binding specificity of the AP site in DNA for a non-hydrogen bond iigand. Ex-perimental results indicate that the AP site should be the preferential binding site for PE The intrinsic binding con-stant of PF for the AP site is one order of magnitude greater than that occurring for PF intercalation. Additionally, the thermostability of the AP site-containing DNA is significantly increased after PF binding. The PF bound to the AP site should adopt a specific binding orientation distinguishable from that by which PF intercalated into base pairs. The results obtained here should be very useful for judging biochemical and biophysical effectiveness of small molecules based on their different binding behavior to DNA.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Solution structure of CCL19 and identification of overlapping CCR7 and PSGL-1 binding sites

    Science.gov (United States)

    Veldkamp, Christopher T.; Kiermaier, Eva; Gabel-Eissens, Skylar J.; Gillitzer, Miranda L.; Lippner, David R.; DiSilvio, Frank A.; Mueller, Casey J.; Wantuch, Paeton L.; Chaffee, Gary R.; Famiglietti, Michael W.; Zgoba, Danielle M.; Bailey, Asha A.; Bah, Yaya; Engebretson, Samantha J.; Graupner, David R.; Lackner, Emily R.; LaRosa, Vincent D.; Medeiros, Tysha; Olson, Michael L.; Phillips, Andrew J.; Pyles, Harley; Richard, Amanda M.; Schoeller, Scott J.; Touzeau, Boris; Williams, Larry G.; Sixt, Michael; Peterson, Francis C.

    2016-01-01

    CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1’s enhancement of resting T-cell recruitment are discussed. PMID:26115234

  17. Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

    Science.gov (United States)

    Gilfoyle, D J; Rodriguez-Lopez, J N; Smith, A T

    1996-03-01

    The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why

  18. Structural basis of Ets1 cooperative binding to widely separated sites on promoter DNA.

    Directory of Open Access Journals (Sweden)

    Nigar D Babayeva

    Full Text Available Ets1 is a member of the Ets family of transcription factors. Ets1 is expressed in autoinhibited form and its DNA binding depends on partner proteins bound to adjacent sequences or the relative positioning of a second Ets-binding site (EBS. The autoinhibition of Ets1 is mediated by structural coupling of regions flanking the DNA-binding domain. The NMR structure of Ets1 revealed that the inhibitory regions comprised of helices HI1 and HI2 and H4 are packed together on the Ets domain to form an inhibitory module. The crystal structure of Ets1 unexpectedly revealed a homodimer in which homodimerisation occurs via swapping of HI1 helices. Modeling of DNA binding indicates that the Ets1 dimer can bind to two antiparallel pieces of DNA. To verify this, we crystallized and solved the structure of the complex comprised of Ets1 dimer and two pieces of DNA. DNA binding by Ets1 dimer resulted in formation of additional intermolecular protein•DNA interactions, implying that the complex formation is cooperative.

  19. Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-04-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

    Science.gov (United States)

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

    Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

  2. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    Science.gov (United States)

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome.

  3. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    Science.gov (United States)

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  4. A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available Kinesin-13s are microtubule (MT depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2 on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

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

    Science.gov (United States)

    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

  6. The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis.

    Science.gov (United States)

    Hannaert, Véronique; Yernaux, Cédric; Rigden, Daniel J; Fothergill-Gilmore, Linda A; Opperdoes, Fred R; Michels, Paul A M

    2002-03-13

    The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.

  7. Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane-cargo apposition during selective autophagy.

    Science.gov (United States)

    Sawa-Makarska, Justyna; Abert, Christine; Romanov, Julia; Zens, Bettina; Ibiricu, Iosune; Martens, Sascha

    2014-05-01

    Autophagy protects cells from harmful substances such as protein aggregates, damaged mitochondria and intracellular pathogens, and has been implicated in a variety of diseases. Selectivity of autophagic processes is mediated by cargo receptors that link cargo to Atg8 family proteins on the developing autophagosomal membrane. To avoid collateral degradation during constitutive autophagic pathways, the autophagic machinery must not only select cargo but also exclude non-cargo material. Here we show that cargo directly activates the cargo receptor Atg19 by exposing multiple Atg8 binding sites. Furthermore, Atg19 mediates tight apposition of the cargo and Atg8-coated membranes in a fully reconstituted system. These properties are essential for the function of Atg19 during selective autophagy in vivo. Our results suggest that cargo receptors contribute to tight membrane bending of the isolation membrane around the cargo.

  8. Changes in 5-HT1A and NMDA binding sites by a single rapid transcranial magnetic stimulation procedure in rats

    NARCIS (Netherlands)

    Kole, MHP; Fuchs, E; Ziemann, U; Paulus, W; Ebert, U

    1999-01-01

    The effects of a single rapid-rate transcranial magnetic stimulation (rTMS) exposure on neurotransmitter binding sites in the rat brain 24 h after the stimulation were examined. Quantification by in vitro-autoradiography showed no differences for H-3-paroxetine binding (5-HT uptake sites) between rT

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

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian

    2014-01-01

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

  10. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter

    DEFF Research Database (Denmark)

    Andersen, Jacob; Ringsted, Kristoffer B; Bang-Andersen, Benny

    2015-01-01

    . Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues...

  11. GALACTOSE-BINDING SITE IN ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN (LT) AND CHOLERA-TOXIN (CT)

    NARCIS (Netherlands)

    MERRITT, EA; SIXMA, TK; KALK, KH; VANZANTEN, BAM; HOL, WGJ

    The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coil is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal

  12. A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Millen, Kat S; Hanek, Ariele P;

    2008-01-01

    Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-pi interaction with the agonist, whereas in GABA(A) receptors, a Tyr performs this role. The glycine receptor binding site, however, contains pr...

  13. GALACTOSE-BINDING SITE IN ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN (LT) AND CHOLERA-TOXIN (CT)

    NARCIS (Netherlands)

    MERRITT, EA; SIXMA, TK; KALK, KH; VANZANTEN, BAM; HOL, WGJ

    1994-01-01

    The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coil is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal str

  14. GALACTOSE-BINDING SITE IN ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN (LT) AND CHOLERA-TOXIN (CT)

    NARCIS (Netherlands)

    MERRITT, EA; SIXMA, TK; KALK, KH; VANZANTEN, BAM; HOL, WGJ

    1994-01-01

    The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coil is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal str

  15. Detection of 3D atomic similarities and their use in the discrimination of small molecule protein-binding sites.

    Science.gov (United States)

    Najmanovich, Rafael; Kurbatova, Natalja; Thornton, Janet

    2008-08-15

    Current computational methods for the prediction of function from structure are restricted to the detection of similarities and subsequent transfer of functional annotation. In a significant minority of cases, global sequence or structural (fold) similarities do not provide clues about protein function. In these cases, one alternative is to detect local binding site similarities. These may still reflect more distant evolutionary relationships as well as unique physico-chemical constraints necessary for binding similar ligands, thus helping pinpoint the function. In the present work, we ask the following question: is it possible to discriminate within a dataset of non-homologous proteins those that bind similar ligands based on their binding site similarities? We implement a graph-matching-based method for the detection of 3D atomic similarities introducing some simplifications that allow us to extend its applicability to the analysis of large allatom binding site models. This method, called IsoCleft, does not require atoms to be connected either in sequence or space. We apply the method to a cognate-ligand bound dataset of non-homologous proteins. We define a family of binding site models with decreasing knowledge about the identity of the ligand-interacting atoms to uncouple the questions of predicting the location of the binding site and detecting binding site similarities. Furthermore, we calculate the individual contributions of binding site size, chemical composition and geometry to prediction performance. We find that it is possible to discriminate between different ligand-binding sites. In other words, there is a certain uniqueness in the set of atoms that are in contact to specific ligand scaffolds. This uniqueness is restricted to the atoms in close proximity of the ligand in which case, size and chemical composition alone are sufficient to discriminate binding sites. Discrimination ability decreases with decreasing knowledge about the identity of the

  16. Mutations at the Qo-Site of the Cytochrome bc1 Complex Strongly Affect Oxygen Binding

    DEFF Research Database (Denmark)

    Husen, Peter; Solov'yov, Ilia A

    2017-01-01

    The homodimeric bc1 protein complex is embedded in membranes of mitochondria and photosynthetic bacteria, where it transports protons across the membrane to maintain an electrostatic potential used to drive ATP synthesis as part of the respiratory or photosynthetic pathways. The reaction cycle...... of the bc1 complex is driven by series of redox processes involving substrate molecules from the membrane, but occasional side reactions between an intermediate semiquinone substrate and molecular oxygen are suspected to be a source of toxic superoxide, which is believed to be a factor in aging. The present...... investigation employs molecular dynamics simulations to study the effect of mutations in the Qo binding sites of the bc1 complex on the ability of oxygen molecules to migrate to and bind at various locations within the complex. It is found that the mutations strongly affect the ability of oxygen to bind...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational...... the pocket, including(2) Val152(3.46) to Ala or Ile, Ser422(8.60) to Ala and Asn157(3.51) to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [(3)H]dopamine uptake inhibition assays and/or [(3)H]CFT competition binding assay. A putative polar interaction of one...... 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....

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

    Science.gov (United States)

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

    2016-08-08

    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.

  19. Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis.

    Science.gov (United States)

    Lindsay, Andrew J; McCaffrey, Mary W

    2004-07-30

    Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.

  20. Selection and characterization of small molecules that bind the HIV-1 frameshift site RNA.

    Science.gov (United States)

    Marcheschi, Ryan J; Mouzakis, Kathryn D; Butcher, Samuel E

    2009-10-16

    HIV-1 requires a -1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of approximately 5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (K(d) = 2.8 microM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 degrees C and decreased translational frameshifting by 28% (+/-14%), as measured in vitro.

  1. Developmental changes in the distribution of cecal lectin-binding sites of Balb-c mice.

    Science.gov (United States)

    Doehrn, S; Breipohl, W; Lierse, W; Romaniuk, K; Young, W

    1992-01-01

    The existence of lectin-binding sites was investigated in the cecum of Balb-c mice at seven developmental stages ranging from 18 days post conception (p.c.) to 8 weeks after birth. Nine horseradish-peroxidase-conjugated lectins (concanavalin A, Triticum vulgaris, Dolichus biflorus, Helix pomatia, Arachis hypogaea, Glycine maximus, Lotus tetragonolobus, Ulex europaeus, Limulus polyphemus) were applied to 5- to 7-microns thin paraffin sections of Bouin-fixed tissue. After DAB staining the sections were evaluated by light microscopy. It was shown that each lectin exhibits a unique developmental pattern. The adult binding patterns were established at the age of 3-4 weeks with only minor changes occurring thereafter. Considerable differences in binding patterns occurred not only between lectins of different groups but also between lectins with the same nominal monosaccharide specificity.

  2. Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

    OpenAIRE

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W.; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I–III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the...

  3. Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP.

    Science.gov (United States)

    Page, Stephen H; Wright, Edward K; Gama, Lucio; Clements, Janice E

    2011-01-01

    CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zeng Zhizhen [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)]. E-mail: zhizhen_zeng@merck.com; Chen, T.-B. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Miller, Patricia J. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Dean, Dennis [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Tang, Y.S. [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Sur, Cyrille [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Williams, David L. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)

    2006-05-15

    We have characterized the interaction of the serotonin transporter ligand [{sup 3}H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [{sup 3}H]-DASB, a tritiated version of the widely used [{sup 11}C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K {sub d}=0.20{+-}0.04 nM). The serotonin transporter density (B {sub max}) obtained for rhesus frontal cortex was found to be 66{+-}8 fmol/mg protein using [{sup 3}H]-DASB, similar to the B {sub max} value obtained using the reference radioligand [{sup 3}H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83{+-}22 fmol/mg protein). Specific binding sites of both [{sup 3}H]-DASB and [{sup 3}H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [{sup 3}H]-citalopram binding in a competition autoradiographic study, with K {sub i} values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [{sup 3}H]-DASB and [{sup 3}H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [{sup 11}C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.

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

    Energy Technology Data Exchange (ETDEWEB)

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  6. γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

    Science.gov (United States)

    Pozdnyakov, Nikolay; Murrey, Heather E; Crump, Christina J; Pettersson, Martin; Ballard, T Eric; Am Ende, Christopher W; Ahn, Kwangwook; Li, Yue-Ming; Bales, Kelly R; Johnson, Douglas S

    2013-04-05

    γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

  7. The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites.

    Science.gov (United States)

    Taylor, James A; Ouimet, Marie-Claude; Wargachuk, Richard; Marczynski, Gregory T

    2011-10-01

    The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control. © 2011 Blackwell Publishing Ltd.

  8. Copper(I)-α-synuclein interaction: structural description of two independent and competing metal binding sites.

    Science.gov (United States)

    Camponeschi, Francesca; Valensin, Daniela; Tessari, Isabella; Bubacco, Luigi; Dell'Acqua, Simone; Casella, Luigi; Monzani, Enrico; Gaggelli, Elena; Valensin, Gianni

    2013-02-04

    The aggregation of α-synuclein (αS) is a critical step in the etiology of Parkinson's disease. Metal ions such as copper and iron have been shown to bind αS, enhancing its fibrillation rate in vitro. αS is also susceptible to copper-catalyzed oxidation that involves the reduction of Cu(II) to Cu(I) and the conversion of O(2) into reactive oxygen species. The mechanism of the reaction is highly selective and site-specific and involves interactions of the protein with both oxidation states of the copper ion. The reaction can induce oxidative modification of the protein, which generally leads to extensive protein oligomerization and precipitation. Cu(II) binding to αS has been extensively characterized, indicating the N terminus and His-50 as binding donor residues. In this study, we have investigated αS-Cu(I) interaction by means of NMR and circular dichroism analysis on the full-length protein (αS(1-140)) and on two, designed ad hoc, model peptides: αS(1-15) and αS(113-130). In order to identify and characterize the metal binding environment in full-length αS, in addition to Cu(I), we have also used Ag(I) as a probe for Cu(I) binding. Two distinct Cu(I)/Ag(I) binding domains with comparable affinities have been identified. The structural rearrangements induced by the metal ions and the metal coordination spheres of both sites have been extensively characterized.

  9. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

    Science.gov (United States)

    Ross, Wilma; Vrentas, Catherine E; Sanchez-Vazquez, Patricia; Gaal, Tamas; Gourse, Richard L

    2013-05-01

    The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    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...... 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...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  12. Soybean. beta. -glucan binding sites display maximal affinity for a heptaglucoside phytoalexin-elicitor

    Energy Technology Data Exchange (ETDEWEB)

    Cosio, E.G.; Waldmueller, T.; Frey, T.; Ebel, J. (Biologisches Institut II der Universitat Freiburg (West Germany))

    1990-05-01

    The affinity of soybean {beta}-glucan-binding sites for a synthetic heptaglucan elicitor was tested in a ligand-competition assay against a {sup 125}I-labeled 1,3-1,6-{beta}-glucan preparation (avg. DP=20). Half-maximal displacement of label (IC{sub 50}) was obtained at 9nM heptaglucan, the highest affinity of all fractions tested to date. Displacement followed a uniform sigmoidal pattern and was complete at 1{mu}M indicating access of heptaglucan to all sites available to the labeled elicitor. A mathematical model was used to predict IC{sub 50} values according to the DP of glucan fragments obtained from fungal cell walls. The lowest IC{sub 50} predicted by this model is 3nM. Binding affinity of the glucans was compared with their elicitor activity in a bioassay.

  13. SNP2TFBS – a database of regulatory SNPs affecting predicted transcription factor binding site affinity

    Science.gov (United States)

    Kumar, Sunil; Ambrosini, Giovanna; Bucher, Philipp

    2017-01-01

    SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/. PMID:27899579

  14. Copper(II) and nickel(II) binding sites of peptide containing adjacent histidyl residues.

    Science.gov (United States)

    Grenács, Ágnes; Sanna, Daniele; Sóvágó, Imre

    2015-10-01

    Copper(II) and nickel(II) complexes of the terminally protected nonapeptide Ac-SGAEGHHQK-NH2 modeling the metal binding sites of the (8-16) domain of amyloid-β have been studied by potentiometric, UV-vis, CD and ESR spectroscopic methods. The studies on the mutants containing only one of the histidyl residues (Ac-SGAEGAHQK-NH2, Ac-SGAEGHAQK-NH2) have also been performed. The formation of imidazole and amide coordinated mononuclear complexes is characteristic of all systems with a preference of nickel(II) binding to the His14 site, while the involvement of both histidines in metal binding is suggested in the corresponding copper(II) complexes. The formation of bis(ligand) and dinuclear complexes has also been observed in the copper(II)-Ac-SGAEGHHQK-NH2 system. The results provide further support for the copper(II) binding ability of the (8-16) domain of amyloid-β and support the previous assumptions that via the bis(ligand) complex formation copper(II) ions may promote the formation of the oligomers of amyloid-β.

  15. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

    Science.gov (United States)

    Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; Montaño, Sherwin P.; Kurosawa, Kohei; Zheng, Yupeng; Akin, Louesa R.; Świst-Rosowska, Kalina M.; Grzybowski, Adrian T.; Koide, Akiko; Krajewski, Krzysztof; Strahl, Brian D.; Kelleher, Neil L.; Ruthenburg, Alexander J.; Koide, Shohei

    2016-01-01

    Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies. PMID:26862167

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Novel Triazole-Quinoline Derivatives as Selective Dual Binding Site Acetylcholinesterase Inhibitors

    Directory of Open Access Journals (Sweden)

    Susimaire P. Mantoani

    2016-02-01

    Full Text Available Alzheimer’s disease (AD is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.

  18. Preferable binding site of gas molecules on graphene nanoribbon with Stone–Wales defect

    Science.gov (United States)

    Auzar, Zuriana; Johari, Zaharah; Sakina, S. H.; Alias, N. E.; Abidin, M. S. Z.

    2017-02-01

    The issue of sensitivity of sensing device has focused on the development of sensing devices by using new materials, such as graphene. The gas molecules in different positions such as on, near and far from the defect are placed in the same binding site in two graphene configurations for fair comparison. The interaction between two different graphene configurations such as (pristine armchair graphene nanoribbon (P-AGNR) and Stone–Wales defect on graphene surface (SW-AGNR)) with gas molecules (e.g. O2, N2 and NH3) have been investigated to observe the preferential position site of adsorbate gas molecules. The preferable position sites are investigated by using Extended-Huckel Theory. It is found that, the electronic properties of each configuration are strongly depends on the position of gas molecules and graphene system. Meanwhile, the binding site of the gas molecules on the defective site of graphene surface is a significant factor in determining the sensing behavior of graphene based gas defection device.

  19. Excitatory amino acid binding sites in the hippocampal region of Alzheimer's disease and other dementias.

    OpenAIRE

    1990-01-01

    Quantitative receptor autoradiography was used to measure muscarinic cholinergic, benzodiazepine, kainate, phencyclidine (PCP), N-methyl-D-aspartate (NMDA) (measured in Tris acetate), quisqualate-sensitive, non-quisqualate-sensitive and total glutamate (measured in Tris chloride buffer) binding sites in adjacent sections of the hippocampal region of 10 Alzheimer's disease, nine control, and six demented, non-Alzheimer's disease postmortem human brains. The measurements were compared to the nu...

  20. Photo-Affinity Labeling of Specific Acetylcholine-Binding Sites on Membranes

    Science.gov (United States)

    Kiefer, Hansruedi; Lindstrom, Jon; Lennox, Edwin S.; Singer, S. J.

    1970-01-01

    Acetylcholinesterase of intact red blood cell membranes and the acetylcholine receptor at the neuromuscular junction of whole-frog sartorius muscle have been irreversibly inactivated by photo-affinity labeling with two quaternary ammonium aryl azides. The inactivation requires that the azides, at the time of their photolytic conversion to highly reactive nitrenes, are reversibly bound to the specific acetylcholine-binding sites. PMID:5275370

  1. Combining features in a graphical model to predict protein binding sites.

    Science.gov (United States)

    Wierschin, Torsten; Wang, Keyu; Welter, Marlon; Waack, Stephan; Stanke, Mario

    2015-05-01

    Large efforts have been made in classifying residues as binding sites in proteins using machine learning methods. The prediction task can be translated into the computational challenge of assigning each residue the label binding site or non-binding site. Observational data comes from various possibly highly correlated sources. It includes the structure of the protein but not the structure of the complex. The model class of conditional random fields (CRFs) has previously successfully been used for protein binding site prediction. Here, a new CRF-approach is presented that models the dependencies of residues using a general graphical structure defined as a neighborhood graph and thus our model makes fewer independence assumptions on the labels than sequential labeling approaches. A novel node feature "change in free energy" is introduced into the model, which is then denoted by ΔF-CRF. Parameters are trained with an online large-margin algorithm. Using the standard feature class relative accessible surface area alone, the general graph-structure CRF already achieves higher prediction accuracy than the linear chain CRF of Li et al. ΔF-CRF performs significantly better on a large range of false positive rates than the support-vector-machine-based program PresCont of Zellner et al. on a homodimer set containing 128 chains. ΔF-CRF has a broader scope than PresCont since it is not constrained to protein subgroups and requires no multiple sequence alignment. The improvement is attributed to the advantageous combination of the novel node feature with the standard feature and to the adopted parameter training method.

  2. Homology inference of protein-protein interactions via conserved binding sites.

    Directory of Open Access Journals (Sweden)

    Manoj Tyagi

    Full Text Available The coverage and reliability of protein-protein interactions determined by high-throughput experiments still needs to be improved, especially for higher organisms, therefore the question persists, how interactions can be verified and predicted by computational approaches using available data on protein structural complexes. Recently we developed an approach called IBIS (Inferred Biomolecular Interaction Server to predict and annotate protein-protein binding sites and interaction partners, which is based on the assumption that the structural location and sequence patterns of protein-protein binding sites are conserved between close homologs. In this study first we confirmed high accuracy of our method and found that its accuracy depends critically on the usage of all available data on structures of homologous complexes, compared to the approaches where only a non-redundant set of complexes is employed. Second we showed that there exists a trade-off between specificity and sensitivity if we employ in the prediction only evolutionarily conserved binding site clusters or clusters supported by only one observation (singletons. Finally we addressed the question of identifying the biologically relevant interactions using the homology inference approach and demonstrated that a large majority of crystal packing interactions can be correctly identified and filtered by our algorithm. At the same time, about half of biological interfaces that are not present in the protein crystallographic asymmetric unit can be reconstructed by IBIS from homologous complexes without the prior knowledge of crystal parameters of the query protein.

  3. Composite Structural Motifs of Binding Sites for Delineating Biological Functions of Proteins

    Science.gov (United States)

    Kinjo, Akira R.; Nakamura, Haruki

    2012-01-01

    Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures. PMID:22347478

  4. Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation.

    Directory of Open Access Journals (Sweden)

    Matti Myllykoski

    Full Text Available The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Pocketome: an encyclopedia of small-molecule binding sites in 4D.

    Science.gov (United States)

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

    2012-01-01

    The importance of binding site plasticity in protein-ligand interactions is well-recognized, and so are the difficulties in predicting the nature and the degree of this plasticity by computational means. To assist in understanding the flexible protein-ligand interactions, we constructed the Pocketome, an encyclopedia of about one thousand experimentally solved conformational ensembles of druggable binding sites in proteins, grouped by location and consistent chain/cofactor composition. The multiplicity of pockets within the ensembles adds an extra, fourth dimension to the Pocketome entry data. Within each ensemble, the pockets were carefully classified by the degree of their pairwise similarity and compatibility with different ligands. The core of the Pocketome is derived regularly and automatically from the current releases of the Protein Data Bank and the Uniprot Knowledgebase; this core is complemented by entries built from manually provided seed ligand locations. The Pocketome website (www.pocketome.org) allows searching for the sites of interest, analysis of conformational clusters, important residues, binding compatibility matrices and interactive visualization of the ensembles using the ActiveICM web browser plugin. The Pocketome collection can be used to build multi-conformational docking and 3D activity models as well as to design cross-docking and virtual ligand screening benchmarks.

  7. NMR and Mutational Identification of the Collagen-Binding Site of the Chaperone Hsp47

    Science.gov (United States)

    Yagi-Utsumi, Maho; Yoshikawa, Sumi; Yamaguchi, Yoshiki; Nishi, Yohei; Kurimoto, Eiji; Ishida, Yoshihito; Homma, Takayuki; Hoseki, Jun; Nishikawa, Yoshimi; Koide, Takaki; Nagata, Kazuhiro; Kato, Koichi

    2012-01-01

    Heat shock protein 47 (Hsp47) acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective 15N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases. PMID:23049894

  8. NMR and mutational identification of the collagen-binding site of the chaperone Hsp47.

    Directory of Open Access Journals (Sweden)

    Maho Yagi-Utsumi

    Full Text Available Heat shock protein 47 (Hsp47 acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective (15N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-12-15

    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.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta......) 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...

  12. Identification of gamma-aminobutyric acid and its binding sites in Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Schaeffer, J.M.; Bergstrom, A.R.

    1988-01-01

    Gamma-aminobutyric acid (GABA), glutamate decarboxylase and GABA-transaminase were identified in the nematode Caenorhabditis elegans. The concentration of GABA in C. elegans is approximately 10-fold lower than the concentration of GABA in rat brain. Glutamate decarboxylase and GABA-transaminase, the GABA anabolic and catabolic enzymes, are also present in C. elegans. Crude membrane fractions were prepared from C. elegans and used to study specific (/sup 3/H) GABA binding sites. GABA binds to C. elegans membranes with high affinity and low capacity. Muscimol is a competitive inhibitor of specific GABA binding with a K/sub I/ value of 120 nM. None of the other GABA agonists or antagonists inhibited greater than 40% of the specific GABA binding at concentrations up to 10/sup -4/M. Thirteen spider venoms were examined as possible GABA agonists or antagonists, the venom from Calilena agelenidae inhibits specific GABA binding with a K/sub I/ value of 6 nl/ml. These results suggest that GABA has a physiological role as a neurotransmitter in C. elegans.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Muthalagi Mani

    2002-06-01

    Full Text Available Abstract Background The serotonin type 3 receptor (5-HT3R is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147 to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152 also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR. Conclusion Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  16. Differential effects of methoxy group on the interaction of curcuminoids with two major ligand binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Hiroki Sato

    Full Text Available Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA clearly indicated that curcumin (Cur, demethylcurcumin (Dmc and bisdemethoxycurcumin (Bdmc bind to both Site I (sub-site Ia and Ib and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds.

  17. Kinetic studies of proton transfer in the microenvironment of a binding site.

    Science.gov (United States)

    Gutman, M; Huppert, D; Nachliel, E

    1982-01-01

    Excitation of 8-hydroxypyrene 1,3,6-trisulfonate to its first electronic singlet state converts the compound from weak base (pK degrees = 7.7) into a strong acid (pK* = 0.5). The dissociation of the proton in water or dilute salt solution is a very fast reaction, K12 = 1 X 10(10) S-1. In concentrated salt solutions the dissociation is slowed as an exponential function of the chemical activity of the water in the solution. This kinetic parameter has been used to gauge the properties of the microenvironment of the binding sites of bovine serum albumin at which this compound is bound. Time-resolved fluorometry reveals two distinct steps: a rapid dissociation of the proton with tau = 300 +/- 40 ps which lasts approximately 0.5 ns, followed by a slower reaction with tau = 3.3 ns. The first rapid phase represents proton dissociation taking place in the binding site. From the rate constant K = 3.3 X 10(9) s-1 we estimate that the ability of the water molecules in the site to hydrate the ejected proton is equivalent to a salt solution with water activity of 0.85. The slow phase represents the escape of the proton from the binding site. The rate of the escape, 1.4 X 10(8) s-1, is significantly slower than diffusion-controlled dissociation. It is concluded that the shape of the site or its lowered proton conductivity do not allow a rapid escape of the proton to the bulk. Still it should be remembered that the escape of the proton is 10(5)-10(6)-times faster than a typical turnover of an enzyme.U

  18. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    Science.gov (United States)

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  19. Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog*

    Science.gov (United States)

    Jayakar, Selwyn S.; Dailey, William P.; Eckenhoff, Roderic G.; Cohen, Jonathan B.

    2013-01-01

    Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [3H]AziPm, and labeled amino acids were identified by Edman degradation. [3H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [3H]AziPm photolabeling at αM2-10′. Propofol inhibited [3H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 μm) than it inhibited ion channel photolabeling (IC50 = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site. PMID:23300078

  20. The distribution of iron between the metal-binding sites of transferrin human serum.

    Science.gov (United States)

    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.

  1. WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

    Science.gov (United States)

    Ojelabi, Ogooluwa A; Lloyd, Kenneth P; Simon, Andrew H; De Zutter, Julie K; Carruthers, Anthony

    2016-12-23

    WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Creation of a putative third metal binding site in type II dihydroorotases significantly enhances enzyme activity.

    Science.gov (United States)

    Huang, Yen-Hua; Huang, Cheng-Yang

    2015-01-01

    Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. DHOase is divided into two types (I and II). Type II DHOase generally contains a binuclear metal center in its active site. Recently, the crystal structure of DHOase domain in human CAD protein (huDHOase) has revealed three metal ions in the protein's active site. However, whether type II DHOase can have the critical third metal ion, as observed in huDHOase, remains unknown. In the present study, the putative third metal binding site in type II enzymes, such as the prokaryotic Salmonella enterica serovar Typhimurium LT2 DHOase (StDHOase) and the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase), was created and identified. StDHOase T198E and ScDHOase T208E mutants had higher activities compared with their wild-type enzymes. The need for a higher DHOase stability and activity may drive creation of the third metal ion binding site in huDHOase, which can be achieved by mutating a highly conserved position T in type II dihydroorotases to E, similar to that in huDHOase.

  3. Improvement of Yarrowia lipolytica lipase enantioselectivity by using mutagenesis targeted to the substrate binding site.

    Science.gov (United States)

    Bordes, F; Cambon, E; Dossat-Létisse, V; André, I; Croux, C; Nicaud, J M; Marty, A

    2009-07-06

    Lip2p lipase from Yarrowia lipolytica was shown to be an efficient catalyst for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. Enantioselectivity of this lipase was improved by site-directed mutagenesis targeted to the substrate binding site. To guide mutagenesis experiments, the three-dimensional model of this lipase was built by homology modelling techniques by using the structures of lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates. On the basis of this structural model, five amino acid residues (T88, V94, D97, V232, V285) that form the hydrophobic substrate binding site of the lipase were selected for site-directed mutagenesis. Position 232 was identified as crucial for the discrimination between enantiomers. Variant V232A displayed an enantioselectivity enhanced by one order of magnitude, whereas variant V232L exhibited a selectivity inversion. To further explore the diversity, position 232 was systematically replaced by the 19 possible amino acids. Screening of this library led to the identification of the V232S variant, which has a tremendously increased E value compared to the parental enzyme for the resolution of 2-bromo-phenylacetic acid ethyl ester (58-fold) and 2-bromo-o-tolylacetic acid ethyl ester (16-fold). In addition to the gain in enantioselectivity, a remarkable increase in velocity was observed (eightfold increase) for both substrates.

  4. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

    Directory of Open Access Journals (Sweden)

    Ju-Chen Chia

    Full Text Available Phytochelatin synthase (PCS uses the substrates glutathione (GSH, γGlu-Cys-Gly and a cadmium (Cd-bound GSH (Cd∙GS2 to produce the shortest phytochelatin product (PC2, (γGlu-Cys2-Gly through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218 by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

  5. Probing the binding sites and the effect of berbamine on the structure of bovine serum albumin

    Science.gov (United States)

    Cheng, Xiao-Xia; Lui, Yi; Zhou, Bo; Xiao, Xiao-He; Liu, Yi

    2009-06-01

    Berbamine, a naturally occurring isoquinoline alkaloid extracted from Berberis sp., is the active constituent of some Chinese herbal medicines and exhibits a variety of pharmacological activities. The effects of berbamine on the structure of bovine serum albumin (BSA) were investigated by circular dichroism, fluorescence and absorption spectroscopy under physiological conditions. Berbamine caused a static quenching of the intrinsic fluorescence of BSA, and the quenching data were analyzed by application of the Stern-Volmer equation. There was a single primary berbamine-binding site on BSA with a binding constant of 2.577 × 10 4 L mol -1 at 298 K. The thermodynamic parameters, enthalpy change (Δ H0) and entropy change (Δ S0) for the reaction were -76.5 kJ mol -1 and -173.4 J mol -1 K -1 according to the van't Hoff equation. The results showed that the hydrogen bond and van der Waals interaction were the predominant forces in the binding process. Competitive experiments revealed a displacement of warfarin by berbamine, indicating that the binding site was located at Drug sites I. The distance r between the donor (BSA) and the acceptor (berbamine) was obtained according to the Förster non-radiation energy transfer theory. The results of three-dimensional fluorescence spectra, UV-vis absorption difference spectra and circular dichroism of BSA in the presence of berbamine showed that the conformation of BSA was changed. The results provide a quantitative understanding of the effect of berbamine on the structure of bovine serum albumin, providing a useful guideline for further drug design.

  6. GFI1B controls its own expression binding to multiple sites.

    Science.gov (United States)

    Anguita, Eduardo; Villegas, Ana; Iborra, Francisco; Hernández, Aurora

    2010-01-01

    Transcription factors play essential roles in both normal and malignant hematopoiesis. This is the case for the growth factor independent 1b (GFI1B) transcription factor, which is required for erythroid and megakaryocytic differentiation and over-expressed in leukemic patients and cell lines. To investigate GFI1B regulation, we searched for multispecies conserved non-coding elements between GFI1B and neighboring genes. We used a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay and DNase1 hypersensitivity to assess the chromatin conformation of these sites. Next, we analyzed transcription factor binding and histone modifications at the GFI1B locus including the conserved non-coding elements by a chromatin immunoprecipitation assay. Finally, we studied the interaction of the GFI1B promoter and the conserved non-coding elements with the chromatin conformation capture technique and used immunofluorescence to evaluate GFI1B levels in individual cells. We localized several conserved non-coding elements containing multiple erythroid specific transcription factor binding sites at the GFI1B locus. In GFI1B-expressing cells a subset of these conserved non-coding elements and the promoter adopt a close spatial conformation, localize with open chromatin sites, harbor chromatin modifications associated with gene activation and bind multiple transcription factors and co-repressors. Conclusions Our findings indicate that GFI1B regulatory elements behave as activators and repressors. Different protein levels within a cell population suggest that cells must activate and repress GFI1B continuously to control its final level. These data are consistent with a model of GFI1B regulation in which GFI1B binds to its own promoter and to the conserved non-coding elements as its levels rise. This would attract repressor complexes that progressively down-regulate the gene. GFI1B expression would decrease until a stage at which the activating complexes predominate and

  7. The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site.

    Science.gov (United States)

    Kanno, Takeshi; Yamamoto, Hideyuki; Yaguchi, Takahiro; Hi, Rika; Mukasa, Takeshi; Fujikawa, Hirokazu; Nagata, Tetsu; Yamamoto, Satoshi; Tanaka, Akito; Nishizaki, Tomoyuki

    2006-06-01

    This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

  8. Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin-binding protein.

    Science.gov (United States)

    Zervosen, Astrid; Herman, Raphael; Kerff, Frédéric; Herman, Alexandre; Bouillez, André; Prati, Fabio; Pratt, R F; Frère, Jean-Marie; Joris, Bernard; Luxen, André; Charlier, Paulette; Sauvage, Eric

    2011-07-20

    Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cβ bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal ......BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C......-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. RESULTS: We now find that both WW binding sites are important for maintaining full Dg...

  10. LIBSA--a method for the determination of ligand-binding preference to allosteric sites on receptor ensembles.

    Science.gov (United States)

    Hocker, Harrison J; Rambahal, Nandini; Gorfe, Alemayehu A

    2014-02-24

    Incorporation of receptor flexibility into computational drug discovery through the relaxed complex scheme is well suited for screening against a single binding site. In the absence of a known pocket or if there are multiple potential binding sites, it may be necessary to do docking against the entire surface of the target (global docking). However no suitable and easy-to-use tool is currently available to rank global docking results based on the preference of a ligand for a given binding site. We have developed a protocol, termed LIBSA for LIgand Binding Specificity Analysis, that analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio (SNR), the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. The method thus facilitates screening libraries of ligand cores against a large library of receptor conformations without prior knowledge of specific pockets, which is especially useful to search for hits that selectively target a particular site. We demonstrate the utility of LIBSA by showing that it correctly identifies known ligand binding sites and predicts the relative preference of a set of related ligands for different pockets on the same receptor.

  11. Development of a protein-ligand-binding site prediction method based on interaction energy and sequence conservation.

    Science.gov (United States)

    Tsujikawa, Hiroto; Sato, Kenta; Wei, Cao; Saad, Gul; Sumikoshi, Kazuya; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

    2016-09-01

    We present a new method for predicting protein-ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score. The performance of our prediction method was evaluated using a non-redundant dataset of 348 ligand-bound and ligand-unbound protein structure pairs, constructed by filtering entries in a ligand-binding site structure database, LigASite. Ligand-bound structure prediction (bound prediction) indicated that 74.0 % of predicted ligand-binding sites overlapped with real ligand-binding sites by over 25 % of their volume. Ligand-unbound structure prediction (unbound prediction) indicated that 73.9 % of predicted ligand-binding residues overlapped with real ligand-binding residues. The amino acid conservation score improved the average prediction accuracy by 17.0 and 17.6 points for the bound and unbound predictions, respectively. These results demonstrate the effectiveness of the combined use of the interaction energy and amino acid conservation in the ligand-binding site prediction.

  12. Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP.

    Science.gov (United States)

    Modi, Niraj; Bárcena-Uribarri, Iván; Bains, Manjeet; Benz, Roland; Hancock, Robert E W; Kleinekathöfer, Ulrich

    2015-02-20

    The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.

  13. Wide-scale analysis of human functional transcription factor binding reveals a strong bias towards the transcription start site.

    Directory of Open Access Journals (Sweden)

    Yuval Tabach

    Full Text Available BACKGROUND: Transcription factors (TF regulate expression by binding to specific DNA sequences. A binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF-DNA binding strength, distance of the binding site from the transcription start site (TSS, DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is a must for identifying functional TF binding sites and for understanding regulation of transcription. METHODOLOGY/PRINCIPAL FINDINGS: We introduce a novel method to screen the promoters of a set of genes with shared biological function (obtained from the functional Gene Ontology (GO classification against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. More than 8,000 human (and 23,000 mouse genes, were assigned to one of 134 GO sets. Their promoters were searched (from 200 bp downstream to 1,000 bp upstream the TSS for 414 known DNA motifs. We optimized the sequence similarity score threshold, independently for every location window, taking into account nucleotide heterogeneity along the promoters of the target genes. The method, combined with binding sequence and location conservation between human and mouse, identifies with high probability functional binding sites for groups of functionally-related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were tested experimentally. CONCLUSIONS/SIGNIFICANCE: We identified reliably functional TF binding sites. This is an essential step towards constructing regulatory networks. The promoter region proximal to the TSS is of central

  14. H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas

    DEFF Res