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Sample records for acid binding sites

  1. Modeling lanthanide series binding sites on humic acid.

    PubMed

    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.

  2. Binding sites of retinol and retinoic acid with serum albumins.

    PubMed

    Belatik, A; Hotchandani, S; Bariyanga, J; Tajmir-Riahi, H A

    2012-02-01

    Retinoids are effectively transported in the bloodstream via serum albumins. We report the complexation of bovine serum albumin (BSA) with retinol and retinoic acid at physiological conditions, using constant protein concentration and various retinoid contents. FTIR, CD and fluorescence spectroscopic methods and molecular modeling were used to analyze retinoid binding site, the binding constant and the effects of complexation on BSA stability and secondary structure. Structural analysis showed that retinoids bind BSA via hydrophilic and hydrophobic interactions with overall binding constants of K(Ret)(-BSA) = 5.3 (±0.8) × 10(6) M(-1) and K(Retac-BSA) = 2.3 (±0.4) × 10(6) M(-1). The number of bound retinoid molecules (n) was 1.20 (±0.2) for retinol and 1.8 (±0.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in retinoid-BSA complexes stabilized by H-bonding network. The retinoid binding altered BSA conformation with a major reduction of α-helix from 61% (free BSA) to 36% (retinol-BSA) and 26% (retinoic acid-BSA) with an increase in turn and random coil structures indicating a partial protein unfolding. The results indicate that serum albumins are capable of transporting retinoids in vitro and in vivo.

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

    SciTech Connect

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

    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.

  4. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

  5. A Sialic Acid Binding Site in a Human Picornavirus

    PubMed Central

    Frank, Martin; Hähnlein-Schick, Irmgard; Ekström, Jens-Ola; Arnberg, Niklas; Stehle, Thilo

    2014-01-01

    The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC. PMID:25329320

  6. Phytotropins: III. NAPHTHYLPHTHALAMIC ACID BINDING SITES ON MAIZE COLEOPTILE MEMBRANES AS POSSIBLE RECEPTOR SITES FOR PHYTOTROPIN ACTION.

    PubMed

    Katekar, G F; Navé, J F; Geissler, A E

    1981-12-01

    Certain members of the phytotropin class of auxin transport inhibitors are shown to bind with high affinity to the known naphthylphthalamic acid binding sites in maize (Zea mays) coleoptiles. The binding site is, thus, a phytotropin binding site. In general, the degree of binding correlates with the phytotropin structure activity rules and with physiological activities of model compounds. It is argued that the binding site may be a receptor, and it also may be the receptor involved in the control of the auxin transport process. The possibility is raised that the binding sites may be intrinsic receptors for endoanalog(s) of the phytotropins.

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

    PubMed Central

    Tonsgard, J H; Meredith, S C

    1991-01-01

    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. PMID:2064600

  8. Probing the Binding Site of Bile Acids in TGR5

    PubMed Central

    2013-01-01

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure–activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

  9. Probing the Binding Site of Bile Acids in TGR5.

    PubMed

    Macchiarulo, Antonio; Gioiello, Antimo; Thomas, Charles; Pols, Thijs W H; Nuti, Roberto; Ferrari, Cristina; Giacchè, Nicola; De Franco, Francesca; Pruzanski, Mark; Auwerx, Johan; Schoonjans, Kristina; Pellicciari, Roberto

    2013-12-12

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure-activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists.

  10. Binding of coumarins to site I of human serum albumin. Effect of the fatty acids.

    PubMed

    Zatón, A M; Ferrer, J M; Ruiz de Gordoa, J C; Marquínez, M A

    1995-07-14

    It is known that binding site I on human serum albumin (HSA) consists of a zone of two overlapping regions: the specific binding region represented by warfarin binding and the specific binding region represented by azapropazone and phenylbutazone binding. In this paper binding parameters to defatted HSA and to HSA with fatty acids (molar ratio of fatty acid/HSA = 4) were compared. High-affinity binding sites for warfarin, 4-chromanol, 4-hydroxycoumarin, coumarin, 3-acetylcoumarin and phenylbutazone (759,549 M-1 > Ka > 67,024 M-1) constitute binding site I on HSA. In this binding area defatted HSA can bind two molecules of warfarin, but the presence of fatty acids diminish the binding capacity of warfarin to HSA (2 > n > 1).

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

    PubMed

    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

  12. GABA sub A (gamma-aminobutyric acid) type binding sites on membranes of spermatozoa

    SciTech Connect

    Erdoe, S.L. ); Wekerle, L. )

    1990-01-01

    The binding of ({sup 3}H) gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABA{sub A} type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa.

  13. Locating the binding sites of folic acid with milk α- and β-caseins.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2012-01-12

    We located the binding sites of folic acid with milk α- and β-caseins at physiological conditions, using constant protein concentration and various folic acid contents. FTIR, UV-visible, and fluorescence spectroscopic methods as well as molecular modeling were used to analyze folic acid binding sites, the binding constant, and the effect of folic acid interaction on the stability and conformation of caseins. Structural analysis showed that folic acid binds caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(folic acid-α-caseins) = 4.8 (±0.6) × 10(4) M(-1) and K(folic acid-β-caseins) = 7.0 (±0.9) × 10(4) M(-1). The number of bound acid molecules per protein was 1.5 (±0.4) for α-casein and 1.4 (±0.3) for β-casein complexes. Molecular modeling showed different binding sites for folic acid on α- and β-caseins. The participation of several amino acids in folic acid-protein complexes was observed, which was stabilized by hydrogen bonding network and the free binding energy of -7.7 kcal/mol (acid-α-casein) and -8.1 kcal/mol (acid-β-casein). Folic acid complexation altered protein secondary structure by the reduction of α-helix from 35% (free α-casein) to 33% (acid-complex) and 32% (free β-casein) to 26% (acid-complex) indicating a partial protein destabilization. Caseins might act as carriers for transportation of folic acid to target molecules.

  14. Characterization of Naphthaleneacetic Acid Binding to Receptor Sites on Cellular Membranes of Maize Coleoptile Tissue 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Characteristics of and optimum conditions for saturable (“specific”) binding of [14C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a KD of 5 to 7 × 10−7m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg2+ or Ca2+ above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor (“supernatant factor”) found in maize tissue. PMID:16659851

  15. Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin.

    PubMed

    Chen, Yan-Min; Guo, Liang-Hong

    2009-03-01

    Binding of five perfluoroalkyl acids with human serum albumin (HSA) was investigated by site-specific fluorescence. Intrinsic fluorescence of tryptophan-214 in HSA was monitored upon addition of the chemicals. Although perfluorobutyl acid (PFBA) and perfluorobutane sulfonate (PFBS) did not cause fluorescence change, perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), and perfluorododecanoic acid (PFDoA) induced fluorescence quenching, from which binding constant of 2.7 x 10(5) M(-1) for PFOA and 2.2 x 10(4) M(-1) for PFOS was calculated. Two fluorescent probes, dansylamide (DA) and dansyl-L: -proline (DP), were employed in fluorescence displacement measurements to study the interaction at two Sudlow's binding sites. At Site I, both PFBA and PFBS displaced DA with binding constants of 1.0 x 10(6) M(-1) and 2.2 x 10(6) M(-1). At Site II, PFBS and PFDoA displaced DP with binding constants of 6.5 x 10(6) M(-1) and 1.2 x 10(6) M(-1), whereas PFBA did not bind. The data were compared with fatty acids to evaluate the potential toxicological effect of these environmental chemicals.

  16. MOLECULAR MODELING AND FUNCTIONAL CONFIRMATION OF A PREDICTED FATTY ACID BINDING SITE OF MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE

    PubMed Central

    Bradbury, Michael W.; Stump, Decherd; Guarnieri, Frank; Berk, Paul D.

    2011-01-01

    Molecular interactions are necessary for proteins to perform their functions. The identification of a putative plasma membrane fatty acid transporter as mitochondrial aspartate aminotransferase indicated that that protein must have a fatty acid binding site. Molecular modeling suggests that such a site exists in the form of a 500 Å3 hydrophobic cleft on the surface of the molecule, and identifies specific amino acid residues likely to be important for binding. The modeling and comparison with the cytosolic isoform indicated that two residues (Arg 201, Ala 219) were likely to be important to the structure and function of the binding site. These residues were mutated to determine if they were essential to that function. Expression constructs with wild-type or mutated cDNAs were produced for bacteria and eukaryotic cells. Proteins expressed in E. coli were tested for oleate binding affinity, which was decreased in the mutant proteins. 3T3 fibroblasts were transfected with expression constructs for both normal and mutated forms. Plasma membrane expression was documented by indirect immunofluorescence before [3H]-oleic acid uptake kinetics were assayed. The Vmax for uptake was significantly increased by over-expression of the wild type protein, but changed little after transfection with mutated proteins, despite their presence on the plasma membrane. The hydrophobic cleft in mitochondrial aspartate aminotransferase can serve as a fatty acid binding site. Specific residues are essential for normal fatty acid binding, without which fatty acid uptake is compromised. These results confirm the function of this protein as a fatty acid binding protein. PMID:21803047

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

    PubMed

    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.

  18. Melamine binding with arachidonic acid binding sites of albumin is a potential mechanism for melamine-induced inflammation.

    PubMed

    Rajpoot, Meenakshi; Bhattacharyya, Rajasri; Banerjee, Dibyajyoti; Sharma, Anil

    2017-07-01

    Melamine adulteration of food is a public health concern. It has been seen that melamine causes disease in many organs. Melamine-induced kidney disease is a well-recognized clinicopathological entity. Inflammation is thought to be important in melamine-induced pathology. Melamine is expected to bind with albumin because it has a positive charge. Albumin binds arachidonic acid. So if binding of melamine with albumin takes place, it has the potential to displace arachidonic acid from the albumin bound state. This phenomenon may be the source of mediators of inflammation in the melamine exposure state. This aspect is investigated in the present study by docking and molecular dynamics simulation. It is observed that melamine binds with some known arachidonic acid binding sites of albumin. This can lead to formation of more free arachidonic acid. It is also observed that melamine does not bind with extracellular signal regulated kinase 2 (ERK2). Therefore, the signal transduction mediated process involving ERK2 is not a likely mechanism of melamine-induced inflammation. Therefore, we think that an increased free arachidonic acid level may contribute more to inflammation in the melamine exposure state. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  19. Characterization of a domoic acid binding site from Pacific razor clam.

    PubMed

    Trainer, Vera L; Bill, Brian D

    2004-08-10

    The Pacific razor clam, Siliqua patula, is known to retain domoic acid, a water-soluble glutamate receptor agonist produced by diatoms of the genus Pseudo-nitzschia. The mechanism by which razor clams tolerate high levels of the toxin, domoic acid, in their tissues while still retaining normal nerve function is unknown. In our study, a domoic acid binding site was solubilized from razor clam siphon using a combination of Triton X-100 and digitonin. In a Scatchard analysis using [3H]kainic acid, the partially-purified membrane showed two distinct receptor sites, a high affinity, low capacity site with a KD (mean +/- S.E.) of 28 +/- 9.4 nM and a maximal binding capacity of 12 +/- 3.8 pmol/mg protein and a low affinity, high capacity site with a mM affinity for radiolabeled kainic acid, the latter site which was lost upon solubilization. Competition experiments showed that the rank order potency for competitive ligands in displacing [3H]kainate binding from the membrane-bound receptors was quisqualate > ibotenate > iodowillardiine = AMPA = fluorowillardiine > domoate > kainate > L-glutamate. At high micromolar concentrations, NBQX, NMDA and ATPA showed little or no ability to displace [3H]kainate. In contrast, Scatchard analysis using [3H]glutamate showed linearity, indicating the presence of a single binding site with a KD and Bmax of 500 +/- 50 nM and 14 +/- 0.8 pmol/mg protein, respectively. These results suggest that razor clam siphon contains both a high and low affinity receptor site for kainic acid and may contain more than one subtype of glutamate receptor, thereby allowing the clam to function normally in a marine environment that often contains high concentrations of domoic acid.

  20. A bidentate Lewis acid with a telluronium ion as an anion-binding site

    NASA Astrophysics Data System (ADS)

    Zhao, Haiyan; Gabbaï, François P.

    2010-11-01

    The search for receptors that can selectively capture small and potentially toxic anions in protic media has sparked a renewed interest in the synthesis and anion-binding properties of polydentate Lewis acids. Seeking new paradigms to enhance the anion affinities of such systems, we synthesized a bidentate Lewis acid that contains a boryl and a telluronium moiety as Lewis acidic sites. Anion-complexation studies indicate that this telluronium borane displays a high affinity for fluoride in methanol. Structural and computational studies show that the unusual fluoride affinity of this bidentate telluronium borane can be correlated with the formation of a B-F --> Te chelate motif supported by a strong lone-pair(F) --> σ*(Te-C) donor-acceptor interaction. These results, which illustrate the viability of heavier chalcogenium centres as anion-binding sites, allow us to introduce a novel strategy for the design of polydentate Lewis acids with enhanced anion affinities.

  1. A bidentate Lewis acid with a telluronium ion as an anion-binding site.

    PubMed

    Zhao, Haiyan; Gabbaï, François P

    2010-11-01

    The search for receptors that can selectively capture small and potentially toxic anions in protic media has sparked a renewed interest in the synthesis and anion-binding properties of polydentate Lewis acids. Seeking new paradigms to enhance the anion affinities of such systems, we synthesized a bidentate Lewis acid that contains a boryl and a telluronium moiety as Lewis acidic sites. Anion-complexation studies indicate that this telluronium borane displays a high affinity for fluoride in methanol. Structural and computational studies show that the unusual fluoride affinity of this bidentate telluronium borane can be correlated with the formation of a B-F → Te chelate motif supported by a strong lone-pair(F) → σ*(Te-C) donor-acceptor interaction. These results, which illustrate the viability of heavier chalcogenium centres as anion-binding sites, allow us to introduce a novel strategy for the design of polydentate Lewis acids with enhanced anion affinities.

  2. Identification of amino acids located in the antibody binding sites of human hepatitis A virus.

    PubMed

    Nainan, O V; Brinton, M A; Margolis, H S

    1992-12-01

    Antigenic mutants of human hepatitis A virus (human-HAV) were isolated by their resistance to neutralizing monoclonal antibodies raised to human-HAV. The nucleotide sequence determined for the capsid regions of 12 mutants identified amino acid changes that clustered in three non-overlapping sites; one in VP3 and two in VP1. All mutants had a change at amino acid residue 70 in VP3, indicating its primary importance for antibody binding. Ten mutants had two amino acid changes occurring in the VP3 site as well as one in one of the two VP1 sites. These data suggest that both sites in VP1 interact with the single VP3 site to form the immunodominant epitope of HAV. The amino acid changes found in the antigenic mutants of human-HAV selected in this study were located in the same positions as changes found in strains of HAV isolated from Old World monkeys. These simian strains of HAV are not recognized by most monoclonal antibodies raised to human-HAV, suggesting that the observed amino acid changes are part of the antibody binding site.

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

    PubMed Central

    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 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. PMID:26681179

  4. Fatty acid binding sites of human and bovine albumins: Differences observed by spin probe ESR

    NASA Astrophysics Data System (ADS)

    Muravsky, Vladimir; Gurachevskaya, Tatjana; Berezenko, Stephen; Schnurr, Kerstin; Gurachevsky, Andrey

    2009-09-01

    Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5-8.0) and temperatures (25-50 °C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.

  5. Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes.

    PubMed Central

    Ying, Q L; Rinehart, A R; Simon, S R; Cheronis, J C

    1991-01-01

    Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids. PMID:1859379

  6. Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site

    PubMed Central

    Huang, Lin-Ya; Patel, Ami; Ng, Robert; Miller, Edward Blake; Halder, Sujata; McKenna, Robert; Asokan, Aravind

    2016-01-01

    ABSTRACT The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants—S472R, V473D, and N500E—escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface “hot spot” dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering. IMPORTANCE The adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect

  7. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36.

    PubMed

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127-279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127-279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions.

  8. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36

    PubMed Central

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A.; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127–279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127–279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions. PMID:24348024

  9. Exploration of binding site pattern in arachidonic acid metabolizing enzymes, Cyclooxygenases and Lipoxygenases.

    PubMed

    Reddy, Kakularam Kumar; Vidya Rajan, Veena Kumari; Gupta, Ashish; Aparoy, Polamarasetty; Reddanna, Pallu

    2015-04-16

    Cyclooxygenase (COXs) and Lipoxygenase (LOXs) pathways are the two major enzymatic pathways in arachidonic acid (AA) metabolism. The term eicosanoid is used to describe biologically active lipid mediators including prostaglandins, thromboxanes, leukotrienes and other oxygenated derivatives, which are produced primarily from AA. Eicosanoids generated in a tissue specific manner play a key role in inflammation and cancer. As AA is the substrate common to variety of COXs and LOXs, inhibition of one pathway results in diversion of the substrate to other pathways, which often is responsible for undesirable side effects. Hence there is need for development of not only isozyme specific inhibitors but also dual/multi enzyme inhibitors. Understanding the interactions of AA and characterizing its binding sites in these enzymes therefore is crucial for developing enzyme specific and multi enzyme inhibitors for enhancing therapeutic efficacy and/or overcoming side effects. AA binding sites in COXs and LOXs are identified and compared by the development of receptor based pharmacophore using MultiBind. Physico chemical properties were compared to understand the details of the binding sites in all the enzymes and to elucidate important amino acids that can be targeted for drug design. The alignment of AA binding sites in the seven enzymes COX-1, COX-2, 5-LOX, 12-LOX, 15-LOX and plant soybean LOX-1 and LOX-3 indicated a common pattern of five common interacting groups. In the same way, comparison of AA binding sites was done pair wise and by multiple alignment in various combinations. It has been identified that aliphatic and aromatic interactions are the most common in all the enzymes. In addition interactions unique to each one of these enzymes were identified. The complete analysis of AA binding sites in the seven enzymes was performed; 120 combinations for the seven enzymes were studied in detail. All the seven enzymes are structurally quite different, yet they share AA as the

  10. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor

    PubMed Central

    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L.; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C.; Schülein, Ralf; Krause, Gerd

    2010-01-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor’s transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.—Kleinau, G., Haas, A.-K., Neumann, S., Worth, C. L., Hoyer, I., Furkert, J., Rutz, Gershengorn, M. C., Schülein, R., Krause, G. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor. PMID:20179143

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

    SciTech Connect

    Lummis, S.C.R.; Johnston, G.A.R. ); Nicoletti, G. ); Holan, G. )

    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.

  12. Identification of metal ion binding sites based on amino acid sequences

    PubMed Central

    Cao, Xiaoyong; Zhang, Xiaojin; Gao, Sujuan; Ding, Changjiang; Feng, Yonge; Bao, Weihua

    2017-01-01

    The identification of metal ion binding sites is important for protein function annotation and the design of new drug molecules. This study presents an effective method of analyzing and identifying the binding residues of metal ions based solely on sequence information. Ten metal ions were extracted from the BioLip database: Zn2+, Cu2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Na+, K+ and Co2+. The analysis showed that Zn2+, Cu2+, Fe2+, Fe3+, and Co2+ were sensitive to the conservation of amino acids at binding sites, and promising results can be achieved using the Position Weight Scoring Matrix algorithm, with an accuracy of over 79.9% and a Matthews correlation coefficient of over 0.6. The binding sites of other metals can also be accurately identified using the Support Vector Machine algorithm with multifeature parameters as input. In addition, we found that Ca2+ was insensitive to hydrophobicity and hydrophilicity information and Mn2+ was insensitive to polarization charge information. An online server was constructed based on the framework of the proposed method and is freely available at http://60.31.198.140:8081/metal/HomePage/HomePage.html. PMID:28854211

  13. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  14. In situ detection of salicylic acid binding sites in plant tissues.

    PubMed

    Liu, Jing-Wen; Deng, Da-Yi; Yu, Ying; Liu, Fang-Fei; Lin, Bi-Xia; Cao, Yu-Juan; Hu, Xiao-Gang; Wu, Jian-Zhong

    2015-02-01

    The determination of hormone-binding sites in plants is essential in understanding the mechanisms behind hormone function. Salicylic acid (SA) is an important plant hormone that regulates responses to biotic and abiotic stresses. In order to label SA-binding sites in plant tissues, a quantum dots (QDs) probe functionalized with a SA moiety was successfully synthesized by coupling CdSe QDs capped with 3-mercaptopropionic acid (MPA) to 4-amino-2-hydroxybenzoic acid (PAS), using 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) as the coupling agent. The probe was then characterized by dynamic light scattering and transmission electron microscopy, as well as UV/vis and fluorescence spectrophotometry. The results confirmed the successful conjugation of PAS to CdSe QDs and revealed that the conjugates maintained the properties of the original QDs, with small core diameters and adequate dispersal in solution. The PAS-CdSe QDs were used to detect SA-binding sites in mung bean and Arabidopsis thaliana seedlings in vitro and in vivo. The PAS-CdSe QDs were effectively transported into plant tissues and specifically bound to SA receptors in vivo. In addition, the effects of the PAS-CdSe QDs on cytosolic Ca(2+) levels in the tips of A. thaliana seedlings were investigated. Both SA and PAS-CdSe QDs had similar effects on the trend in cytosolic-free Ca(2+) concentrations, suggesting that the PAS-CdSe QDs maintained the bioactivity of SA. To summarize, PAS-CdSe QDs have high potential as a fluorescent probe for the in vitro/in vivo labeling and imaging of SA receptors in plants.

  15. Identification of gamma-aminobutyric acid and its binding sites in Caenorhabditis elegans

    SciTech Connect

    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.

  16. CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

    PubMed Central

    Kellett-Clarke, Helena; Stegmann, Monika; Barclay, A. Neil; Metcalfe, Clive

    2015-01-01

    CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies. PMID:26379032

  17. Surface binding site analysis of Ca2+-homoionized clay-humic acid complexes.

    PubMed

    Martinez, Raul E; Sharma, Prasesh; Kappler, Andreas

    2010-12-15

    Clay-humic substance complexes play a major role in controlling the mobility of toxic metals in contaminated soils. However, our understanding of the underlying mechanisms is limited. Binding site analysis of clay and clay-mineral-humic composites, in this study, revealed an enhanced surface reactivity for the clay surface by the sorbed humic substances. Kaolinite and illite had three binding sites with pK(a) values ranging from ∼4.5 to 9.6 at Ca(2+) concentrations of 0.01 and 0.1 M respectively. In the presence of peat humic acid (PHA), four or five binding sites were observed for humics sorbed kaolinite surface at Ca(2+) concentrations of 0.01 and 0.1 M respectively. pK(a) values ranged from ∼4.4 to 9.6 for humic acid concentration of 0.01 and 0.1 mg/mL. For illite, four or five binding sites were found with pK(a)s ranging from ∼4.1 to 9.4. From zeta potential measurements of PHA-kaolinite or PHA-illite suspensions, the already negative potential decreased by 30 mV from pH 4 to 7, and by 10 mV for pH values greater than 7. For illite the initial negative surface potential decreased by 15 mV up to a pH of 9. Above this pH, the potential decrease diminished to 2 or 5 mV. These changes in surface potential confirm the adsorption of PHA to the clay mineral surface. FTIR measurements of clay samples were able to identify the kaolinite and illite phases. In addition, FTIR absorption bands found in the range of 1950-1800 cm(-1), suggest the interaction of PHA with kaolinite and illite surfaces. The results of this study indicate that the sorption of humic substances increases the availability of clay surface functional groups for deprotonation and potential sorption of toxic metal cations. Copyright © 2010. Published by Elsevier Inc.

  18. Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase.

    PubMed

    Rubach, Jon K; Plapp, Bryce V

    2003-03-18

    Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.

  19. Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor*

    PubMed Central

    Rosen, Daniel; Lewis, Alexander M.; Mizote, Akiko; Thomas, Justyn M.; Aley, Parvinder K.; Vasudevan, Sridhar R.; Parkesh, Raman; Galione, Antony; Izumi, Minoru; Ganesan, A.; Churchill, Grant C.

    2009-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca2+ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca2+ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca2+ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca2+ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor. PMID:19826006

  20. Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: evidence for a phospholipid binding site which overlaps the calmodulin-binding site.

    PubMed

    Meneghelli, Silvia; Fusca, Tiziana; Luoni, Laura; De Michelis, Maria Ida

    2008-09-01

    The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate > phosphatidylserine > phosphatidylcholine approximately = phosphatidylethanolamine approximately = 0. Acidic phospholipids increased V(max-Ca2+) and lowered the value of K(0.5-Ca2+) below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K(0.5-Ca2+) value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Delta74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.

  1. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

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

    SciTech Connect

    Frost, S.J.; Raja, R.H.; Weigel, P.H. )

    1990-11-13

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

  3. Putative binding sites for arachidonic acid on the human cardiac Kv1.5 channel

    PubMed Central

    Bai, Jia‐Yu; Ding, Wei‐Guang; Kojima, Akiko; Seto, Tomoyoshi

    2015-01-01

    Background and Purpose In human heart, the Kv1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)‐induced inhibition of the human Kv1.5 (hKv1.5) channel. Experimental Approach Site‐directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv1.5 channel. Whole‐cell patch‐clamp method was used to record membrane currents through wild type and mutant hKv1.5 channels heterologously expressed in CHO cells. Computer docking simulation was conducted to predict the putative binding site(s) of AA in an open‐state model of the Kv1.5 channel. Key Results The hKv1.5 current was minimally affected at the onset of depolarization but was progressively reduced during depolarization by the presence of AA, suggesting that AA acts as an open‐channel blocker. AA itself affected the channel at extracellular sites independently of its metabolites and signalling pathways. The blocking effect of AA was attenuated at pH 8.0 but not at pH 6.4. The blocking action of AA developed rather rapidly by co‐expression of Kvβ1.3. The AA‐induced block was significantly attenuated in H463C, T480A, R487V, I502A, I508A, V512A and V516A, but not in T462C, A501V and L510A mutants of the hKv1.5 channel. Docking simulation predicted that H463, T480, R487, I508, V512 and V516 are potentially accessible for interaction with AA. Conclusions and Implications AA itself interacts with multiple amino acids located in the pore domain of the hKv1.5 channel. These findings may provide useful information for future development of selective blockers of hKv1.5 channels. PMID:26292661

  4. Computational prediction and in vitro analysis of potential physiological ligands of the bile acid binding site in cytochrome c oxidase†

    PubMed Central

    Buhrow, Leann; Hiser, Carrie; Van Voorst, Jeffrey R.; Ferguson-Miller, Shelagh; Kuhn, Leslie A.

    2013-01-01

    A conserved bile acid site has been crystallographically defined in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome c oxidase (RsCcO). Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme a and a3 cofactors by blocking the K proton uptake path. Current studies identify physiologically relevant ligands for the bile acid site using a novel three-pronged computational approach: ROCS comparison of ligand shape and electrostatics, SimSite3D comparison of ligand binding site features, and SLIDE screening of potential ligands by docking. Identified candidate ligands include steroids, nicotinamides, flavins, nucleotides, retinoic acid, and thyroid hormones, which are predicted to make key protein contacts with the residues involved in bile acid binding. In vitro oxygen consumption and ligand competition assays on RsCcO wildtype and its Glu101Ala mutant support regulatory activity and specificity of some of these ligands. An ATP analog and GDP inhibit RsCcO under low substrate conditions, while fusidic acid, cholesteryl hemisuccinate, retinoic acid, and T3 thyroid hormone are more potent inhibitors under both high and low substrate conditions. The sigmoidal kinetics of RsCcO inhibition in the presence of certain nucleotides is reminiscent of previously reported ATP inhibition of mammalian CcO, suggesting regulation involving the conserved core subunits of both mammalian and bacterial oxidases. Ligand binding to the bile acid site is non-competitive with respect to cytochrome c and appears to arrest CcO in a semi-oxidized state with some resemblance to the “resting” state of the enzyme. PMID:24073649

  5. Computational prediction and in vitro analysis of potential physiological ligands of the bile acid binding site in cytochrome c oxidase.

    PubMed

    Buhrow, Leann; Hiser, Carrie; Van Voorst, Jeffrey R; Ferguson-Miller, Shelagh; Kuhn, Leslie A

    2013-10-08

    A conserved bile acid site has been crystallographically defined in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome c oxidase (RsCcO). Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme a and a3 cofactors by blocking the K proton uptake path. Current studies identify physiologically relevant ligands for the bile acid site using a novel three-pronged computational approach: ROCS comparison of ligand shape and electrostatics, SimSite3D comparison of ligand binding site features, and SLIDE screening of potential ligands by docking. Identified candidate ligands include steroids, nicotinamides, flavins, nucleotides, retinoic acid, and thyroid hormones, which are predicted to make key protein contacts with the residues involved in bile acid binding. In vitro oxygen consumption and ligand competition assays on RsCcO wildtype and its Glu101Ala mutant support regulatory activity and specificity of some of these ligands. An ATP analog and GDP inhibit RsCcO under low substrate conditions, while fusidic acid, cholesteryl hemisuccinate, retinoic acid, and T3 thyroid hormone are more potent inhibitors under both high and low substrate conditions. The sigmoidal kinetics of RsCcO inhibition in the presence of certain nucleotides is reminiscent of previously reported ATP inhibition of mammalian CcO, suggesting regulation involving the conserved core subunits of both mammalian and bacterial oxidases. Ligand binding to the bile acid site is noncompetitive with respect to cytochrome c and appears to arrest CcO in a semioxidized state with some resemblance to the "resting" state of the enzyme.

  6. Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization

    PubMed Central

    Lemloh, Marie-Louise; Altintoprak, Klara; Wege, Christina; Weiss, Ingrid M.; Rothenstein, Dirk

    2017-01-01

    Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO2 mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals. PMID:28772478

  7. Trans-gamma-hydroxycrotonic acid binding sites in brain: evidence for a subpopulation of gamma-hydroxybutyrate sites.

    PubMed

    Hechler, V; Schmitt, M; Bourguignon, J J; Maitre, M

    1990-03-02

    Trans-gamma-hydroxycrotonate (THCA), a compound naturally present in rat brain, possesses high-affinity binding sites with a heterogeneous distribution which are superimposable with those for gamma-hydroxybutyrate (GHB). Binding studies of THCA on rat brain membranes revealed two binding components, one of high affinity (Kd1, 7 nM, Bmax1 42 fmol/mg protein) and the other of low affinity (Kd2, 2 microM, Bmax2 13 pmol/mg protein). Displacement curves of [3H]THCA by THCA and GHB or of [3H]GHB by THCA are in favour of the existence of a specific high affinity site for THCA. Quantitative autoradiography with image analysis of [3H]THCA binding in rat brain slices indicated that [3H]THCA high affinity binding was displaced at a lower potency by GHB. THCA showed also some selectivity in displacing [3H]GHB from its high affinity binding site (Kd = 95 nM). This mutual overlap favours a subpopulation of GHB receptors, which have THCA as a natural ligand, showing partial agonistic properties compared to GHB. The functional significance of this result remains unknown.

  8. Iodine 125-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells

    SciTech Connect

    Yagaloff, K.A.; Hartig, P.R.

    1985-12-01

    /sup 125/I-Lysergic acid diethylamide (/sup 125/I-LSD) binds with high affinity to serotonergic sites on rat choroid plexus. These sites were localized to choroid plexus epithelial cells by use of a novel high resolution stripping film technique for light microscopic autoradiography. In membrane preparations from rat choroid plexus, the serotonergic site density was 3100 fmol/mg of protein, which is 10-fold higher than the density of any other serotonergic site in brain homogenates. The choroid plexus site exhibits a novel pharmacology that does not match the properties of 5-hydroxytryptamine-1a (5-HT1a), 5-HT1b, or 5-HT2 serotonergic sites. /sup 125/I-LSD binding to the choroid plexus site is potently inhibited by mianserin, serotonin, and (+)-LSD. Other serotonergic, dopaminergic, and adrenergic agonists and antagonists exhibit moderate to weak affinities for this site. The rat choroid plexus /sup 125/I-LSD binding site appears to represent a new type of serotonergic site which is located on non-neuronal cells in this tissue.

  9. In situ fluorescence labelling of jasmonic acid binding sites in plant tissues with cadmium-free quantum dots.

    PubMed

    Liao, Qiumei; Yu, Ying; Cao, Yujuan; Lin, Bixia; Wei, Jingjing

    2015-02-01

    The fluorescence labelling of plant hormone binding sites is an important analytical technique in research on the molecular mechanisms of plant hormone activities. The authors synthesised a jasmonic acid (JA)-conjugated ZnS:Mn quantum dot (QD) probe, with a cubic structure and average hydrodynamic sizes of about 17.0 nm. The maximum fluorescence emission of the probe was recorded at about 585 nm. The probe was used for fluorescence labelling of JA binding sites in mung bean seedling tissues. Analysis revealed that the probe exhibited high selectivity to JA binding sites and good performance in eliminating interference from background fluorescence in plant tissues. In addition, the probe did not exhibit any apparent biotoxicity, and is much more suitable than probes constructed from CdTe QDs for the analysis of biological samples.

  10. Subnucleosomes and their relationships to the arrangement of histone binding sites along nucleosome deoxyribonucleic acid

    SciTech Connect

    Nelson, D.A.; Mencke, A.J.; Chambers, S.A.; Oosterhof, D.K.; Rill, R.L.

    1982-01-01

    Micrococcal nuclease cleaves within nucleosomes at sites spaced about 10.4 base pairs (bp) apart. Cleavages at sites equivalent to 30-35 bp from the ends of 146-bp cores cause spontaneous loss of an H2a-H2b pair associated with 30-40 bp length DNA. Cleavages at certain other sites do not affect the nucleosome integrity unless a solvent perturbant such as urea is added. Chromatin moderately digested with micrococcal nuclease, when fractionated by sedimentation or electrophoresis in the presence of 3 M urea, yielded four previously unobserved subnucleosomes with the following histone/DNA compositions: (H3)/sub 2/(H4)/sub 2/(H2a)(H2b)/95-115 bp; (H3)(H4)/70-80 bp DNA; (H2a)(H2b)/50-60 bp DNA; and (H1)/60-70 bp DNA. All but the latter subnucleosome were also obtained upon DNase I digestion of purified nucleosome cores labeled on the 5' ends with /sup 32/P. Only subnucleosomes that retained H2a and H2b also retained labeled ends. These results show that H2a and H2b are paired on the terminal 30-40 bp of core DNA, as suggested from analyses of histone-DNA cross-link products by Mirzabekov and coworkers. Considerations of the orgins and compositions of subnucleosomes and of cross-linking data suggest an expanded model for the locations of histone binding sites along nucleosome core DNA. The principal features of this model are (i) strong electrostatic binding sites of H2a and H2b occur at positions approximately 20-30 bp from the core ends, (ii) strong electrostatic binding sites of H3 and H4 occur primarily on the central 40 bp of core DNA, (iii) strong nonelectrostatic, urea-sensitive binding sites of H3 and H4 occur at positions approximately 30-50 bp from the core ends, and (iv) urea-sensitive binding sites of H2a or H2b may occur on the terminal 10-20 bp of core DNA.

  11. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

    PubMed

    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  12. Evidence for a Single Naphthylphthalamic Acid Binding Site on the Zucchini Plasma Membrane.

    PubMed Central

    Muday, G. K.; Brunn, S. A.; Haworth, P.; Subramanian, M.

    1993-01-01

    The binding of [2,3,4,5,(n)-3H]N-1-napthylphthalamicacid ([3H]-NPA) to zucchini (Cucurbita pepo L.) plasma membranes was examined in detail using two different filtration assays and the results were rigorously analyzed by saturation curves, double-reciprocal plots, Scatchard plots, Hill plots, and the computer program Ligand (P.J. Munson, D. Rodbard [1980] Anal Biochem 107: 220-239). To facilitate these analyses, a new assay that allows rapid and quantitative analysis of [3H]NPA binding with high reproducibility and ease of manipulation has been developed. These detailed kinetic analyses indicate that only one binding site for [3H]NPA (Kd = 16 nM) was associated with the zucchini plasma membrane. Analysis of [3H]NPA dissociation by several auxin transport inhibitors revealed similar dissociation constants with both plasma and microsomal membrane. Collectively, these data indicate the presence of only one binding site for NPA associated with the zucchini plasma membrane. PMID:12231953

  13. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    PubMed

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  14. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

    PubMed

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

    2016-01-19

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding.

  15. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor

    PubMed Central

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K.; Gorska, Magdalena; Tuszynski, Jack A.; Wozniak, Michal

    2016-01-01

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  16. Gas phase acidity measurement of local acidic groups in multifunctional species: controlling the binding sites in hydroxycinnamic acids.

    PubMed

    Guerrero, Andres; Baer, Tomas; Chana, Antonio; González, Javier; Dávalos, Juan Z

    2013-07-03

    The applicability of the extended kinetic method (EKM) to determine the gas phase acidities (GA) of different deprotonable groups within the same molecule was tested by measuring the acidities of cinnamic, coumaric, and caffeic acids. These molecules differ not only in the number of acidic groups but in their nature, intramolecular distances, and calculated GAs. In order to determine independently the GA of groups within the same molecule using the EKM, it is necessary to selectively prepare pure forms of the hydrogen-bound heterodimer. In this work, the selectivity was achieved by the use of solvents of different vapor pressure (water and acetonitrile), as well as by variation of the drying temperature in the ESI source, which affected the production of heterodimers with different solvation energies and gas-phase dissociation energies. A particularly surprising finding is that the calculated solvation enthalpies of water and the aprotic acetonitrile are essentially identical, and that the different gas-phase products generated are apparently the result of their different vapor pressures, which affects the drying mechanism. This approach for the selective preparation of heterodimers, which is based on the energetics, appears to be quite general and should prove useful for other studies that require the selective production of heterodimers in ESI sources. The experimental results were supported by density functional theory (DFT) calculations of both gas-phase and solvated species. The experimental thermochemical parameters (deprotonation ΔG, ΔH, and ΔS) are in good agreement with the calculated values for the monofunctional cinnamic acid, as well as the multifunctional coumaric and caffeic acids. The measured GA for cinnamic acid is 334.5 ± 2.0 kcal/mol. The measured acidities for the COOH and OH groups of coumaric and caffeic acids are 332.7 ± 2.0, 318.7 ± 2.1, 332.2 ± 2.0, and 317.3 ± 2.2 kcal/mol, respectively.

  17. Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors.

    PubMed

    Liu, Zewen; Han, Zhaojun; Liu, Shuhua; Zhang, Yixi; Song, Feng; Yao, Xiangmei; Gu, Jianhua

    2008-07-01

    Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the alpha subunits and another three loops (D, E, and F) of the non-alpha subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-alpha subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two alpha subunits (Nlalpha1 and Nlalpha3). In Drosophila S2 cells and Xenopus oocytes, Nlalpha1 can form functional receptors with rat beta2 subunit. However, the same thing was not observed in Nlalpha3. In the present paper, by exchanging the corresponding regions between Nlalpha1 and Nlalpha3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.

  18. Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

    PubMed Central

    Wolny, Marcin; Grzybek, Michał; Bok, Ewa; Chorzalska, Anna; Lenoir, Marc; Czogalla, Aleksander; Adamczyk, Klaudia; Kolondra, Adam; Diakowski, Witold; Overduin, Michael; Sikorski, Aleksander F.

    2011-01-01

    It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton. PMID:21738695

  19. Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site.

    PubMed

    Nieto-Posadas, Andrés; Picazo-Juárez, Giovanni; Llorente, Itzel; Jara-Oseguera, Andrés; Morales-Lázaro, Sara; Escalante-Alcalde, Diana; Islas, León D; Rosenbaum, Tamara

    2011-11-20

    Since 1992, there has been growing evidence that the bioactive phospholipid lysophosphatidic acid (LPA), whose amounts are increased upon tissue injury, activates primary nociceptors resulting in neuropathic pain. The TRPV1 ion channel is expressed in primary afferent nociceptors and is activated by physical and chemical stimuli. Here we show that in control mice LPA produces acute pain-like behaviors, which are substantially reduced in Trpv1-null animals. Our data also demonstrate that LPA activates TRPV1 through a unique mechanism that is independent of G protein-coupled receptors, contrary to what has been widely shown for other ion channels, by directly interacting with the C terminus of the channel. We conclude that TRPV1 is a direct molecular target of the pain-producing molecule LPA and that this constitutes, to our knowledge, the first example of LPA binding directly to an ion channel to acutely regulate its function.

  20. Neurologic syndrome associated with homozygous mutation at MAG sialic acid binding site.

    PubMed

    Roda, Ricardo H; FitzGibbon, Edmond J; Boucekkine, Houda; Schindler, Alice B; Blackstone, Craig

    2016-08-01

    The MAG gene encodes myelin-associated glycoprotein (MAG), an abundant protein involved in axon-glial interactions and myelination during nerve regeneration. Several members of a consanguineous family with a clinical syndrome reminiscent of Pelizaeus-Merzbacher disease and demyelinating leukodystrophy on brain MRI were recently found to harbor a homozygous missense p.Ser133Arg MAG mutation. Here, we report two brothers from a nonconsanguineous family afflicted with progressive cognitive impairment, neuropathy, ataxia, nystagmus, and gait disorder. Exome sequencing revealed the homozygous missense mutation p.Arg118His in MAG. This Arg118 residue in immunoglobulin domain 1 is critical for sialic acid binding, providing a compelling mechanistic basis for disease pathogenesis.

  1. SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.

    PubMed

    Seamon, Kyle J; Sun, Zhiqiang; Shlyakhtenko, Luda S; Lyubchenko, Yuri L; Stivers, James T

    2015-07-27

    The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3'-5' exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Characterization of the S1 binding site of the glutamic acid-specific protease from Streptomyces griseus.

    PubMed Central

    Stennicke, H. R.; Birktoft, J. J.; Breddam, K.

    1996-01-01

    The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1. PMID:8931145

  3. Inclusion of nucleic acid contribution into assessment of protein 3d-1D compatibility and prediction of binding sites for nucleic acids.

    PubMed

    Fujii, S; Yamagata, Y

    1997-01-01

    The novel three dimensional structure(3D)-amino acid sequence(1D) compatibility program, 3d-1D method has developed and expanded into protein-nucleic acid system. The environment characteristics are determined using the coordinates of backbone atoms and C beta atom. This simplified estimation allows the expanded application to monitor the change of environment class depending on different sequence alignment. This method has improved the detective performance of the different protein into the similar folds. If protein has a wide contact with nucleic acid molecule, the compatibility profile goes down in the contact region without ligand molecule. This phenomenon becomes useful to monitor the binding sites of the bulky ligand such as nucleic acid and to model the protein-nucleic acid complex.

  4. Probing the acidic residue within the integrin binding site of laminin-511 that interacts with the metal ion-dependent adhesion site of α6β1 integrin.

    PubMed

    Taniguchi, Yukimasa; Li, Shaoliang; Takizawa, Mamoru; Oonishi, Eriko; Toga, Junko; Yagi, Emiko; Sekiguchi, Kiyotoshi

    2017-06-03

    Laminins are major cell-adhesive proteins of basement membranes that interact with integrins in a divalent cation-dependent manner. Laminin-511 consists of α5, β1, and γ1 chains, of which three laminin globular domains of the α5 chain (α5/LG1-3) and a Glu residue in the C-terminal tail of chain γ1 (γ1-Glu1607) are required for binding to integrins. However, it remains unsettled whether the Glu residue in the γ1 tail is involved in integrin binding by coordinating the metal ion in the metal ion-dependent adhesion site of β1 integrin (β1-MIDAS), or by stabilizing the conformation of α5/LG1-3. To address this issue, we examined whether α5/LG1-3 contain an acidic residue required for integrin binding that is as critical as the Glu residue in the γ1 tail; to achieve this, we undertook exhaustive alanine substitutions of the 54 acidic residues present in α5/LG1-3 of the E8 fragment of laminin-511 (LM511E8). Most of the alanine mutants possessed α6β1 integrin binding activities comparable with wild-type LM511E8. Alanine substitution for α5-Asp3198 and Asp3219 caused mild reduction in integrin binding activity, and that for α5-Asp3218 caused severe reduction, possibly resulting from conformational perturbation of α5/LG1-3. When α5-Asp3218 was substituted with asparagine, the resulting mutant possessed significant binding activity to α6β1 integrin, indicating that α5-Asp3218 is not directly involved in integrin binding through coordination with the metal ion in β1-MIDAS. Given that substitution of γ1-Glu1607 with glutamine nullified the binding activity to α6β1 integrin, these results, taken together, support the possibility that the critical acidic residue coordinating the metal ion in β1-MIDAS is Glu1607 in the γ1 tail, but no such residue is present in α5/LG1-3. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Characterization of epoxyeicosatrienoic acid binding site in U937 membranes using a novel radiolabeled agonist, 20-125i-14,15-epoxyeicosa-8(Z)-enoic acid.

    PubMed

    Yang, Wenqi; Tuniki, Venugopal Raju; Anjaiah, Siddam; Falck, J R; Hillard, Cecilia J; Campbell, William B

    2008-03-01

    Epoxyeicosatrienoic acids (EETs) are important regulators of vascular tone and homeostasis. Whether they initiate signaling through membrane receptors is unclear. We developed 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), a radiolabeled EET agonist, to characterize EET binding to membranes of U937 cells. 20-I-14,15-EE8ZE stimulated cAMP production in U937 cells with similar potency, but it decreased efficacy compared with 11,12-EET. Maximum cAMP production increased 4.2-fold, with an EC(50) value of 9 muM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC(50) value. Both 20-I-14,15-EE8ZE agonist activities were blocked by the EET antagonist 14,15-epoxyeicosa-5(Z)enoic acid (14,15-EE5ZE). Specific 20-(125)I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4 degrees C. The binding was saturable, reversible, and exhibited K(D) and B(max) values of 11.8 +/- 1.1 nM and 5.8 +/- 0.2 pmol/mg protein, respectively. Pretreatment of the membranes with guanosine 5'-O-(3-thio)triphosphate reduced the B(max) in a concentration-related manner. 20-(125)I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE approximately 14,15-EET > 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-(125)I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.

  6. Americium binding to humic acid.

    PubMed

    Peters, A J; Hamilton-Taylor, J; Tipping, E

    2001-09-01

    The binding of americium (Am) by peat humic acid (PHA) has been investigated at Am concentrations between 10(-1) and 10(-7) M at pH approximately 2.6 in the presence and absence of Cu as a competing ion. Cu-PHA binding was also investigated in order to derive independent binding constants for use in modeling the competitive binding studies. Humic ion-binding model VI was used to compare the acquired data with previously published binding data and to investigate the importance of high-affinity binding sites in metal-PHA binding. Am was not observed to bind to high-affinity, low-concentration binding sites. The model VI parameter deltaLK2 takes into accountthe small number of strong sites in PHA and was found to be important for Cu-PHA binding but not for Am-PHA binding, regardless of whether Cu was present. Analysis of the PHA sample revealed that it contained a considerable quantity of Fe not removed by the extraction procedure, much of which is believed to be present as Fe(III). Model VI was then used to investigate the possible importance of the presence of Fe(III) in the Am-PHA binding experiments. When Fe(III) was assumed to be present, improved descriptions of the data by model VI were obtained by assuming that all of the metals [Am, Cu, and Fe(III)] undergo strong binding. This highlights the importance of Fe(III) competition in metal-PHA binding studies and possible shortcomings in the extraction procedure used to extract PHA.

  7. Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1•Nup98)

    PubMed Central

    Quan, Beili; Seo, Hyuk-Soo; Blobel, Günter; Ren, Yi

    2014-01-01

    mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the β-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore. PMID:24927547

  8. Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1 • Nup98).

    PubMed

    Quan, Beili; Seo, Hyuk-Soo; Blobel, Günter; Ren, Yi

    2014-06-24

    mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the β-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

  9. Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1∙Nup98)

    SciTech Connect

    Quan, Beili; Seo, Hyuk-Soo; Blobel, Günter; Ren, Yi

    2014-07-01

    mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the β-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

  10. Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis

    SciTech Connect

    Feild, M.J.

    1988-01-01

    The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.

  11. Expression of non-angiotensin II -125I-CGP 42112 binding sites on activated microglia after kainic acid induced neurodegeneration.

    PubMed

    Jöhren, O; Viswanathan, M; Saavedra, J M

    1995-12-08

    [125I]CGP 42112, first developed to identify angiotensin II receptor subtype 2 (AT2), was recently shown to bind to a novel non-angiotensin binding site in injured rat brain tissue. We addressed the question whether non-angiotensin [125I]CGP 42112 binding appears after kainic acid induced hippocampal neurodegeneration, a process of neuronal cell death at a distance from the toxin injection site. After intraventricular kainic acid injection, we found non-angiotensin [125I]CGP 42112 binding in the hippocampal areas CA3 (4 and 14 days after injection), CA1 and CA4 and the subiculum (14 days after injection). In addition, 14 days after kainic acid injection, [125I]CGP 42112 binding was found in 50% of the animals, in the thalamus, amygdala and piriform cortex, areas receiving projections from the hippocampus and suffering kainic acid induced delayed neurodegeneration. The loss of neurons in these regions was accompanied by an accumulation of activated microglia as demonstrated by immunostaining with the specific antibodies OX-42 and ED1. The time course and regional pattern of OX-42/ED1 positive immunostaining was identical with the appearance and distribution of the non-angiotensin [125I]CGP 42112 binding site. The non-angiotensin [125I]CGP 42112 binding was not detected in brain regions unaffected by kainic acid injection. Our findings indicate the expression of a novel [125I]CGP 42112 binding site on activated microglia. This site appears at a distance from the lesion and may be of importance in the process of neuronal death and brain tissue repair.

  12. The different iron binding sites of bovine spleen purple acid phosphatase

    NASA Astrophysics Data System (ADS)

    Cichutek, Klaus; Witzel, Herbert; Parak, Fritz

    1988-02-01

    The purple acid phosphatase contains two inequivalent irons which can be removed subsequently. The Mössbauer spectrum of the purple inactive enzyme (oxidized) indicates two high spin ferric irons with antiparallel coupling giving zero effective spin. The active pink enzyme (partly reduced) is obtained by a one electron transfer to the iron which is less stable bound in the protein. The Mössbauer spectra indicate a high spin Fe(2+) antiparallel spin coupled to high spin Fe(3+).

  13. Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.

    PubMed

    Anguizola, Jeanethe; Debolt, Erin; Suresh, D; Hage, David S

    2016-05-15

    The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

  14. Efficient hydrolysis of cellulose over a novel sucralose-derived solid acid with cellulose-binding and catalytic sites.

    PubMed

    Hu, Shuanglan; Smith, Thomas John; Lou, Wenyong; Zong, Minhua

    2014-02-26

    A new sucralose-derived solid acid catalyst (SUCRA-SO3H), containing -Cl and -SO3H functional groups, has been shown to be highly effective for hydrolyzing β-1,4-glucans, completely hydrolyzing cellobiose (1) into glucose (2) in 3 h and converting the microcrystalline cellulose pretreated by the ionic liquid into glucose (2) with a yield of around 55% and a selectivity of 98% within 24 h at a relatively moderate temperature (393K). The enhanced adsorption capacity that the catalyst has for glucan by virtue of the presence of chloride groups that act as cellulose-binding sites offers the possibility of resolving the existing bottleneck in heterogeneous catalysis to hydrolyze cellulose, namely, the low accessibility of cellulose to the reaction position in typical solid catalysts. The apparent activation energy for hydrolysis of cellobiose (1) with SUCRA-SO3H was 94 kJ/mol, which was much lower than that with sulfuric acid (133 kJ/mol) and the corresponding sucrose-derived catalyst (SUCRO-SO3H) without chlorine groups (114 kJ/mol).

  15. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  16. Aspartic Acid 397 in Subunit B of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Forms Part of a Sodium-binding Site, Is Involved in Cation Selectivity, and Affects Cation-binding Site Cooperativity

    PubMed Central

    Shea, Michael E.; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

    2013-01-01

    The Na+-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC). PMID:24030824

  17. ETMB-RBF: Discrimination of Metal-Binding Sites in Electron Transporters Based on RBF Networks with PSSM Profiles and Significant Amino Acid Pairs

    PubMed Central

    Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

    2013-01-01

    Background Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation–reduction reactions. In these oxidation–reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. Methods We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. Results We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. Conclusions We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins

  18. ETMB-RBF: discrimination of metal-binding sites in electron transporters based on RBF networks with PSSM profiles and significant amino acid pairs.

    PubMed

    Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

    2013-01-01

    Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation-reduction reactions. In these oxidation-reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins.

  19. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    SciTech Connect

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2008-12-01

    A water network stabilizes the structure of cellular retionic acid binding protein II. The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the α2 helix at its entrance. This chain of interactions acts as a ‘pillar’ that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the α2 helix adopting an altered conformation compared with wild-type CRABPII.

  20. Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

    PubMed Central

    Tullius, M. V.; Vann, W. F.; Gibson, B. W.

    1999-01-01

    Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP. PMID:10091669

  1. All-trans-retinoic acid and retinol binding to the FA1 site of human serum albumin competitively inhibits heme-Fe(III) association.

    PubMed

    Di Muzio, Elena; Polticelli, Fabio; di Masi, Alessandra; Fanali, Gabriella; Fasano, Mauro; Ascenzi, Paolo

    2016-01-15

    Retinoids are a class of chemicals derived from vitamin A metabolism, playing important and diverse functions. Vitamin A, also named all-trans-retinol (all-trans-ROL), is coverted into two classes of biologically active retinoids, i.e. 11-cis-retinoids and acidic retinoids. Among acidic retinoids, all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid (9-cis-RA) represent the main metabolic products. Specific and aspecific proteins solubilize, protect, and detoxify retinoids in the extracellular environment. The retinoid binding protein 4 (RBP4), the epididymal retinoid-binding protein (ERBP), and the interphotoreceptor matrix retinoid-binding protein (IRBP) play a central role in ROL transport, whereas lipocalin-type prostaglandin D synthase (also named β-trace) and human serum albumin (HSA) transport preferentially all-trans-RA. Here, the modulatory effect of all-trans-RA and all-trans-ROL on ferric heme (heme-Fe(III)) binding to HSA is reported. All-trans-RA and all-trans-ROL binding to the FA1 site of HSA competitively inhibit heme-Fe(III) association. Docking simulations and local structural comparison of HSA with all-trans-RA- and all-trans-ROL-binding proteins support functional data indicating the preferential binding of all-trans-RA and all-trans-ROL to the FA1 site of HSA. Present results may be relevant in vivo, in fact HSA could act as a secondary carrier of retinoids in human diseases associated with reduced levels of RBP4 and IRBP. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Characterization of the N-Acetyl-5-neuraminic Acid-binding Site of the Extracytoplasmic Solute Receptor (SiaP) of Nontypeable Haemophilus influenzae Strain 2019

    SciTech Connect

    Johnston, Jason W.; Coussens, Nathan P.; Allen, Simon; Houtman, Jon C.D.; Turner, Keith H.; Zaleski, Anthony; Ramaswamy, S.; Gibson, Bradford W.; Apicella, Michael A.

    2012-11-14

    Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4 {angstrom} resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.

  3. Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analog†

    PubMed Central

    Chiara, David C.; Dostalova, Zuzana; Jayakar, Selwyn S.; Zhou, Xiaojuan; Miller, Keith W.; Cohen, Jonathan B.

    2012-01-01

    The γ-aminobutyric acid type-A receptor (GABAAR) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABAARs purified from bovine brain, [3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABAAR transmembrane domain at the interface between the β and α subunits. To further define GABAAR etomidate binding sites, we now use [3H]TDBzl-etomidate, an aryl diazirine with broader amino acid side-chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABAARs and obtain a more extensive identification of photolabeled GABAAR amino acids. [3H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [3H]azietomidate, while no photolabeling was detected of amino acids in the αM2 or βM2 helices that also border the etomidate binding site. The location of these photolabeled amino acids in GABAAR homology models derived from the recently solved structures of prokaryote (GLIC) or invertebrate (GluCl) homologs and the results of computational docking studies predict the orientation of [3H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABAAR intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [3H]TDBzl-etomidate and [3H]azietomidate also provides evidence of an homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABAAR. PMID:22243422

  4. Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs{sup +}-selective binding site

    SciTech Connect

    Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Shibazaki, Chie; Shimizu, Rumi; Yamada, Mitsugu; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

    2015-03-01

    The tertiary structure of a β-lactamase derived from the halobacterium Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr{sup 2+}-binding sites and one Cs{sup +}-binding site were discovered in the HaBLA molecule. Environmentally friendly absorbents are needed for Sr{sup 2+} and Cs{sup +}, as the removal of the radioactive Sr{sup 2+} and Cs{sup +} that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs{sup +} or Sr{sup 2+}. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P3{sub 1} using X-ray crystallography. Moreover, the locations of bound Sr{sup 2+} and Cs{sup +} ions were identified by anomalous X-ray diffraction. The location of one Cs{sup +}-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na{sup +} (90 mM Na{sup +}/10 mM Cs{sup +}). From an activity assay using isothermal titration calorimetry, the bound Sr{sup 2+} and Cs{sup +} ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs{sup +}-binding site provides important information that is useful for the design of artificial Cs{sup +}-binding sites that may be useful in the bioremediation of radioactive isotopes.

  5. Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog*♦

    PubMed Central

    Jayakar, Selwyn S.; Zhou, Xiaojuan; Chiara, David C.; Dostalova, Zuzana; Savechenkov, Pavel Y.; Bruzik, Karol S.; Dailey, William P.; Miller, Keith W.; Eckenhoff, Roderic G.; Cohen, Jonathan B.

    2014-01-01

    Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [3H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([3H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [3H]AziPm photoincorporation into GABAAR subunits maximally by ∼50%. When the amino acids photolabeled by [3H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [3H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [3H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[3H]mTFD-MPAB. The propofol-inhibitable [3H]AziPm photolabeling in the GABAAR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the

  6. Predicted metal binding sites for phytoremediation.

    PubMed

    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. Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site.

    PubMed

    Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Shibazaki, Chie; Shimizu, Rumi; Yamada, Mitsugu; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

    2015-03-01

    Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes.

  8. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    SciTech Connect

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2009-09-02

    The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the {alpha} 2 helix at its entrance. This chain of interactions acts as a 'pillar' that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the {alpha}2 helix adopting an altered conformation compared with wild-type CRABPII.

  9. Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

    PubMed Central

    Rey-Burusco, M. Florencia; Ibáñez-Shimabukuro, Marina; Gabrielsen, Mads; Franchini, Gisela R.; Roe, Andrew J.; Griffiths, Kate; Zhan, Bin; Cooper, Alan; Kennedy, Malcolm W.; Córsico, Betina; Smith, Brian O.

    2015-01-01

    Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein–ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male. PMID:26318523

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

    PubMed

    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.

  11. Cl-/Ca2+-dependent L-glutamate binding sites do not correspond to 2-amino-4-phosphonobutanoate-sensitive excitatory amino acid receptors.

    PubMed Central

    Fagg, G. E.; Lanthorn, T. H.

    1985-01-01

    A series of phosphono and phosphino analogues of glutamate were used to compare the pharmacological properties of (a) Cl-/Ca2+-dependent, 2-amino-4-phosphonobutanoate (AP4)-sensitive L-[3H]-glutamate binding sites in rat brain synaptic plasma membranes (SPMs) and (b) AP4-sensitive excitatory synaptic responses by use of electrophysiological techniques. In the presence of Cl- and Ca2+, L-[3H]-glutamate bound to SPMs with Kd 804 nM and Bmax 53 pmol mg-1 protein. The AP4-sensitive (Ki 7.3 microM) population of binding sites represented 61% of L-glutamate specifically bound. omega-Substituted analogues of AP4 were potent inhibitors of L-[3H]-glutamate binding (Ki values 2.4-38 microM), whereas N-substituted compounds or propionic acid derivatives were inactive. Experiments with AP4 alone and in combination with other analogues demonstrated that the primary target of all substances was the AP4-sensitive population of L-glutamate binding sites. In the hippocampal slice in vitro, AP4 antagonized lateral perforant path-evoked field potentials with an IC50 of 2.7 microM. In contrast to their actions at AP4-sensitive L-glutamate binding sites, all other compounds (except for the omega-carboxymethylphosphino analogue, IC50 19 microM) were weak or inactive as antagonists of this synaptic response (IC50 values greater than 100 microM). Inactive compounds which exhibited activity in the binding assay did not reverse the synaptic depressant effects of AP4, indicating that they were neither agonists nor antagonists at AP4-sensitive synapses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2998527

  12. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

    PubMed

    Kanje, Sara; Hober, Sophia

    2015-04-01

    Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

  13. Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

    PubMed

    Thuresson, E D; Lakkides, K M; Rieke, C J; Sun, Y; Wingerd, B A; Micielli, R; Mulichak, A M; Malkowski, M G; Garavito, R M; Smith, W L

    2001-03-30

    Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually

  14. Substitution in the murine nectin1 receptor of a single conserved amino acid at a position distal from the herpes simplex virus gD binding site confers high-affinity binding to gD.

    PubMed

    Menotti, Laura; Casadio, Rita; Bertucci, Carlo; Lopez, Marc; Campadelli-Fiume, Gabriella

    2002-06-01

    By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC'C" surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of beta-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD.

  15. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  16. A single site in human β-hexosaminidase A binds both 6-sulfate-groups on hexosamines and the sialic acid moiety of GM2 ganglioside

    PubMed Central

    Sharma, Rohita; Bukovac, Scott; Callahan, John; Mahuran, Don

    2010-01-01

    Human β-hexosaminidase A (Hex A) (αβ) is composed of two subunits whose primary structures are ~60% identical. Deficiency of either subunit results in severe neurological disease due to the storage of GM2 ganglioside; Tay–Sachs disease, α deficiency, and Sandhoff disease, β deficiency. Whereas both subunits contain active sites only the α-site can efficiently bind negatively charged 6-sulfated hexosamine substrates and GM2 ganglioside. We have recently identified the αArg424 as playing a critical role in the binding of 6-sulfate-containing substrates, and βAsp452 as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an αArg424Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[3H]-GM2 ganglioside complex as a substrate. The mutant showed a ~3-fold increase in its Km for the complex. Next a form of Hex B (ββ) containing a double mutation, βAspLeu453 AsnArg (duplicating the α-aligning sequences), was expressed. As compared to the wild type (WT), the mutant exhibited a >30-fold increase in its ability to hydrolyze a 6-sulfated substrate and was now able to hydrolyze GM2 ganglioside when the GM2 activator protein was replaced by sodium taurocholate. Thus, this α-site is critical for binding both types of negatively charge substrates. PMID:12527415

  17. Quercetin inhibits acid-sensing ion channels through a putative binding site in the central vestibular region.

    PubMed

    Mukhopadhyay, Mohona; Singh, Anurag; Sachchidanand, S; Bera, Amal Kanti

    2017-02-22

    Acid-sensing ion channels (ASICs) are associated with many pathophysiological processes, such as neuronal death during ischemic stroke, epileptic seizure and nociception. However, there is a dearth of ASIC-specific therapeutic blockers. Here we report that quercetin, a plant flavonoid, which is known for its neuroprotective effect, reversibly inhibits homomeric rat ASIC1a, ASIC2a and ASIC3 with an IC50 of about 2µM. Also, quercetin prevents low pH-induced intracellular calcium rise and cell death in HEK-293 cells, which have endogenous expression of ASIC1a and 2a. The inhibitory effect of quercetin on ASICs is not due to membrane perturbation, as it did not have any effect on other channels, like NMDA receptor, GABAA receptor and P2X4 receptor. Unlike quercetin, another flavonoid resveratrol had no effect on ASIC1a. Computational analysis revealed that quercetin binds to the channel in a cavity at the central vestibule, lined by several charged residues like Q276, R369, E373 and E416 in ASIC1a. Mutation of Arg369 to Ala or Glu416 to Gln abolished the inhibitory effect of quercetin on rat ASIC1a completely, while Glu373 to Gln showed reduced sensitivity. Our results raise the possibility of using quercetin for targeting ASICs in vivo.

  18. The role of a basic amino acid cluster in target site selection and non-specific binding of bZIP peptides to DNA.

    PubMed Central

    Metallo, S J; Paolella, D N; Schepartz, A

    1997-01-01

    The ability of a transcription factor to locate and bind its cognate DNA site in the presence of closely related sites and a vast array of non-specific DNA is crucial for cell survival. The CREB/ATF family of transcription factors is an important group of basic region leucine zipper (bZIP) proteins that display high affinity for the CRE site and low affinity for the closely related AP-1 site. Members of the CREB/ATF family share in common a cluster of basic amino acids at the N-terminus of their bZIP element. This basic cluster is necessary and sufficient to cause the CRE site to bend upon binding of a CREB/ATF protein. The possibility that DNA bending and CRE/AP-1 specificity were linked in CREB/ATF proteins was investigated using chimeric peptides derived from human CRE-BP1 (a member of the CREB/ATF family) and yeast GCN4, which lacks both a basic cluster and CRE/AP-1 specificity. Gain of function and loss of function experiments demonstrated that the basic cluster was not responsible for the CRE/AP-1 specificity displayed by all characterized CREB/ATF proteins. The basic cluster was, however, responsible for inducing very high affinity for non- specific DNA. It was further shown that basic cluster-containing peptides bind non-specific DNA in a random coil conformation. We postulate that the high non- specific DNA affinities of basic cluster-containing peptides result from cooperative electrostatic interactions with the phosphate backbone that do not require peptide organization. PMID:9224594

  19. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    PubMed

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  20. Crystal Structures of the Staphylococcal Toxin SSL5 in Complex With Sialyl-Lewis X Reveal a Conserved Binding Site That Shares Common Features With Viral And Bacterial Sialic Acid-Binding Proteins

    SciTech Connect

    Baker, H.M.; Basu, I.; Chung, M.C.; Caradoc-Davies, T.; Fraser, J.D.; Baker, E.N.

    2009-06-02

    Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (Fc alphaRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe(X)), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5-sLe(X) complex at resolutions of 1.65 and 2.75 A for crystals at two pH values. In both structures, sLe(X) bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.

  1. Crystal structures of the staphylococcal toxin SSL5 in complex with sialyl Lewis X reveal a conserved binding site that shares common features with viral and bacterial sialic acid binding proteins.

    PubMed

    Baker, Heather M; Basu, Indira; Chung, Matthew C; Caradoc-Davies, Tom; Fraser, John D; Baker, Edward N

    2007-12-14

    Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (Fc alphaRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe(X)), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5-sLe(X) complex at resolutions of 1.65 and 2.75 A for crystals at two pH values. In both structures, sLe(X) bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.

  2. Data of protein-RNA binding sites.

    PubMed

    Lee, Wook; Park, Byungkyu; Choi, Daesik; Han, Kyungsook

    2017-02-01

    Despite the increasing number of protein-RNA complexes in structure databases, few data resources have been made available which can be readily used in developing or testing a method for predicting either protein-binding sites in RNA sequences or RNA-binding sites in protein sequences. The problem of predicting protein-binding sites in RNA has received much less attention than the problem of predicting RNA-binding sites in protein. The data presented in this paper are related to the article entitled "PRIdictor: Protein-RNA Interaction predictor" (Tuvshinjargal et al. 2016) [1]. PRIdictor can predict protein-binding sites in RNA as well as RNA-binding sites in protein at the nucleotide- and residue-levels. This paper presents four datasets that were used to test four prediction models of PRIdictor: (1) model RP for predicting protein-binding sites in RNA from protein and RNA sequences, (2) model RaP for predicting protein-binding sites in RNA from RNA sequence alone, (3) model PR for predicting RNA-binding sites in protein from protein and RNA sequences, and (4) model PaR for predicting RNA-binding sites in protein from protein sequence alone. The datasets supplied in this article can be used as a valuable resource to evaluate and compare different methods for predicting protein-RNA binding sites.

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

    PubMed

    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.

  4. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site. [R

    SciTech Connect

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

    1986-01-01

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

  5. A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

    PubMed

    Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

    2014-09-01

    Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

  6. Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress

    PubMed Central

    Mira, Nuno P.; Henriques, Sílvia F.; Keller, Greg; Teixeira, Miguel C.; Matos, Rute G.; Arraiano, Cecília M.; Winge, Dennis R.; Sá-Correia, Isabel

    2011-01-01

    The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5′-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3′. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (KD of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (KD of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (KD of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5′-(G/C)(A/C)GG(G/C)G-3′. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed. PMID:21586585

  7. Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

    SciTech Connect

    Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.; Yu, Zhanqian; Powers, Evan T.; Kelly, Jeffery W.; Lieberman, Raquel L.

    2013-03-07

    Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

  8. Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95.

    PubMed

    Cousins, Sarah L; Stephenson, F Anne

    2012-04-13

    N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.

  9. Identification of N-Methyl-d-aspartic Acid (NMDA) Receptor Subtype-specific Binding Sites That Mediate Direct Interactions with Scaffold Protein PSD-95*

    PubMed Central

    Cousins, Sarah L.; Stephenson, F. Anne

    2012-01-01

    N-methyl-d-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149–1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins. PMID:22375001

  10. Identification of protein-protein binding sites by incorporating the physicochemical properties and stationary wavelet transforms into pseudo amino acid composition.

    PubMed

    Jia, Jianhua; Liu, Zi; Xiao, Xuan; Liu, Bingxiang; Chou, Kuo-Chen

    2016-09-01

    With the explosive growth of protein sequences entering into protein data banks in the post-genomic era, it is highly demanded to develop automated methods for rapidly and effectively identifying the protein-protein binding sites (PPBSs) based on the sequence information alone. To address this problem, we proposed a predictor called iPPBS-PseAAC, in which each amino acid residue site of the proteins concerned was treated as a 15-tuple peptide segment generated by sliding a window along the protein chains with its center aligned with the target residue. The working peptide segment is further formulated by a general form of pseudo amino acid composition via the following procedures: (1) it is converted into a numerical series via the physicochemical properties of amino acids; (2) the numerical series is subsequently converted into a 20-D feature vector by means of the stationary wavelet transform technique. Formed by many individual "Random Forest" classifiers, the operation engine to run prediction is a two-layer ensemble classifier, with the 1st-layer voting out the best training data-set from many bootstrap systems and the 2nd-layer voting out the most relevant one from seven physicochemical properties. Cross-validation tests indicate that the new predictor is very promising, meaning that many important key features, which are deeply hidden in complicated protein sequences, can be extracted via the wavelets transform approach, quite consistent with the facts that many important biological functions of proteins can be elucidated with their low-frequency internal motions. The web server of iPPBS-PseAAC is accessible at http://www.jci-bioinfo.cn/iPPBS-PseAAC , by which users can easily acquire their desired results without the need to follow the complicated mathematical equations involved.

  11. Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

    PubMed Central

    Ahmed, Ahmed H.; Oswald, Robert E.

    2010-01-01

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

  12. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    PubMed

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  13. An alternate binding site for PPARγ ligands

    PubMed Central

    Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

    2014-01-01

    PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

  14. LIBRA: LIgand Binding site Recognition Application.

    PubMed

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

    2015-12-15

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

  15. Antigenic Variation of Clade 2.1 H5N1 Virus Is Determined by a Few Amino Acid Substitutions Immediately Adjacent to the Receptor Binding Site

    PubMed Central

    Koel, Björn F.; van der Vliet, Stefan; Burke, David F.; Bestebroer, Theo M.; Bharoto, Eny E.; Yasa, I. Wayan W.; Herliana, Inna; Laksono, Brigitta M.; Xu, Kemin; Skepner, Eugene; Russell, Colin A.; Rimmelzwaan, Guus F.; Perez, Daniel R.; Osterhaus, Albert D. M. E.; Smith, Derek J.; Prajitno, Teguh Y.

    2014-01-01

    ABSTRACT Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition. PMID:24917596

  16. Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA.

    PubMed

    Hatamoto, Masashi; Nakai, Kazufumi; Ohashi, Akiyoshi; Imachi, Hiroyuki

    2009-10-01

    Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 microM of the peptide-PNAs, cell-free ss-galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate.

  17. Circannual variations in the binding of [3H]lysergic acid diethylamide to serotonin2A receptors and of [3H]paroxetine to serotonin uptake sites in platelets from healthy volunteers.

    PubMed

    Spigset, O; Allard, P; Mjörndal, T

    1998-05-15

    Circannual variations occur in several serotonergic parameters, including platelet serotonin uptake and platelet [3H]imipramine binding. Binding of [3H]lysergic acid diethylamide ([3H]LSD) to platelet serotonin (5-HT)2A receptors and binding of [3H]paroxetine to platelet serotonin uptake sites were studied longitudinally for 1 year in 12 healthy volunteers. For [3H]LSD, the number of binding sites (Bmax) showed no significant seasonal variation (two-way analysis of variance), although Bmax was significantly higher during the months October through February than during the months April through August (32.6 vs. 29.8 fmol/mg protein; p = .015). For [3H]paroxetine, Bmax showed a significant seasonal variation (p = .003) with maximum in August (1322 fmol/mg protein) and minimum in February (1168 fmol/mg protein). The affinity constant (Kd) showed a significant seasonal variation for [3H]LSD binding (p = .046), but not for [3H]paroxetine binding. The seasonal fluctuations in [3H]LSD binding and in paroxetine binding tended to be inversely correlated for Bmax (r = -.70; p = .08) and were significantly negatively correlated for Kd (r = -.88; p = .009). The present study demonstrates a seasonal effect on platelet serotonin uptake site binding and indicates a possible seasonal effect on 5-HT2A receptor binding. The results imply that circannual fluctuations should be taken into account when these platelet serotonin markers are studied.

  18. Chloride binding site of neurotransmitter sodium symporters

    PubMed Central

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

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion 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. PMID:23641004

  19. Chloride binding site of neurotransmitter sodium symporters.

    PubMed

    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.

  20. (/sup 3/)tetrahydrotrazodone binding. Association with serotonin binding sites

    SciTech Connect

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-05-01

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

  1. The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

    PubMed Central

    Santos, Kira L.; Vento, Megan A; Wright, John W.; Speth, Robert C.

    2013-01-01

    A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 μM) and PD123319 (10 μM) plus 100 μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50’s ~1–20 μM This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1 mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [1] near the active site of neurolysin. It is proposed that the

  2. The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin.

    PubMed

    Santos, Kira L; Vento, Megan A; Wright, John W; Speth, Robert C

    2013-06-10

    A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 μM) and PD123319 (10 μM) plus 100 μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1-20 μM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the

  3. Ethylene binding site affinity in ripening apples

    SciTech Connect

    Blankenship, S.M. . Dept. of Horticultural Science); Sisler, E.C. )

    1993-09-01

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

  4. Determination of binding affinities of retinoids to retinoic acid-binding protein and serum albumin

    PubMed Central

    Sani, Brahma P.; Titus, Belinda C.; Banerjee, Chandra K.

    1978-01-01

    Binding affinities of retinoic acid and its synthetic analogues to intracellular retinoic acid-binding protein, which is a possible candidate for mediating their biological function, and to serum albumin, the plasma transport protein, were evaluated. A quantitative method involving elimination of interfering serum albumin by immunoprecipitation was developed to measure the binding efficiency of these retinoids, some of which are active in modifying epithelial differentiation and preventing tumorigenesis. Two cyclopentenyl analogues of retinoic acid and 13-cis-retinoic acid showed, like retinoic acid, a binding efficiency of 100% for the cellular binding protein. With the phenyl, dichlorophenyl and trimethylmethoxyphenyl analogues of retinoic acid, the binding efficiency increased as the substituents on the aromatic ring increased; thus the trimethylmethoxyphenyl analogue binds almost as efficiently as retinoic acid itself. However, the trimethylmethoxyphenyl analogue with a sulphur atom on the side chain has a much decreased binding affinity. The correlation noticed between the binding efficiency of these retinoids and their biological activity in differentiation and/or in the control of tumorigenesis particularly enhances the confidence in the present method of determining the relative binding efficiencies. None of the vitamins, hormones and cofactors tested, showed appreciable affinity for the retinoic acid-binding site. Studies on binding of retinoic acid and its analogues to serum albumin indicate that no correlation exists between binding affinity for albumin and their biological potency. PMID:666734

  5. Localization of the chaperone binding site

    NASA Technical Reports Server (NTRS)

    Boyle, D.; Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.

  6. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    SciTech Connect

    Taguchi, J.; Kuriyama, K. )

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  7. Identification of second arginine-glycine-aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection.

    PubMed

    T, Prasada Rao; T, Lakshmi Prasanth; R, Parvathy; S, Murugavel; Devi, Karuna; Joshi, Paritosh

    2017-02-02

    Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix interacts with complement C9. This interaction may modulate innate immunity. Details of Vn-C9 interaction are limited. An assessment of Vn-C9 interaction was made employing goat homologous system. Vn binding to C9 was observed in three different assays. Using recombinant fragments, the C9 binding was mapped to the N-terminus of Vn. Site directed mutagenesis was performed to alter the second RGD sequence (RGD-2) of Vn. Change of R to G or D to A in RGD-2 caused significant decrease in Vn binding to C9 whereas change of R to G in the first RGD motif (RGD-1) had no effect on Vn binding to C9. These results imply that the RGD-2 of goat Vn is involved in C9 binding. In competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 in terms of bacterial pathogenesis was also evaluated. Serum dependent inhibition of E. coli growth was significantly reverted when Vn or its N-fragment were included in the assay. The C-fragment, which did not support C9 binding, also partly nullified serum dependent inhibition of bacterial growth probably through other serum component(s).

  8. Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase.

    PubMed

    Rangaswamy, Udaya S; Wang, Weijia; Cheng, Xing; McTamney, Patrick; Carroll, Danielle; Jin, Hong

    2017-08-15

    Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses.IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the

  9. alpha 2-Macroglobulin binding to cultured fibroblasts. Solubilization and partial purification of binding sites.

    PubMed

    Hanover, J A; Cheng, S; Willingham, M C; Pastan, I H

    1983-01-10

    Binding sites having the characteristics of receptors for "activated" alpha 2-macroglobulin (alpha 2M) have been solubilized with octyl-beta-D-glucoside from fibroblast membranes. When the detergent was removed by dialysis, the resulting insoluble extract was shown to bind 125I-alpha 2M specifically. Analysis of the binding data using a nonlinear curve-fitting program suggests that the solubilized preparation contains two classes of binding sites (KD = 0.34 nM and KD = 104 nM). Membranes or solubilized extracts from KB cells which lack alpha 2M binding sites did not specifically bind 125I-alpha 2M. The solubilized binding sites from fibroblasts were inactivated by boiling and trypsin treatment, and required Ca+2 for maximal binding. In addition, the high affinity binding of 125I-alpha 2M to the solubilized receptor was inhibited by bacitracin and by alpha-bromo-5-iodo-4-hydroxy-3-nitroacetophenone, two agents which interfere with the uptake of alpha 2M in cultured fibroblasts. Using a combination of ion exchange and gel permeation chromatography, we have purified the high affinity alpha 2M binding site approximately 100-fold from membrane derived from NIH-3T3 (spontaneously transformed) fibroblasts grown as tumors in mice. The receptor is apparently an acidic protein and the receptor octyl-beta-D-glucoside complex has a Stokes radius of 45-50 A as measured by gel filtration.

  10. A citrate-binding site in calmodulin.

    PubMed

    Neufeld, T; Eisenstein, M; Muszkat, K A; Fleminger, G

    1998-01-01

    Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low-affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg106-His107. Owing to the tight proximity of this site to the third Ca(2+)-binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein.

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

    PubMed

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

    2017-02-17

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

  12. Action at a distance: amino acid substitutions that affect binding of the phosphorylated CheY response regulator and catalysis of dephosphorylation can be far from the CheZ phosphatase active site.

    PubMed

    Freeman, Ashalla M; Mole, Beth M; Silversmith, Ruth E; Bourret, Robert B

    2011-09-01

    Two-component regulatory systems, in which phosphorylation controls the activity of a response regulator protein, provide signal transduction in bacteria. For example, the phosphorylated CheY response regulator (CheYp) controls swimming behavior. In Escherichia coli, the chemotaxis phosphatase CheZ stimulates the dephosphorylation of CheYp. CheYp apparently binds first to the C terminus of CheZ and then binds to the active site where dephosphorylation occurs. The phosphatase activity of the CheZ(2) dimer exhibits a positively cooperative dependence on CheYp concentration, apparently because the binding of the first CheYp to CheZ(2) is inhibited compared to the binding of the second CheYp. Thus, CheZ phosphatase activity is reduced at low CheYp concentrations. The CheZ21IT gain-of-function substitution, located far from either the CheZ active site or C-terminal CheY binding site, enhances CheYp binding and abolishes cooperativity. To further explore mechanisms regulating CheZ activity, we isolated 10 intragenic suppressor mutations of cheZ21IT that restored chemotaxis. The suppressor substitutions were located along the central portion of CheZ and were not allele specific. Five suppressor mutants tested biochemically diminished the binding of CheYp and/or the catalysis of dephosphorylation, even when the suppressor substitutions were distant from the active site. One suppressor mutant also restored cooperativity to CheZ21IT. Consideration of results from this and previous studies suggests that the binding of CheYp to the CheZ active site (not to the C terminus) is rate limiting and leads to cooperative phosphatase activity. Furthermore, amino acid substitutions distant from the active site can affect CheZ catalytic activity and CheYp binding, perhaps via the propagation of structural or dynamic perturbations through a helical bundle.

  13. Synthesis and characterization of water-soluble silk peptides and recombinant silk protein containing polyalanine, the integrin binding site, and two glutamic acids at each terminal site as a possible candidate for use in bone repair materials.

    PubMed

    Asakura, Tetsuo; Suzuki, Yu; Nagano, Aya; Knight, David; Kamiya, Masakatsu; Demura, Makoto

    2013-10-14

    The recombinant proteins [EE(A)12EETGRGDSPAAS]n (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E2(A)nE2TGRGDSPAE2(A)nE2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO(-) to COOH of the E2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)12EETGRGDSPAAS]n (n = 5,10) by expression in Escherichia coli . The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)12EETGRGDSPAAS]10 has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material.

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

    PubMed

    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.

  15. Serotonin binding sites of human blood platelets

    SciTech Connect

    Kim, B.K.; Steiner, M.; Baldini, M.G.

    1980-07-15

    The possible use of formaldehyde-fixed platelets to characterize and enumerate the specific receptor sites for 5-hydroxytryptamine was investigated. Equilibrium, pH-dependent capacity and specificity of 5-hydroxytryptamine binding by formaldehyde-fixed platelets were demonstrated. Analysis of binding data revealed two different sites: (1) high affinity with low capacity, and (2) low affinity with high capacity. The results of binding studies using nonfixed control platelets were comparable with those of formaldehyde-fixed platelets. The versatility of formaldehyde fixation for studies of surface receptors was also shown by demonstrating nearly equal binding affinity for PGE/sub 1/ in control and formaldehyde-treated platelets. Our results indicate that formaldehyde fixation is a useful tool for the study of membrane receptor sites especially when active transport of the ligand such as serotonin is a problem.

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

    PubMed

    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.

  17. Influence of sulfhydryl sites on metal binding by bacteria

    NASA Astrophysics Data System (ADS)

    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

  18. Predicting Ca(2+)-binding sites in proteins.

    PubMed

    Nayal, M; Di Cera, E

    1994-01-18

    The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins.

  19. Prediction of FAD binding sites in electron transport proteins according to efficient radial basis function networks and significant amino acid pairs.

    PubMed

    Le, Nguyen-Quoc-Khanh; Ou, Yu-Yen

    2016-07-30

    Cellular respiration is a catabolic pathway for producing adenosine triphosphate (ATP) and is the most efficient process through which cells harvest energy from consumed food. When cells undergo cellular respiration, they require a pathway to keep and transfer electrons (i.e., the electron transport chain). Due to oxidation-reduction reactions, the electron transport chain produces a transmembrane proton electrochemical gradient. In case protons flow back through this membrane, this mechanical energy is converted into chemical energy by ATP synthase. The convert process is involved in producing ATP which provides energy in a lot of cellular processes. In the electron transport chain process, flavin adenine dinucleotide (FAD) is one of the most vital molecules for carrying and transferring electrons. Therefore, predicting FAD binding sites in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. We used an independent data set to evaluate the performance of the proposed method, which had an accuracy of 69.84 %. We compared the performance of the proposed method in analyzing two newly discovered electron transport protein sequences with that of the general FAD binding predictor presented by Mishra and Raghava and determined that the accuracy of the proposed method improved by 9-45 % and its Matthew's correlation coefficient was 0.14-0.5. Furthermore, the proposed method enabled reducing the number of false positives significantly and can provide useful information for biologists. We developed a method that is based on PSSM profiles and SAAPs for identifying FAD binding sites in newly discovered electron transport protein sequences. This approach achieved a significant improvement after we added SAAPs to PSSM features to analyze FAD binding proteins in the electron transport chain. The proposed method can serve as an effective tool for predicting FAD binding sites in electron

  20. Electrostatic Steering at Acetylcholine Binding Sites

    PubMed Central

    Meltzer, Robert H.; Thompson, Errol; Soman, Kizhake V.; Song, Xing-Zhi; Ebalunode, Jerry O.; Wensel, Theodore G.; Briggs, James M.; Pedersen, Steen E.

    2006-01-01

    The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+-chelates revealed net potentials of −20 mV at the nAChR agonist sites and −14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the αδ-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be −14 mV; the calculated potential at the αγ-site was approximately threefold stronger than at the αδ-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the αγ-site of the nAChR. We conclude that long-range electrostatic interactions contribute −0.3 and −1 kcal/mol to the binding energy at the nAChR αδ- and αγ-sites due to an increase in association rates. PMID:16751247

  1. Electrostatic steering at acetylcholine binding sites.

    PubMed

    Meltzer, Robert H; Thompson, Errol; Soman, Kizhake V; Song, Xing-Zhi; Ebalunode, Jerry O; Wensel, Theodore G; Briggs, James M; Pedersen, Steen E

    2006-08-15

    The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.

  2. Mapping the binding site pocket of the serotonin 5-Hydroxytryptamine2A receptor. Ser3.36(159) provides a second interaction site for the protonated amine of serotonin but not of lysergic acid diethylamide or bufotenin.

    PubMed

    Almaula, N; Ebersole, B J; Zhang, D; Weinstein, H; Sealfon, S C

    1996-06-21

    Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.

  3. Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function

    PubMed Central

    Desai, Aditya J.; Dong, Maoqing; Harikumar, Kaleeckal G.

    2015-01-01

    Dysfunction of the type 1 cholecystokinin (CCK) receptor (CCK1R) as a result of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore the possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that, similar to the impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had a negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes but was protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant Y140A was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in a high-cholesterol environment. This mechanism may contribute to the beneficial effect of ursodeoxycholic acid in reducing cholesterol gallstone formation. PMID:26138469

  4. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

    In this issue of Molecular Pharmacology, Tränkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tränkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tränkle et al. should be of considerable general interest.

  5. Bridging lectin binding sites by multivalent carbohydrates.

    PubMed

    Wittmann, Valentin; Pieters, Roland J

    2013-05-21

    Carbohydrate-protein interactions are involved in a multitude of biological recognition processes. Since individual protein-carbohydrate interactions are usually weak, multivalency is often required to achieve biologically relevant binding affinities and selectivities. Among the possible mechanisms responsible for binding enhancement by multivalency, the simultaneous attachment of a multivalent ligand to several binding sites of a multivalent receptor (i.e. chelation) has been proven to have a strong impact. This article summarizes recent examples of chelating lectin ligands of different size. Covered lectins include the Shiga-like toxin, where the shortest distance between binding sites is ca. 9 Å, wheat germ agglutinin (WGA) (shortest distance between binding sites 13-14 Å), LecA from Pseudomonas aeruginosa (shortest distance 26 Å), cholera toxin and heat-labile enterotoxin (shortest distance 31 Å), anti-HIV antibody 2G12 (shortest distance 31 Å), concanavalin A (ConA) (shortest distance 72 Å), RCA120 (shortest distance 100 Å), and Erythrina cristagalli (ECL) (shortest distance 100 Å). While chelating binding of the discussed ligands is likely, experimental proof, for example by X-ray crystallography, is limited to only a few cases.

  6. Phosphate binding sites identification in protein structures

    PubMed Central

    Parca, Luca; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Ausiello, Gabriele

    2011-01-01

    Nearly half of known protein structures interact with phosphate-containing ligands, such as nucleotides and other cofactors. Many methods have been developed for the identification of metal ions-binding sites and some for bigger ligands such as carbohydrates, but none is yet available for the prediction of phosphate-binding sites. Here we describe Pfinder, a method that predicts binding sites for phosphate groups, both in the form of ions or as parts of other non-peptide ligands, in proteins of known structure. Pfinder uses the Query3D local structural comparison algorithm to scan a protein structure for the presence of a number of structural motifs identified for their ability to bind the phosphate chemical group. Pfinder has been tested on a data set of 52 proteins for which both the apo and holo forms were available. We obtained at least one correct prediction in 63% of the holo structures and in 62% of the apo. The ability of Pfinder to recognize a phosphate-binding site in unbound protein structures makes it an ideal tool for functional annotation and for complementing docking and drug design methods. The Pfinder program is available at http://pdbfun.uniroma2.it/pfinder. PMID:20974634

  7. Binding of [(3)H]lysergic acid diethylamide to serotonin 5-HT(2A) receptors and of [(3)H]paroxetine to serotonin uptake sites in platelets from healthy children, adolescents and adults.

    PubMed

    Sigurdh, J; Spigset, O; Allard, P; Mjörndal, T; Hägglöf, B

    1999-11-01

    Possible age effects on binding of [(3)H]lysergic acid diethylamide ([(3)H]LSD) to serotonin 5-HT(2A) receptors and of [(3)H]paroxetine to serotonin uptake sites were studied in platelets from healthy children (11-12 years of age), adolescents (16-17 years of age) and adults. Significant overall age effects were found both for the number of binding sites (B(max)) for [(3)H]LSD binding (p < 0.001), the affinity constant (K(d)) for [(3)H]LSD binding (p < 0.001), B(max) for [(3)H]paroxetine binding (p < 0.001) and K(d) for [(3)H] paroxetine binding (p = 0.006). In general, there was a decrease in B(max) with increasing age, which predominantly occurred between the ages 11-12 years and 16-17 years for the 5-HT(2A) receptor, and after 16-17 years of age for the serotonin uptake site. These developmental changes might have an impact on the effect of treatment with serotonergic drugs in children and adolescents. When the platelet serotonin variables investigated are employed in studies in children or adolescents, age matching or, alternatively, introduction of age control in the statistical analysis should be performed.

  8. Interactions between Sindbis virus RNAs and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding site.

    PubMed Central

    Weiss, B; Geigenmüller-Gnirke, U; Schlesinger, S

    1994-01-01

    In previous studies of encapsidation of Sindbis virus RNA, we identified a 570nt fragment (nt 684-1253) from the 12 kb genome that binds to the viral capsid protein with specificity and is required for packaging of Sindbis virus defective interfering RNAs. We now show that the capsid binding activity resides in a highly structured 132nt fragment (nt 945-1076). We had also demonstrated that a 68 amino acid peptide derived from the capsid protein retained most of the binding activity of the original protein and have now developed an RNA mobility shift assay with this peptide fused to glutathione-S-transferase. We have used this assay in conjunction with the original assay in which the intact capsid protein was immobilized on nitrocellulose to analyze more extensive deletions in the 132-mer. All of the deletions led to a reduction in binding, but the binding of a 5' 67-mer was enhanced by the addition of nonspecific flanking sequences. This result suggests that the stability of a particular structure within the 132nt sequence may be important for capsid recognition. Images PMID:8139918

  9. Specificity of Auxin-binding Sites on Maize Coleoptile Membranes as Possible Receptor Sites for Auxin Action 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Dissociation coefficients of auxin-binding sites on maize (Zea mays L.) coleoptile membranes were measured, for 48 auxins and related ring compounds, by competitive displacement of 14C-naphthaleneacetic acid from the binding sites. The sites bind with high affinity several ring compounds with acidic side chains 2 to 4 carbons long, and much more weakly bind neutral ring compounds and phenols related to these active acids, most phenoxyalkylcarboxylic acids, and arylcarboxylic acids except benzoic acid, which scarcely binds, and triiodobenzoic acids, which bind strongly. Specificity of the binding is narrowed in the presence of a low molecular weight “supernatant factor” that occurs in maize and other tissues. Activity of many of the analogs as auxin agonists or antagonists in the cell elongation response was determined with maize coleoptiles. These activities on the whole roughly parallel the affinities of the binding sites for the same compounds, especially affinities measured in the presence of supernatant factor, but there are some quantitative discrepancies, especially among phenoxyalkylcarboxylic acids. In view of several factors that can cause receptor affinity and biological activity values to diverge quantitatively among analogs, the findings appear to support the presumption that the auxin-binding sites may be receptors for auxin action. PMID:16660143

  10. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β− Interface*

    PubMed Central

    Jayakar, Selwyn S.; Zhou, Xiaojuan; Savechenkov, Pavel Y.; Chiara, David C.; Desai, Rooma; Bruzik, Karol S.; Miller, Keith W.; Cohen, Jonathan B.

    2015-01-01

    In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ+-β− subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β+-α− subunit interfaces. GABA inhibits S-[3H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[3H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ+-β− site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators. PMID:26229099

  11. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.

    PubMed

    Jayakar, Selwyn S; Zhou, Xiaojuan; Savechenkov, Pavel Y; Chiara, David C; Desai, Rooma; Bruzik, Karol S; Miller, Keith W; Cohen, Jonathan B

    2015-09-18

    In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343-19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ(+)-β(-) subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β(+)-α(-) subunit interfaces. GABA inhibits S-[(3)H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2-15') in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[(3)H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ(+)-β(-) site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators. © 2015 by The American Society for Biochemistry and

  12. Derivatives of cinnamic acid interact with the nucleotide binding site of mitochondrial aldehyde dehydrogenase. Effects on the dehydrogenase reaction and stimulation of esterase activity by nucleotides.

    PubMed

    Poole, R C; Bowden, N J; Halestrap, A P

    1993-04-22

    A wide variety of cinnamic acid derivatives are inhibitors of the low Km mitochondrial aldehyde dehydrogenase. Two of the most potent inhibitors are alpha-cyano-3,4-dihydroxythiocinnamamide (Ki0.6 microM) and alpha-cyano-3,4,5-trihydroxycinnamonitrile (Ki2.6 microM). With propionaldehyde as substrate the inhibition by these compounds was competitive with respect to NAD+. alpha-Fluorocinnamate was a much less effective inhibitor of the enzyme, with mixed behaviour towards NAD+, but with a major competitive component. These cinnamic acid derivatives were ineffective as inhibitors of the aldehyde dehydrogenase-catalysed hydrolysis of p-nitrophenyl acetate, but inhibited the ability of NAD+ and NADH to activate this activity. Inhibition of the stimulation of esterase activity was competitive with respect to NAD+ and NADH, and the derived Ki values were the same as for inhibition of dehydrogenase activity. NAD+, but not acetaldehyde, could elute the low Km aldehyde dehydrogenase from alpha-cyanocinnamate-Sepharose, to which the enzyme binds specifically (Poole RC and Halestrap AP, Biochem J 259: 105-110, 1989). The cinnamic acid derivatives have little effect on lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase or a high Km aldehyde dehydrogenase present in rat liver mitochondria. It is concluded that some cinnamic acid derivatives are potent inhibitors of the low Km aldehyde dehydrogenase, by competing with NAD+/NADH for binding to the enzyme. They are much less effective as inhibitors of other NAD(+)-dependent dehydrogenases.

  13. Regulation of the Function of the Human ABCG2 Multidrug Transporter by Cholesterol and Bile Acids: Effects of Mutations in Potential Substrate and Steroid Binding Sites

    PubMed Central

    Telbisz, Ágnes; Hegedüs, Csilla; Váradi, András; Sarkadi, Balázs

    2014-01-01

    ABCG2 (ATP-binding cassette, subfamily G, member 2) is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity, and by examining purified ABCG2 reconstituted in proteoliposomes we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol dependence, but changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555–558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Regarding the effect of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, which did not significantly affect substrate-stimulated ATPase activity. These ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate were not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacologic modulation of ABCG2 function. PMID:24384916

  14. Determination of the Substrate Binding Mode to the Active Site Iron of (S)-2-Hydroxypropylphosphonic Acid Epoxidase Using 17O-Enriched Substrates and Substrate Analogues†

    PubMed Central

    Yan, Feng; Moon, Sung-Ju; Liu, Pinghua; Zhao, Zongbao; Lipscomb, John D.; Liu, Aimin; Liu, Hung-wen

    2009-01-01

    (S)-2-hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio-and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogs designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analog compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogs lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron

  15. Radiation inactivation reveals discrete cation binding sites that modulate dihydropyridine binding sites

    SciTech Connect

    Bolger, G.T.; Skolnick, P.; Kempner, E.S. )

    1989-08-01

    In low ionic strength buffer (5 mM Tris.HCl), the binding of (3H) nitrendipine to dihydropyridine calcium antagonist binding sites of mouse forebrain membranes is increased by both Na{sup +} and Ca{sup 2+}. Radiation inactivation was used to determine the target size of ({sup 3}H)nitrendipine binding sites in 5 mM Tris.HCl buffer, in the presence and absence of these cations. After irradiation, ({sup 3}H) nitrendipine binding in buffer with or without Na+ was diminished, due to a loss of binding sites and also to an increase in Kd. After accounting for radiation effects on the dissociation constant, the target size for the nitrendipine binding site in buffer was 160-170 kDa and was 170-180 kDa in the presence of sodium. In the presence of calcium ions, ({sup 3}H)nitrendipine binding showed no radiation effects on Kd and yielded a target size of 150-170 kDa. These findings suggest, as in the case of opioid receptors, the presence of high molecular weight membrane components that modulate cation-induced alterations in radioligand binding to dihydropyridine binding sites.

  16. Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.

    PubMed

    Futaki, S; Ueno, H; Martinez del Pozo, A; Pospischil, M A; Manning, J M; Ringe, D; Stoddard, B; Tanizawa, K; Yoshimura, T; Soda, K

    1990-12-25

    In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild

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

    PubMed Central

    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. PMID:26540053

  18. 5-(Piperidin-4-yl)-3-hydroxypyrazole: A novel scaffold for probing the orthosteric γ-aminobutyric acid type A receptor binding site.

    PubMed

    Krall, Jacob; Kongstad, Kenneth T; Nielsen, Birgitte; Sørensen, Troels E; Balle, Thomas; Jensen, Anders A; Frølund, Bente

    2014-11-01

    A series of bioisosteric N1- and N2 -substituted 5-(piperidin-4-yl)-3-hydroxypyrazole analogues of the partial GABAA R agonists 4-PIOL and 4-PHP have been designed, synthesized, and characterized pharmacologically. The unsubstituted 3-hydroxypyrazole analogue of 4-PIOL (2 a; IC50 ∼300 μM) is a weak antagonist at the α1 β2 γ2 GABAA R, whereas substituting the N1- or N2- position with alkyl or aryl substituents resulted in antagonists with binding affinities in the high nanomolar to low micromolar range at native rat GABAA Rs. Docking studies using a α1 β2 γ2 GABAA R homology model along with the obtained SAR indicate that the N1 -substituted analogues of 4-PIOL and 4-PHP, 2 a-k, and previously reported 3-substituted 4-PHP analogues share a common binding mode to the orthosteric binding site in the receptor. Interestingly, the core scaffold of the N2 -substituted analogues of 4-PIOL and 4-PHP, 3 b-k, are suggested to flip 180° thereby adapting to the binding pocket and addressing a cavity situated above the core scaffold. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Binding of [3H]paroxetine to serotonin uptake sites and of [3H]lysergic acid diethylamide to 5-HT2A receptors in platelets from women with premenstrual dysphoric disorder during gonadotropin releasing hormone treatment.

    PubMed

    Bixo, M; Allard, P; Bäckström, T; Mjörndal, T; Nyberg, S; Spigset, O; Sundström-Poromaa, I

    2001-08-01

    Changes in serotonergic parameters have been reported in psychiatric conditions such as depression but also in the premenstrual dysphoric disorder (PMDD). In addition, hormonal effects on serotonergic activity have been established. In the present study, binding of [3H]paroxetine to platelet serotonin uptake sites and binding of [3H]lysergic acid diethylamide ([3H]LSD) to platelet serotonin (5-HT)2A receptors were studied in patients with PMDD treated with a low dose of a gonadotropin releasing hormone (GnRH) agonist (buserelin) or placebo and compared to controls. The PMDD patients were relieved of premenstrual symptoms like depression and irritability during buserelin treatment. The number of [3H]paroxetine binding sites (Bmax) were significantly higher in the follicular phase in untreated PMDD patients compared to controls. When treated with buserelin the difference disappeared. No differences in [3H]LSD binding between the three groups were shown. The present study demonstrated altered platelet [3H]paroxetine binding characteristics in women with PMDD compared to controls. Furthermore, [3H]paroxetine binding was affected by PMDD treatment with a low dose of buserelin. The results are consistent with the hypothesis that changes in serotonergic transmission could be a trait in the premenstrual dysphoric disorder.

  20. Preferred Metal Binding Site of Aniline

    NASA Astrophysics Data System (ADS)

    Kumari, Sudesh; Sohnlein, Brad; Yang, Dong-Sheng

    2012-06-01

    Group III metal-aniline complexes, M-aniline (M = Sc, Y, and La), were produced by interactions between laser-vaporized metal atoms and aniline vapor in a pulsed molecular beam source, identified by photoionization time-of-flight mass spectrometry, and studied by pulsed-field ionization zero electron kinetic energy (ZEKE) spectroscopy and density functional theory calculations. Adiabatic ionization energies and several vibrational intervals were measured from the ZEKE spectra. Metal binding sites and electronic states were determined by combining the ZEKE measurements and theoretical calculations. Although aniline has various possible sites for metal coordination, the preferred site was determined to be phenyl ring. The metal binding with the phenyl ring yields syn and anti conformers. In these conformers, the neutral complexes are in doublet ground states and the corresponding singly charged cations in singlet states.

  1. Reversible binding of ethacrynic acid to human serum albumin: difference circular dichroism study.

    PubMed

    Bertucci, C; Nanni, B; Salvadori, P

    1999-01-01

    The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anti-cooperative) and to site III (cooperative).

  2. Avermectin binding in Caenorhabditis elegans. A two-state model for the avermectin binding site.

    PubMed

    Schaeffer, J M; Haines, H W

    1989-07-15

    Specific binding sites for ivermectin (IVM; 22,23-dihydroavermectin-B1) were identified and characterized in a crude membrane fraction prepared from the nematode, Caenorhabditis elegans (C. elegans). Specific [3H]IVM binding was saturable with an apparent dissociation constant, Kd, of 0.26 nM and a receptor concentration of 3.53 pmol/mg protein. [3H]IVM binding in C. elegans was linear with tissue protein concentration, and optimal binding occurred within a pH range of 7.3 to 7.6. Kinetic analysis of the binding showed that the reaction proceeded by a two-step mechanism. Initially, a rapidly reversible complex was formed and, after additional incubation, this complex was transformed to a much more slowly reversible complex. Stereospecificity of [3H]IVM binding to C. elegans membranes was demonstrated by competition with a series of avermectin derivatives. The in vivo effects of IVM and its derivatives on C. elegans motility were concentration dependent and correlated well with their relative binding affinities. Several putative neurotransmitters including gamma-aminobutyric acid (GABA), carbamyl choline, taurine, glutamate and dopamine were tested and found to have no effect on IVM binding. Specific IVM binding sites were also identified in rat brain; however, the affinity was approximately 100-fold lower than that observed in C. elegans and stereospecificity studies demonstrated structural differences in the two binding sites. These results are the first direct demonstration of a specific IVM binding site in nematodes and thus are important in furthering our understanding of its mode of action.

  3. Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases.

    PubMed

    Saxena, Ashima; Fedorko, James M; Vinayaka, C R; Medhekar, Rohit; Radić, Zoran; Taylor, Palmer; Lockridge, Oksana; Doctor, Bhupendra P

    2003-11-01

    E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.

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

    PubMed

    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.

  5. Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype.

    PubMed

    Chennathukuzhi, V M; Kurihara, Y; Bray, J D; Yang, J; Hecht, N B

    2001-11-01

    The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.

  6. Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor*♦

    PubMed Central

    Chiara, David C.; Jayakar, Selwyn S.; Zhou, Xiaojuan; Zhang, Xi; Savechenkov, Pavel Y.; Bruzik, Karol S.; Miller, Keith W.; Cohen, Jonathan B.

    2013-01-01

    GABA type A receptors (GABAAR), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β+-α− subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[3H]mTFD-MPAB did not photolabel the etomidate sites at the β+-α− subunit interfaces. Instead, it photolabeled sites at the α+-β− and γ+-β− subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β+-α− interface relative to the α+-β−/γ+-β− interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics. PMID:23677991

  7. Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.

    PubMed

    Chiara, David C; Jayakar, Selwyn S; Zhou, Xiaojuan; Zhang, Xi; Savechenkov, Pavel Y; Bruzik, Karol S; Miller, Keith W; Cohen, Jonathan B

    2013-07-05

    GABA type A receptors (GABAAR), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β(+)-α(-) subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[(3)H]mTFD-MPAB did not photolabel the etomidate sites at the β(+)-α(-) subunit interfaces. Instead, it photolabeled sites at the α(+)-β(-) and γ(+)-β(-) subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (-)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β(+)-α(-) interface relative to the α(+)-β(-)/γ(+)-β(-) interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.

  8. Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

    PubMed Central

    Pineda-Molina, Estela; Reyes-Darias, José-Antonio; Lacal, Jesús; Ramos, Juan L.; García-Ruiz, Juan Manuel; Gavira, Jose A.; Krell, Tino

    2012-01-01

    Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate. PMID:23112148

  9. DBSI: DNA-binding site identifier

    PubMed Central

    Zhu, Xiaolei; Ericksen, Spencer S.; Mitchell, Julie C.

    2013-01-01

    In this study, we present the DNA-Binding Site Identifier (DBSI), a new structure-based method for predicting protein interaction sites for DNA binding. DBSI was trained and validated on a data set of 263 proteins (TRAIN-263), tested on an independent set of protein-DNA complexes (TEST-206) and data sets of 29 unbound (APO-29) and 30 bound (HOLO-30) protein structures distinct from the training data. We computed 480 candidate features for identifying protein residues that bind DNA, including new features that capture the electrostatic microenvironment within shells near the protein surface. Our iterative feature selection process identified features important in other models, as well as features unique to the DBSI model, such as a banded electrostatic feature with spatial separation comparable with the canonical width of the DNA minor groove. Validations and comparisons with established methods using a range of performance metrics clearly demonstrate the predictive advantage of DBSI, and its comparable performance on unbound (APO-29) and bound (HOLO-30) conformations demonstrates robustness to binding-induced protein conformational changes. Finally, we offer our feature data table to others for integration into their own models or for testing improved feature selection and model training strategies based on DBSI. PMID:23873960

  10. DBSI: DNA-binding site identifier.

    PubMed

    Zhu, Xiaolei; Ericksen, Spencer S; Mitchell, Julie C

    2013-09-01

    In this study, we present the DNA-Binding Site Identifier (DBSI), a new structure-based method for predicting protein interaction sites for DNA binding. DBSI was trained and validated on a data set of 263 proteins (TRAIN-263), tested on an independent set of protein-DNA complexes (TEST-206) and data sets of 29 unbound (APO-29) and 30 bound (HOLO-30) protein structures distinct from the training data. We computed 480 candidate features for identifying protein residues that bind DNA, including new features that capture the electrostatic microenvironment within shells near the protein surface. Our iterative feature selection process identified features important in other models, as well as features unique to the DBSI model, such as a banded electrostatic feature with spatial separation comparable with the canonical width of the DNA minor groove. Validations and comparisons with established methods using a range of performance metrics clearly demonstrate the predictive advantage of DBSI, and its comparable performance on unbound (APO-29) and bound (HOLO-30) conformations demonstrates robustness to binding-induced protein conformational changes. Finally, we offer our feature data table to others for integration into their own models or for testing improved feature selection and model training strategies based on DBSI.

  11. Synthetic human serum albumin Sudlow I binding site mimics.

    PubMed

    Karlsson, Björn C G; Rosengren, Annika M; Näslund, Inga; Andersson, Per Ola; Nicholls, Ian A

    2010-11-25

    Here, we report the design, synthesis, and characterization of molecularly imprinted polymer (MIP) derived mimics of the human serum albumin (HSA) Sudlow I site-the binding site for the anticoagulant warfarin. MIP design was based upon a combination of experimental ((1)H NMR) and computational (molecular dynamics) methods. Two MIPs and corresponding nonimprinted reference polymers were synthesized and characterized (scanning electron microscopy; nitrogen sorption; and Fourier transform infrared spectroscopy). MIP-ligand recognition was examined using radioligand binding studies, where the largest number of selective sites was found in a warfarin-imprinted methacrylic acid-ethylene dimethacrylate copolymer (MAA-MIP). The warfarin selectivity of this MIP was confirmed using radioligand displacement and zonal chromatographic studies. A direct comparison of MIP-warfarin binding characteristics with those of the HSA Sudlow I binding site was made, and similarities in site population (per gram polymer or protein) and affinities were observed. The warfarin selectivity of the MIP suggests its potential for use as a recognition element in a MIP-based warfarin sensor and even as a model to aid in understanding and steering blood-plasma protein-regulated transport processes or even for the development of warfarin sensors.

  12. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

    Background Studies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks. Results We developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation. Conclusions We provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation. PMID:24238150

  13. Nimesulide binding site in the B0AT1 (SLC6A19) amino acid transporter. Mechanism of inhibition revealed by proteoliposome transport assay and molecular modelling.

    PubMed

    Pochini, Lorena; Seidita, Angela; Sensi, Cristina; Scalise, Mariafrancesca; Eberini, Ivano; Indiveri, Cesare

    2014-06-01

    The effect of pharmaceutical compounds on the rat kidney B0AT1 transporter in proteoliposomes has been screened. To this aim, inhibition of the transport activity by the different compounds was measured on Na(+)-[(3)H]glutamine co-transport in the presence of membrane potential positive outside. Most of the tested drugs had no effect on the transport activity. Some compounds exhibited inhibitory effects from 5 to 88% at concentration of 300μM. Among the tested compounds, only the anti-inflammatory drug nimesulide exerted potent inhibition on B0AT1. From dose response analysis, an IC50 value of 23μM was found. Inhibition kinetic analysis was performed: noncompetitive inhibition of the glutamine transport was observed while competitive behaviour was found when the inhibition was analyzed with respect to the Na(+) concentration. Several molecules harbouring functional groups of nimesulide (analogues) were tested as inhibitors. None among the tested molecules has the capacity to inhibit the transport with the exception of the compound NS-398, whose chemical structure is very close to that of whole nimesulide. The IC50 for this compound was 131μM. Inhibition kinetics showed behaviour of NS-398 identical to that of nimesulide, i.e., noncompetitive inhibition respect to glutamine and competitive inhibition respect to Na(+). Molecular docking of nimesulide suggested that this drug is able to bind B0AT1 in an external dedicated binding site and that its binding produces a steric hindrance effect of the protein translocation path abolishing the transporter activity.

  14. MetalS2: a tool for the structural alignment of minimal functional sites in metal-binding proteins and nucleic acids.

    PubMed

    Andreini, Claudia; Cavallaro, Gabriele; Rosato, Antonio; Valasatava, Yana

    2013-11-25

    We developed a new software tool, MetalS(2), for the structural alignment of Minimal Functional Sites (MFSs) in metal-binding biological macromolecules. MFSs are 3D templates that describe the local environment around the metal(s) independently of the larger context of the macromolecular structure. Such local environment has a determinant role in tuning the chemical reactivity of the metal, ultimately contributing to the functional properties of the whole system. On our example data sets, MetalS(2) unveiled structural similarities that other programs for protein structure comparison do not consistently point out and overall identified a larger number of structurally similar MFSs. MetalS(2) supports the comparison of MFSs harboring different metals and/or with different nuclearity and is available both as a stand-alone program and a Web tool ( http://metalweb.cerm.unifi.it/tools/metals2/).

  15. Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors

    PubMed Central

    Bulyk, Martha L.; Johnson, Philip L. F.; Church, George M.

    2002-01-01

    We can determine the effects of many possible sequence variations in transcription factor binding sites using microarray binding experiments. Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently. This indicates that the current practice of characterizing transcription factor binding sites by mutating individual positions of binding sites one base pair at a time does not provide a true picture of the sequence specificity. Similarly, current bioinformatic practices using either just a consensus sequence, or even mononucleotide frequency weight matrices to provide more complete descriptions of transcription factor binding sites, are not accurate in depicting the true binding site specificities, since these methods rely upon the assumption that the nucleotides of binding sites exert independent effects on binding affinity. Our results stress the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences. We also show results suggesting that microarray binding data using particular subsets of all possible binding sites can be used to extrapolate the relative binding affinities of all possible full-length binding sites, given a known binding site for use as a starting sequence for site preference refinement. PMID:11861919

  16. Oxytocin binding sites in bovine mammary tissue

    SciTech Connect

    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.

  17. Binding site and inhibitory mechanism of the mambalgin-2 pain-relieving peptide on acid-sensing ion channel 1a.

    PubMed

    Salinas, Miguel; Besson, Thomas; Delettre, Quentin; Diochot, Sylvie; Boulakirba, Sonia; Douguet, Dominique; Lingueglia, Eric

    2014-05-09

    Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the β-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and β-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.

  18. Distinct oxidative cleavage and modification of bovine [Cu- Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule.

    PubMed

    Uehara, Hiroshi; Luo, Shen; Aryal, Baikuntha; Levine, Rodney L; Rao, V Ashutosh

    2016-05-01

    We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction.

  19. Signal transducer and activator of transcription (stat) binding sites but not stat3 are required for fasting-induced transcription of agouti-related protein messenger ribonucleic acid.

    PubMed

    Kaelin, Christopher B; Gong, Lijie; Xu, Allison Wanting; Yao, Fayi; Hockman, Kristin; Morton, Gregory J; Schwartz, Michael W; Barsh, Gregory S; MacKenzie, Robert G

    2006-10-01

    Energy homeostasis depends on the regulation of hypothalamic neurons by leptin, an adipocyte hormone whose circulating levels communicate body energy stores. Leptin activates the transcription factor signal transducer and activator of transcription 3 (Stat3) in hypothalamic neurons, including neuronal subtypes producing Agouti-related protein (Agrp), a neuropeptide that stimulates feeding. Previous studies have suggested a model in which high levels of Agrp transcription during fasting represent a default state that is actively repressed by phospho-Stat3 induced by leptin signaling in the fed state. We identify putative Stat3 binding elements in the Agrp promoter that have been highly conserved during vertebrate evolution. Using a reporter assay in transgenic mice that faithfully recapitulates normal regulation of Agrp, we show that these sites are required, but in a way opposite to that predicted by the existing model: mutation of the sites leads to a default state characterized by a low level of Agrp transcription and insensitivity to fasting. We also find that removing activatable Stat3 from Agrp neurons has no detectable effect on steady-state levels of Agrp mRNA in the fed or fasted state. These results suggest a new model for transcriptional regulation of orexigenic neuropeptides in which the default level of expression is low in the fed state, and transcriptional activation in response to fasting is mediated by factors other than Stat3.

  20. Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.

    PubMed Central

    Fraser, C M; Chung, F Z; Wang, C D; Venter, J C

    1988-01-01

    By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR. Images PMID:2840663

  1. Mass spectrometry and NMR analysis of ligand binding by human liver fatty acid binding protein.

    PubMed

    Santambrogio, C; Favretto, F; D'Onofrio, M; Assfalg, M; Grandori, R; Molinari, H

    2013-08-01

    Human liver fatty acid binding protein (hL-FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a 'head-in' or 'head-out' orientation of the carboxylate end. Electrospray-ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head-out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Binding of perfluorooctanoic acid to rat and human plasma proteins.

    PubMed

    Han, Xing; Snow, Timothy A; Kemper, Raymond A; Jepson, Gary W

    2003-06-01

    Perfluorooctanoic acid (PFOA) is a commercially important organic fluorochemical and is considered to have a long half-life in human blood. In this paper, PFOA binding to rat and human plasma proteins was investigated. On the basis of results from size-exclusion chromatography and ligand blotting, most PFOA was in protein-bound form in male and female rat plasma, and the primary PFOA binding protein in plasma was serum albumin. PFOA binding to rat serum albumin (RSA) in the gas phase was observed by electrospray ionization MS. (19)F NMR experiments revealed that binding to RSA caused peak broadening and chemical shift changes of PFOA resonances, and on the basis of this observation, the dissociation constant was determined to be approximately 0.3 mM. The dissociation constants for PFOA binding to RSA and human serum albumin (HSA) and the numbers of PFOA binding sites on RSA and HSA were also determined by a separation method using microdesalting columns. No significant difference was found between PFOA binding to RSA and PFOA binding to HSA. The dissociation constants for binding of PFOA to RSA or HSA and the numbers of PFOA binding sites were in the range of 0.3-0.4 mM and 6-9, respectively. On the basis of these binding parameters and the estimated plasma concentration of serum albumin, greater than 90% of PFOA would be bound to serum albumin in both rat and human blood.

  3. Detection of Binding Site Molecular Interaction Field Similarities.

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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 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. Images Fig. 1. Fig. 2. Fig. 3. Fig. 6. Fig. 7. Fig. 9. PMID:1898347

  6. Distinct OGT-Binding Sites Promote HCF-1 Cleavage

    PubMed Central

    Bhuiyan, Tanja; Waridel, Patrice; Kapuria, Vaibhav; Zoete, Vincent; Herr, Winship

    2015-01-01

    Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity. PMID:26305326

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

    PubMed

    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.

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

    PubMed Central

    Gadiraju, Sashidhar; Vyhlidal, Carrie A; Leeder, J Steven; Rogan, Peter K

    2003-01-01

    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. Conclusions Delila-Genome was used to

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

    PubMed

    Gadiraju, Sashidhar; Vyhlidal, Carrie A; Leeder, J Steven; Rogan, Peter K

    2003-09-08

    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. 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. Delila-Genome was used to scan the human genome sequence

  10. FCA does not bind abscisic acid.

    PubMed

    Risk, Joanna M; Macknight, Richard C; Day, Catherine L

    2008-12-11

    The RNA-binding protein FCA promotes flowering in Arabidopsis. Razem et al. reported that FCA is also a receptor for the phytohormone abscisic acid (ABA). However, we find that FCA does not bind ABA, suggesting that the quality of the proteins assayed and the sensitivity of the ABA-binding assay have led Razem et al. to erroneous conclusions. Because similar assays have been used to characterize other ABA receptors, our results indicate that the ABA-binding properties of these proteins should be carefully re-evaluated and that alternative ABA receptors are likely to be discovered.

  11. Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean.

    PubMed

    Battistuzzi, G; Dietrich, M; Löcke, R; Witzel, H

    1997-05-01

    The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (Mr 111000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted 1H NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives.

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

    PubMed Central

    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

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

    PubMed

    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. Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

    PubMed

    Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-03-25

    Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology and designing enzymes with new functional capabilities. This article is protected by copyright. All rights reserved.

  15. Competitive binding between mercury and copper for reduced sulfur binding sites on dissolved organic matter from the Florida Everglades

    NASA Astrophysics Data System (ADS)

    Gerbig, C. A.; Aiken, G. R.; Ryan, J. N.

    2007-12-01

    The interaction of mercury and dissolved organic matter (DOM) strongly influences the biogeochemistry of mercury in the Florida Everglades. Previous laboratory-based studies of simple systems at environmentally relevant concentrations of mercury(II) (a soft Lewis acid) and DOM found strong conditional binding constants (log KHgL' = 28-31). These large constants result from the interaction of mercury(II) with reduced sulfur (a soft Lewis base) sites on DOM. Reported conditional binding constants for other metals with DOM (e.g. log KCuL' = 11-14), suggest that metals of borderline Lewis acidity would not compete with mercury(II) for the strongest binding sites at environmentally relevant concentrations. However, the small proportion of strong binding sites responsible for mercury(II) binding have proven to be susceptible to competitive effects from borderline metals. Equilibrium dialysis experiments using organic matter isolated from the Florida Everglades were designed to determine the effects of competitive binding between copper(II) and mercury(II) on DOM binding sites. These experiments demonstrated that copper(II), a borderline Lewis acid, effectively competed for strong DOM sites at concentrations only 1-2 orders of magnitude greater than experimental mercury(II) concentrations (which ranged from 0.05 to 0.2nM). Our results indicate that the reduced sulfur sites responsible for Hg(II) binding on DOM also have high affinities for borderline metals. Interactions of copper(II) and DOM were also investigated in the absence of mercury(II). These results further substantiate the significance of a small concentration of strong binding sites on DOM. At low copper(II) to DOM ratios, preliminary results indicate that the binding interactions between copper(II) and DOM are significantly greater than previously reported and are close to those measured for DOM-mercury(II) binding. We conclude that currently available binding constants for metals of interest (borderline

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

    PubMed Central

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

    2015-01-01

    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. PMID:26655740

  17. Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

    PubMed Central

    Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

    1999-01-01

    A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

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

    PubMed

    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.

  19. Characterization of hydroxycinnamic acid derivatives binding to bovine serum albumin.

    PubMed

    Jin, Xiao-Ling; Wei, Xia; Qi, Feng-Ming; Yu, Sha-Sha; Zhou, Bo; Bai, Shi

    2012-05-07

    Hydroxycinnamic acid derivatives (HCAs) are a group of naturally occurring polyphenolic compounds which possess various pharmacological activities. In this work, the interactions of bovine serum albumin (BSA) with six HCA derivatives, including chlorogenic acid (CHA), caffeic acid (CFA), m-coumaric acid (m-CA), p-coumaric acid (p-CA), ferulic acid (FA) and sinapic acid (SA) have been investigated by NMR spectroscopic techniques in combination with fluorescence and molecular modeling methods. Competitive STD NMR experiments using warfarin sodium and L-tryptophan as site-selective probes indicated that HCAs bind to site I in the subdomain IIA of BSA. From the analysis of the STD NMR-derived binding epitopes and molecular docking models, it was deduced that CHA, CFA, m-CA and p-CA show similar binding modes and orientation, in which the phenyl ring is in close contact with protein surface, whereas carboxyl group points out of the protein. However, FA and SA showed slightly different binding modes, due to the steric hindrance of methoxy-substituents on the phenyl ring. Relaxation experiments provided detailed information about the relationship between the affinity and structure of HCAs. The binding affinity was the strongest for CHA and ranked in the order CHA > CFA > m-CA ≥ p-CA > FA > SA, which agreed well with the results from fluorescence experiments. Based on our experimental results, we also conclude that HCAs bind to BSA mainly by hydrophobic interaction and hydrogen bonding. This study therefore provides valuable information for elucidating the mechanisms of BSA-HCAs interaction.

  20. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  1. Predicting Ca2+-binding Sites Using Refined Carbon Clusters

    PubMed Central

    Zhao, Kun; Wang, Xue; Wong, Hing C.; Wohlhueter, Robert; Kirberger, Michael P.; Chen, Guantao; Yang, Jenny J.

    2012-01-01

    Identifying Ca2+-binding sites in proteins is the first step towards understanding the molecular basis of diseases related to Ca2+-binding proteins. Currently, these sites are identified in structures either through X-ray crystallography or NMR analysis. However, Ca2+-binding sites are not always visible in X-ray structures due to flexibility in the binding region or low occupancy in a Ca2+-binding site. Similarly, both Ca2+ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca2+-binding sites in both X-ray and NMR structures, we report a new graph theory algorithm (MUGC) to predict Ca2+-binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side-chain oxygen ligand coordinates, MUGC is able to achieve 94% sensitivity with 76% selectivity on a dataset of X-ray structures comprised of 43 Ca2+-binding proteins. Additionally, prediction of Ca2+-binding sites in NMR structures were obtained by MUGC using a different set of parameters determined by analysis of both Ca2+-constrained and unconstrained Ca2+-loaded structures derived from NMR data. MUGC identified 20 out of 21 Ca2+-binding sites in NMR structures inferred without the use of Ca2+ constraints. MUGC predictions are also highly-selective for Ca2+-binding sites as analyses of binding sites for Mg2+, Zn2+, and Pb2+ were not identified as Ca2+-binding sites. These results indicate that the geometric arrangement of the second-shell carbon cluster is sufficient for both accurate identification of Ca2+-binding sites in NMR and X-ray structures, and for selective differentiation between Ca2+ and other relevant divalent cations. PMID:22821762

  2. Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

    PubMed Central

    Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

  3. Mu opioid receptor binding sites in human brain

    SciTech Connect

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand (/sup 3/H)DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of (/sup 3/H)DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas.

  4. Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding▿

    PubMed Central

    Wilda, Maximiliano; Lopez, Nora; Casabona, Juan Cruz; Franze-Fernandez, Maria T.

    2008-01-01

    Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. The TacV genome encodes four proteins: the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a RING finger protein (Z). Using a reverse genetics system, we demonstrated that TacV N and L are sufficient to drive transcription and replication mediated by TacV-like RNAs and that Z is a powerful inhibitor of these processes (Lopez et al., J. Virol. 65:12241-12251, 2001). More recently, we provided the first evidence of an interaction between Z and L and showed that Z's inhibitory activity was dependent on its ability to bind to L (Jácamo et al., J. Virol. 77:10383-10393, 2003). In the present study, we mapped the TacV Z-binding sites on the 2,210-amino-acid L polymerase. To that end, we performed deletion analysis and point mutations of L and studied the Z-L interaction by coimmunoprecipitation with specific sera. We found that the C-terminal region of L was not essential for the interaction and identified two noncontiguous regions that were critical for binding: one at the N-terminus of L between residues 156 and 292 and a second one in the polymerase domain (domain III). The importance of domain III in binding was revealed by substitutions in D1188 and H1189 within motif A and in each residue of the conserved SDD sequence (residues 1328, 1329, and 1330) within motif C. Our results showed that of the substituted residues, only H1189 and D1329 appeared to be critically involved in binding Z. PMID:18799569

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

    PubMed

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

    2016-03-01

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

  6. Mapping fatty acid binding to beta-lactoglobulin: Ligand binding is restricted by modification of Cys 121.

    PubMed Central

    Narayan, M.; Berliner, L. J.

    1998-01-01

    Native beta-lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 microM for the primary fatty acid binding site. Chemical modification of Cys 121, which lies at the external putative hydrophobic binding site of Blg, does not affect retinol or 4,4'-bis 1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) binding to the protein, indicating that the incorporated appendages do not perturb the internal hydrophobic site within the beta-barrel of Blg (i.e., the retinoid site is unaffected). On the other hand, methylation of Cys 121, reduces the affinity of Blg for palmitic acid by 10-fold as monitored by intrinsic fluorescence. Modification of the Cys 121 with methylmethanethiosulfonate or a thiol-specific spin label appears to either further weaken or totally eliminate fatty acid binding, respectively, due to steric hindrance. Furthermore, this binding pattern has been independently verified using a spin labeled fatty acid analog and monitoring ESR as well as by bis-ANS fluorescence when bound to the protein. These results suggest that fatty acids bind at the "external site" of beta-lactoglobulin, between the sole alpha-helix and the beta-barrel. In addition, structural stability studies of native and chemically modified Blg appear to confirm this observation as well. PMID:9514270

  7. M-ORBIS: mapping of molecular binding sites and surfaces.

    PubMed

    Albou, Laurent-Philippe; Poch, Olivier; Moras, Dino

    2011-01-01

    M-ORBIS is a Molecular Cartography approach that performs integrative high-throughput analysis of structural data to localize all types of binding sites and associated partners by homology and to characterize their properties and behaviors in a systemic way. The robustness of our binding site inferences was compared to four curated datasets corresponding to protein heterodimers and homodimers and protein-DNA/RNA assemblies. The Molecular Cartographies of structurally well-detailed proteins shows that 44% of their surfaces interact with non-solvent partners. Residue contact frequencies with water suggest that ∼86% of their surfaces are transiently solvated, whereas only 15% are specifically solvated. Our analysis also reveals the existence of two major binding site families: specific binding sites which can only bind one type of molecule (protein, DNA, RNA, etc.) and polyvalent binding sites that can bind several distinct types of molecule. Specific homodimer binding sites are for instance nearly twice as hydrophobic than previously described and more closely resemble the protein core, while polyvalent binding sites able to form homo and heterodimers more closely resemble the surfaces involved in crystal packing. Similarly, the regions able to bind DNA and to alternatively form homodimers, are more hydrophobic and less polar than previously described DNA binding sites.

  8. Substrate and drug binding sites in LeuT.

    PubMed

    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.

  9. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  10. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  11. Kinetics of ligand binding to nucleic acids.

    PubMed

    Arakelyan, V B; Babayan, S Y; Tairyan, V I; Arakelyan, A V; Parsadanyan, M A; Vardevanyan, P O

    2006-02-01

    Ligand binding to nucleic acids (NA) is considered as a stationary Markov process. It is shown that the probabilistic description of ligand-NA binding allows one to describe not only the kinetics of the change of number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. The general analysis of absorption isotherms and kinetics of ligand binding to NA make it possible to determine of rate constants of ligand-NA complex formation and dissociation.

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

    PubMed

    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.

  13. Acid rain stone test sites

    NASA Astrophysics Data System (ADS)

    Sherwood, Susan I.; Doe, Bruce R.

    1984-04-01

    As a part of the United States National Acid Precipitation Assessment Program, Task Group G: Effects on Materials and Cultural Resources, which is chaired by Ray Herrmann, the National Park Service has established four test sites for 10-year testing of two kinds of dimension stone used in buildings and monuments. The four sites are (from south to north) Research Triangle Park near Raleigh, N.C. (activated May 25, 1984); the roof of the West End Branch of the Washington, D.C. Library (activated August 11, 1984); the Department of Energy Compound at the Environmental Measurements Laboratory of Bell Telephone Laboratories near Chester, N.J. (activated June 5, 1984); and Huntington Wildlife Forest in the Adirondack Mountains, Newcomb, N.Y. (activated June 19, 1984).

  14. A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Southerland, William M.

    1994-04-01

    The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.

  15. Defining the bacteroides ribosomal binding site.

    PubMed

    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.

  16. Defining the Bacteroides Ribosomal Binding Site

    PubMed Central

    Horn, Nikki; Carding, Simon R.

    2013-01-01

    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. PMID:23335775

  17. Type II oestrogen binding sites in human colorectal carcinoma.

    PubMed Central

    Piantelli, M; Ricci, R; Larocca, L M; Rinelli, A; Capelli, A; Rizzo, S; Scambia, G; Ranelletti, F O

    1990-01-01

    Seven cases of colorectal adenocarcinomas were investigated for the presence of oestrogen receptors and progesterone receptors. The tumours specifically bound oestradiol. This binding almost exclusively resulted from the presence of high numbers of type II oestrogen binding sites. Oestrogen receptors were absent or present at very low concentrations. Immunohistochemical investigation of nuclear oestrogen receptors gave negative results. This indicates that antioestrogen receptor antibodies recognise oestrogen receptors but not type II oestrogen binding sites. The presence of specific type II oestrogen binding sites and progesterone binding offers further evidence for a potential role for these steroids and their receptors in colorectal carcinoma. PMID:2266171

  18. Lipid binding to the carotenoid binding site in photosynthetic reaction centers.

    PubMed

    Deshmukh, Sasmit S; Tang, Kai; Kálmán, László

    2011-10-12

    Lipid binding to the carotenoid binding site near the inactive bacteriochlorophyll monomer was probed in the reaction centers of carotenoid-less mutant, R-26 from Rhodobacter sphaeroides. Recently, a marked light-induced change of the local dielectric constant in the vicinity of the inactive bacteriochlorophyll monomer was reported in wild type that was attributed to structural changes that ultimately lengthened the lifetime of the charge-separated state by 3 orders of magnitude (Deshmukh, S. S.; Williams, J. C.; Allen, J. P.; Kalman, L. Biochemistry 2011, 50, 340). Here in the R-26 reaction centers, the combination of light-induced structural changes and lipid binding resulted in a 5 orders of magnitude increase in the lifetime of the charge-separated state involving the oxidized dimer and the reduced primary quinone in proteoliposomes. Only saturated phospholipids with fatty acid chains of 12 and 14 carbon atoms long were bound successfully at 8 °C by cooling the reaction center protein slowly from room temperature. In addition to reporting a dramatic increase of the lifetime of the charge-separated state at physiologically relevant temperatures, this study reveals a novel lipid binding site in photosynthetic reaction center. These results shed light on a new potential application of the reaction center in energy storage as a light-driven biocapacitor since the charges separated by ∼30 Å in a low-dielectric medium can be prevented from recombination for hours.

  19. Why Transcription Factor Binding Sites Are Ten Nucleotides Long

    PubMed Central

    Stewart, Alexander J.; Hannenhalli, Sridhar; Plotkin, Joshua B.

    2012-01-01

    Gene expression is controlled primarily by transcription factors, whose DNA binding sites are typically 10 nt long. We develop a population-genetic model to understand how the length and information content of such binding sites evolve. Our analysis is based on an inherent trade-off between specificity, which is greater in long binding sites, and robustness to mutation, which is greater in short binding sites. The evolutionary stable distribution of binding site lengths predicted by the model agrees with the empirical distribution (5–31 nt, with mean 9.9 nt for eukaryotes), and it is remarkably robust to variation in the underlying parameters of population size, mutation rate, number of transcription factor targets, and strength of selection for proper binding and selection against improper binding. In a systematic data set of eukaryotic and prokaryotic transcription factors we also uncover strong relationships between the length of a binding site and its information content per nucleotide, as well as between the number of targets a transcription factor regulates and the information content in its binding sites. Our analysis explains these features as well as the remarkable conservation of binding site characteristics across diverse taxa. PMID:22887818

  20. IP3 receptor binds to and sensitizes TRPV4 channel to osmotic stimuli via a calmodulin-binding site.

    PubMed

    Garcia-Elias, Anna; Lorenzo, Ivan M; Vicente, Rubén; Valverde, Miguel A

    2008-11-14

    Activation of the non-selective cation channel TRPV4 by mechanical and osmotic stimuli requires the involvement of phospholipase A2 and the subsequent production of the arachidonic acid metabolites, epoxieicosatrienoic acids (EET). Previous studies have shown that inositol trisphosphate (IP3) sensitizes TRPV4 to mechanical, osmotic, and direct EET stimulation. We now search for the IP3 receptor-binding site on TRPV4 and its relevance to IP3-mediated sensitization. Three putative sites involved in protein-protein interactions were evaluated: a proline-rich domain (PRD), a calmodulin (CaM)-binding site, and the last four amino acids (DAPL) that show a PDZ-binding motif-like. TRPV4-DeltaCaM-(Delta812-831) channels preserved activation by hypotonicity, 4alpha-phorbol 12,13-didecanoate, and EET but lost their physical interaction with IP3 receptor 3 and IP3-mediated sensitization. Deletion of a PDZ-binding motif-like (TRPV4-DeltaDAPL) did not affect channel activity or IP3-mediated sensitization, whereas TRPV4-DeltaPRD-(Delta132-144) resulted in loss of channel function despite correct trafficking. We conclude that IP3-mediated sensitization requires IP3 receptor binding to a TRPV4 C-terminal domain that overlaps with a previously described calmodulin-binding site.

  1. Drug-binding properties of rat alpha 1-foetoprotein. Binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid.

    PubMed Central

    Hervé, F; Rajkowski, K; Martin, M T; Dessen, P; Cittanova, N

    1984-01-01

    As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found. PMID:6206846

  2. Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses.

    PubMed

    Wang, Xiaoquan; Ilyushina, Natalia A; Lugovtsev, Vladimir Y; Bovin, Nicolai V; Couzens, Laura K; Gao, Jin; Donnelly, Raymond P; Eichelberger, Maryna C; Wan, Hongquan

    2017-01-15

    Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the United States in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses.

  3. Impact of germline and somatic missense variations on drug binding sites.

    PubMed

    Yan, C; Pattabiraman, N; Goecks, J; Lam, P; Nayak, A; Pan, Y; Torcivia-Rodriguez, J; Voskanian, A; Wan, Q; Mazumder, R

    2017-03-01

    Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein's gene. To identify the nsSNVs that may affect drug binding, protein-drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein-drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein-drug binding sites. Using this method we identified 12 993 amino acid-drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid-drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid-drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid-drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein-drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein-drug binding predicted drug target proteins and prevalence of both somatic and

  4. Autoradiographic localization of endothelin-1 binding sites in porcine skin

    SciTech Connect

    Zhao, Y.D.; Springall, D.R.; Wharton, J.; Polak, J.M. )

    1991-01-01

    Autoradiographic techniques and {sup 125}I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with the known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed

    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.

  7. Iodine binding to humic acid.

    PubMed

    Bowley, H E; Young, S D; Ander, E L; Crout, N M J; Watts, M J; Bailey, E H

    2016-08-01

    The rate of reactions between humic acid (HA) and iodide (I(-)) and iodate (IO3(-)) have been investigated in suspensions spiked with (129)I at concentrations of 22, 44 and 88 μg L(-1) and stored at 10 °C. Changes in the speciation of (129)I(-), (129)IO3(-) and mixed ((129)I(-) + (129)IO3(-)) spikes were monitored over 77 days using liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS). In suspensions spiked with (129)I(-) 25% of the added I(-) was transformed into organic iodine (Org-(129)I) within 77 days and there was no evidence of (129)IO3(-) formation. By contrast, rapid loss of (129)IO3(-) and increase in both (129)I(-) and Org-(129)I was observed in (129)IO3(-)-spiked suspensions. However, the rate of Org-(129)I production was greater in mixed systems compared to (129)IO3(-)-spiked suspensions with the same total (129)I concentration, possibly indicating IO3(-)I(-) redox coupling. Size exclusion chromatography (SEC) demonstrated that Org-(129)I was present in both high and low molecular weight fractions of the HA although a slight preference to bond with the lower molecular weight fractions was observed indicating that, after 77 days, the spiked isotope had not fully mixed with the native (127)I pool. Iodine transformations were modelled using first order rate equations and fitted rate coefficients determined. However, extrapolation of the model to 250 days indicated that a pseudo-steady state would be attained after ∼200 days but that the proportion of (129)I incorporated into HA was less than that of (127)I indicating the presence of a recalcitrant pool of (127)I that was unavailable for isotopic mixing.

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

    PubMed

    Lin, Chin-Yo; Vega, Vinsensius B; Thomsen, Jane S; Zhang, Tao; Kong, Say Li; Xie, Min; Chiu, Kuo Ping; Lipovich, Leonard; Barnett, Daniel H; Stossi, Fabio; Yeo, Ailing; George, Joshy; Kuznetsov, Vladimir A; Lee, Yew Kok; Charn, Tze Howe; Palanisamy, Nallasivam; Miller, Lance D; Cheung, Edwin; Katzenellenbogen, Benita S; Ruan, Yijun; Bourque, Guillaume; Wei, Chia-Lin; Liu, Edison T

    2007-06-01

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

  9. Whole-Genome Cartography of Estrogen Receptor α Binding Sites

    PubMed Central

    Thomsen, Jane S; Zhang, Tao; Kong, Say Li; Xie, Min; Chiu, Kuo Ping; Lipovich, Leonard; Barnett, Daniel H; Stossi, Fabio; Yeo, Ailing; George, Joshy; Kuznetsov, Vladimir A; Lee, Yew Kok; Charn, Tze Howe; Palanisamy, Nallasivam; Miller, Lance D; Cheung, Edwin; Katzenellenbogen, Benita S; Ruan, Yijun; Bourque, Guillaume; Wei, Chia-Lin; Liu, Edison T

    2007-01-01

    Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor α (ERα) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERα binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5′ and 3′ ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERα binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERα-positive from ERα-negative breast tumors. The expression dynamics of the genes adjacent to ERα binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERα appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERα target genes. Unexpectedly, we found that only 22%–24% of the bona fide human ERα binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERα binding and gene regulation. PMID:17542648

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

    PubMed Central

    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

  11. Common recognition principles across diverse sequence and structural families of sialic acid binding proteins.

    PubMed

    Bhagavat, Raghu; Chandra, Nagasuma

    2014-01-01

    Sialic acids form a large family of 9-carbon monosaccharides and are integral components of glycoconjugates. They are known to bind to a wide range of receptors belonging to diverse sequence families and fold classes and are key mediators in a plethora of cellular processes. Thus, it is of great interest to understand the features that give rise to such a recognition capability. Structural analyses using a non-redundant data set of known sialic acid binding proteins was carried out, which included exhaustive binding site comparisons and site alignments using in-house algorithms, followed by clustering and tree computation, which has led to derivation of sialic acid recognition principles. Although the proteins in the data set belong to several sequence and structure families, their binding sites could be grouped into only six types. Structural comparison of the binding sites indicates that all sites contain one or more different combinations of key structural features over a common scaffold. The six binding site types thus serve as structural motifs for recognizing sialic acid. Scanning the motifs against a non-redundant set of binding sites from PDB indicated the motifs to be specific for sialic acid recognition. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. As an example analysis, a genome-wide scan for the motifs in structures of Mycobacterium tuberculosis proteome identified 17 hits that contain combinations of the features, suggesting a possible function of sialic acid binding by these proteins.

  12. Sizes of Mn-binding sites in spinach thylakoids.

    PubMed

    Takahashi, M; Asada, K

    1986-12-25

    The sizes of the Mn-binding sites in spinach thylakoids were estimated by target size analysis, assaying the membrane-bound Mn that was resistant to EDTA washing after radiation inactivation. The inactivation curve showed well the inactivation of two independent Mn-binding sites of different sizes: about two-thirds of the Mn coordinated to a binding site of 65 kDa, and the rest bound to a much smaller site of only about 3 kDa. In the large site, there was about 1 g atom of Mn/110 mol of chlorophyll in spinach thylakoids, which was constant in normally grown plants, although the Mn level in the small site depended on culture conditions. Thylakoids that had been incubated with hydroxylamine or in 0.8 M Tris lost Mn exclusively from the large binding site.

  13. Sizes of Mn-binding sites in spinach thylakoids

    SciTech Connect

    Takahashi, M.; Asada, K.

    1986-12-25

    The sizes of the Mn-binding sites in spinach thylakoids were estimated by target size analysis, assaying the membrane-bound Mn that was resistant to EDTA washing after radiation inactivation. The inactivation curve showed well the inactivation of two independent Mn-binding sites of different sizes: about two-thirds of the Mn coordinated to a binding site of 65 kDa, and the rest bound to a much smaller site of only about 3 kDa. In the large site, there was about 1 g atom of Mn/110 mol of chlorophyll in spinach thylakoids, which was constant in normally grown plants, although the Mn level in the small site depended on culture conditions. Thylakoids that had been incubated with hydroxylamine or in 0.8 M Tris lost Mn exclusively from the large binding site.

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

    PubMed

    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.

  15. Modulatory effects of unsaturated fatty acids on the binding of glucocorticoids to rat liver glucocorticoid receptors.

    PubMed

    Vallette, G; Vanet, A; Sumida, C; Nunez, E A

    1991-09-01

    Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.

  16. Druggability of methyl-lysine binding sites

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  17. Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding

    SciTech Connect

    Krudy, G.A.; Brito, R.M.M.; Putkey, J.A.; Rosevear, P.R. )

    1992-02-18

    Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with {sup 15}N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca{sup 2+}-binding loops II, III, and IV, as well a tightly hydrogen-bonded amides within the short antiparallel {beta}-sheets between pairs of Ca{sup 2+}-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca{sup 2+}-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca{sup 2+}-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated the Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca{sup 2+}-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca{sup 2+}-binding sites. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain {beta}-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca{sup 2+}-binding loops in each domain of Ca{sup 2+}-saturated cTnC3. In the absence of Ca{sup 2+}, no strong hydrogen bonds were detected between the {beta}-strands in the N-terminal domain of cTnC3. Thus, Ca{sup 2+} binding at site II results in a tightening of the Ca{sup 2+}-binding loop and formation of one strong hydrogen bond between {beta}-strands in the N-terminal domain. These changes may initiate movement of helices in the N-terminal domain responsible for the interaction of TnC with troponin I.

  18. Viral receptor-binding site antibodies with diverse germline origins

    PubMed Central

    Schmidt, Aaron G.; Therkelsen, Matthew D.; Stewart, Shaun; Kepler, Thomas B.; Liao, Hua-Xin; Moody, M. Anthony; Haynes, Barton F.; Harrison, Stephen C.

    2015-01-01

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by eleven different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B-cell targets. PMID:25959776

  19. Analysis of (/sup 3/H) Kainic acid binding with rat and Frog brain membranes

    SciTech Connect

    Zharkovskii, A.M.; Zharkovskaya, T.A.

    1985-10-01

    This paper analyzes the binding of (H 3)-KA with membrances in vitro and the effect of various neuroactive amino acids, suggested as endogenous ligands for binding sites of (H 3)-KA, on binding. Experiments were carried out on male albino rats and on winter frogs. Choice of the frog's brain was determined by the high density of high-affinity binding sites of (H 3)-KA. The concentrations of substances inhibiting binding (H 3)-KA by 50% were calculated by logit-probit analysis, and inhibition constants were also calculated. It is shown that although L-glutamate and folic acid inhibit binding of (H 3)-KA, they do not satisfy the criteria to be met by endogenous ligands, and this inhibition of binding is noncompetitive in character. This suggests that KA binding sites and glutamate receptors are not identical, although they may perhaps be subunits of a single complex.

  20. Physicochemical aspects of the energetics of binding of sulphanilic acid with bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Banipal, Tarlok S.; Kaur, Amandeep; Banipal, Parampaul K.

    2017-01-01

    The thermodynamic study of the binding of sulphanilic acid with model transport protein bovine serum albumin is a promising approach in the area of synthesizing new sulfa drugs with improved therapeutic effect. Thus, such binding studies play an important role in the rational drug design process. The binding between sulphanilic acid and bovine serum albumin has been studied using calorimetry, light scattering in combination with spectroscopic and microscopic techniques. The calorimetric data reveals the presence of two sequential nature of binding sites where the first binding site has stronger affinity ( 104 M- 1) and second binding site has weaker affinity ( 103 M- 1). However, the spectroscopic (absorption and fluorescence) results suggest the presence of single low affinity binding site ( 103 M- 1) on protein. The contribution of polar and non-polar interactions to the binding process has been explored in the presence of various additives. It is found that sulphanilic acid binds with high affinity at Sudlow site II and with low affinity at Sudlow site I of protein. Light scattering and circular dichroism measurements have been used to study the effect on the molecular topology and conformation of protein, respectively. Thus these studies provide important insights into the binding of sulphanilic acid with bovine serum albumin both quantitatively and qualitatively.

  1. Physicochemical aspects of the energetics of binding of sulphanilic acid with bovine serum albumin.

    PubMed

    Banipal, Tarlok S; Kaur, Amandeep; Banipal, Parampaul K

    2017-01-05

    The thermodynamic study of the binding of sulphanilic acid with model transport protein bovine serum albumin is a promising approach in the area of synthesizing new sulfa drugs with improved therapeutic effect. Thus, such binding studies play an important role in the rational drug design process. The binding between sulphanilic acid and bovine serum albumin has been studied using calorimetry, light scattering in combination with spectroscopic and microscopic techniques. The calorimetric data reveals the presence of two sequential nature of binding sites where the first binding site has stronger affinity (~10(4)M(-1)) and second binding site has weaker affinity (~10(3)M(-1)). However, the spectroscopic (absorption and fluorescence) results suggest the presence of single low affinity binding site (~10(3)M(-1)) on protein. The contribution of polar and non-polar interactions to the binding process has been explored in the presence of various additives. It is found that sulphanilic acid binds with high affinity at Sudlow site II and with low affinity at Sudlow site I of protein. Light scattering and circular dichroism measurements have been used to study the effect on the molecular topology and conformation of protein, respectively. Thus these studies provide important insights into the binding of sulphanilic acid with bovine serum albumin both quantitatively and qualitatively. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.

    PubMed

    Albani, J R

    2006-11-06

    Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.

  4. Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses

    PubMed Central

    Wang, Xiaoquan; Ilyushina, Natalia A.; Lugovtsev, Vladimir Y.; Bovin, Nicolai V.; Couzens, Laura K.; Gao, Jin

    2016-01-01

    ABSTRACT Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the United States in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses. IMPORTANCE Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the United States since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the U

  5. Three different binding sites of Cks1 are required for p27-ubiquitin ligation.

    PubMed

    Sitry, Danielle; Seeliger, Markus A; Ko, Tun K; Ganoth, Dvora; Breward, Sadie E; Itzhaki, Laura S; Pagano, Michele; Hershko, Avram

    2002-11-01

    Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound.

  6. Benzene selectivity in competitive arene hydrogenation: Effects of single-site catalyst···acidic oxide surface binding geometry

    DOE PAGES

    Gu, Weixing; Stalzer, Madelyn Marie; Nicholas, Christopher P.; ...

    2015-04-17

    Organozirconium complexes are chemisorbed on Brønsted acidic sulfated ZrO2 (ZrS), sulfated Al2O3 (AlS), and ZrO2–WO3 (ZrW). Under mild conditions (25 °C, 1 atm H2), the supported Cp*ZrMe3, Cp*ZrBz3, and Cp*ZrPh3 catalysts are very active for benzene hydrogenation with activities declining with decreasing acidity, ZrS >> AlS ≈ ZrW, arguing that more Brønsted acidic oxides (those having weaker corresponding conjugate bases) yield stronger surface organometallic electrophiles and for this reason have higher benzene hydrogenation activity. Benzene selective hydrogenation, a potential approach for carcinogenic benzene removal from gasoline, is probed using benzene/toluene mixtures, and selectivities for benzene hydrogenation vary with catalyst asmore » ZrBz3+/ZrS–, 83% > Cp*ZrMe2+/ZrS–, 80% > Cp*ZrBz2+/ZrS–, 67% > Cp*ZrPh2+/ZrS–, 57%. For Cp*ZrBz2+/ZrS–, which displays the highest benzene hydrogenation activity with moderate selectivity in benzene/toluene mixtures. Other benzene/arene mixtures are examined, and benzene selectivities vary with arene as mesitylene, 99%, > ethylbenzene, 86% > toluene, 67%. Structural and computational studies by solid-state NMR spectroscopy, XAS, and periodic DFT methods applied to supported Cp*ZrMe3 and Cp*ZrBz3 indicate that larger Zr···surface distances are present in more sterically encumbered Cp*ZrBz2+/AlS– vs Cp*ZrMe2+/AlS–. Furthermore, the combined XAS, solid state NMR, and DFT data argue that the bulky catalyst benzyl groups expand the “cationic” metal center–anionic sulfated oxide surface distances, and this separation/weakened ion-pairing enables the activation/insertion of more sterically encumbered arenes and influences hydrogenation rates and selectivity patterns.« less

  7. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  8. Location of platinum binding sites on bacteriorhodopsin by electron diffraction

    SciTech Connect

    Dumont, M.E.; Wiggins, J.W.; Hayward, S.B.

    1981-05-01

    A platinum-containing derivative of bacteriorhodopsin has been prepared by treating purple membranes with glycyl-L-methionatoplatinum. Low-dose electron diffraction was used to identify Pt binding sites in the 5.6 A resolution reconstruction of the bacteriorhodopsin unit cell in projection. This is a necessary first step in the use of the Pt derivative for identifying the parts of the amino acid sequence corresponding to the ..cap alpha.. helices in the bacteriorhodopsin structure and for obtaining phases for reflections out to 3.5 A resolution by the method of heavy-atom isomorphous replacement. The largest peak in a Fourier difference map between platinum-labeled and native purple membrane is larger than thespurious features expected to arise from errors in measurements of diffraction intensities.

  9. The number of nucleotide binding sites in cytochrome C oxidase.

    PubMed

    Rieger, T; Napiwotzki, J; Hüther, F J; Kadenbach, B

    1995-12-05

    The binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), [35S]ATP alpha S and 8-azido-[gamma-32P]ATP to isolated cytochrome c oxidase of bovine heart and liver and to the two-subunit enzyme of Paracoccus dentrificans was studied by measuring the fluorescence change or bound radioactivity, respectively. With TNP-ATP three binding sites were determined at cytochrome c oxidase from bovine heart and liver, both with two dissociation constants Kd of about 0.2 and 0.9 microM. Trypsin treatment of the enzyme from bovine heart, resulted in one binding site with a Kd of 0.3 microM. The two-subunit enzyme of Paracoccus dentrificans had only one binding site with a Kd of 3.6 microM. The binding of [35S]ATP alpha S to cytochrome c oxidase was studied by equilibrium dialysis. With the enzyme of bovine heart seven and the enzyme of liver six high-affinity binding sites with apparent Kd's of 7.5 and 12 microM, respectively, were obtained. The two-subunit enzyme of Paracoccus denitrificans had one binding site with a Kd of 20 microM. The large number of binding sites at cytochrome c oxidase from bovine heart, mainly at nuclear coded subunits, was verified by photoaffinity labelling with 8-azido-[gamma-32P]ATP.

  10. Predicting peptide binding sites on protein surfaces by clustering chemical interactions.

    PubMed

    Yan, Chengfei; Zou, Xiaoqin

    2015-01-05

    Short peptides play important roles in cellular processes including signal transduction, immune response, and transcription regulation. Correct identification of the peptide binding site on a given protein surface is of great importance not only for mechanistic investigation of these biological processes but also for therapeutic development. In this study, we developed a novel computational approach, referred to as ACCLUSTER, for predicting the peptide binding sites on protein surfaces. Specifically, we use the 20 standard amino acids as probes to globally scan the protein surface. The poses forming good chemical interactions with the protein are identified, followed by clustering with the density-based spatial clustering of applications with noise technique. Finally, these clusters are ranked based on their sizes. The cluster with the largest size is predicted as the putative binding site. Assessment of ACCLUSTER was performed on a diverse test set of 251 nonredundant protein-peptide complexes. The results were compared with the performance of POCASA, a pocket detection method for ligand binding site prediction. Peptidb, another protein-peptide database that contains both bound structures and unbound or homologous structures was used to test the robustness of ACCLUSTER. The performance of ACCLUSTER was also compared with PepSite2 and PeptiMap, two recently developed methods developed for identifying peptide binding sites. The results showed that ACCLUSTER is a promising method for peptide binding site prediction. Additionally, ACCLUSTER was also shown to be applicable to nonpeptide ligand binding site prediction. © 2014 Wiley Periodicals, Inc.

  11. Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity.

    PubMed Central

    Arnold, P H; Blake, D G; Grindley, N D; Boocock, M R; Stark, W M

    1999-01-01

    Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules. PMID:10064606

  12. Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

    SciTech Connect

    Sloan, J.W.

    1984-01-01

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

  13. Elucidating the influence of gold nanoparticles on the binding of salvianolic acid B and rosmarinic acid to bovine serum albumin.

    PubMed

    Peng, Xin; Qi, Wei; Huang, Renliang; Su, Rongxin; He, Zhimin

    2015-01-01

    Salvianolic acid B and rosmarinic acid are two main water-soluble active ingredients from Salvia miltiorrhiza with important pharmacological activities and clinical applications. The interactions between salvianolic acid B (or rosmarinic acid) and bovine serum albumin (BSA) in the presence and absence of gold nanoparticles (Au NPs) with three different sizes were investigated by using biophysical methods for the first time. Experimental results proved that two components quenched the fluorescence of BSA mainly through a static mechanism irrespective of the absence or presence of Au NPs. The presence of Au NPs decreased the binding constants of salvianolic acid B with BSA from 27.82% to 10.08%, while Au NPs increased the affinities of rosmarinic acid for BSA from 0.4% to 14.32%. The conformational change of BSA in the presence of Au NPs (caused by a noncompetitive binding between Au NPs and drugs at different albumin sites) induced changeable affinity and binding distance between drugs and BSA compared with no Au NPs. The competitive experiments revealed that the site I (subdomain IIA) of BSA was the primary binding site for salvianolic acid B and rosmarinic acid. Additionally, two compounds may induce conformational and micro-environmental changes of BSA. The results would provide valuable binding information between salvianolic acid B (or rosmarinic acid) and BSA, and also indicated that the Au NPs could alter the interaction mechanism and binding capability of drugs to BSA, which might be beneficial to understanding the pharmacokinetics and biological activities of the two drugs.

  14. Structural signatures of antibiotic binding sites on the ribosome

    PubMed Central

    David-Eden, Hilda; Mankin, Alexander S.; Mandel-Gutfreund, Yael

    2010-01-01

    The ribosome represents a major target for antibacterial drugs. Being a complex molecular machine, it offers many potential sites for functional interference. The high-resolution structures of ribosome in complex with various antibiotics provide a unique data set for understanding the universal features of drug-binding pockets on the ribosome. In this work, we have analyzed the structural and evolutionary properties of 65 antibiotic binding sites (ABSs) in the ribosome. We compared these sites to similar-size computed pockets extracted from the small and large ribosomal subunits. Based on this analysis, we defined properties of the known drug-binding sites, which constitute the signature of a ‘druggable’ site. The most noticeable properties of the ABSs are prevalence of non-paired bases, a strong bias in favor of unusual syn conformation of the RNA bases and an unusual sugar pucker. We propose that despite the different geometric and chemical properties of diverse antibiotics, their binding sites tend to have common attributes that possibly reflect the potency of the pocket for binding small molecules. Finally, we utilized the ensemble of properties to derive a druggability index, which can be used in conjunction with site functionality information to identify new drug-binding sites on the ribosome. PMID:20494981

  15. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  16. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  17. Models of metal binding structures in fulvic acid from the Suwannee River, Georgia

    USGS Publications Warehouse

    Leenheer, J.A.; Brown, G.K.; MacCarthy, P.; Cabaniss, S.E.

    1998-01-01

    Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca2+ ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The 'metal binding' fraction was characterized by quantitative 13C NMR, 1H NMR, and FT-1R spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short- chain aliphatic dibasic acid structures. The Ca2+ binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca2+ ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The `metal binding' fraction was characterized by quantitative 13C NMR, 1H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that

  18. Structural Consensus among Antibodies Defines the Antigen Binding Site

    PubMed Central

    Kunik, Vered; Peters, Bjoern; Ofran, Yanay

    2012-01-01

    The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ∼20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification. PMID:22383868

  19. 65-kilodalton protein phosphorylated by interleukin 2 stimulation bears two putative actin-binding sites and two calcium-binding sites

    SciTech Connect

    Zu, Youli; Shigesada, Katsuya; Hanaoka, Masao; Namba, Yuziro ); Nishida, Eisuke ); Kubota, Ichiro ); Kohno, Michiaki )

    1990-09-11

    The authors have previously characterized a 65-kilodalton protein (p65) as an interleukin 2 stimulated phosphoprotein in human T cells and showed that three endopeptide sequences of p65 are present in the sequence of l-plastin. In this paper, they present the complete primary structure of p65 based on the cDNA isolated from a human T lymphocyte (KUT-2) cDNA library. Analysis of p65 sequences and the amino acid composition of cleaved p65 N-terminal peptide indicated that the deduced p65 amino acid sequence exactly coincides with that of l-plastin over the C-terminal 580 residues and has a 57-residue extension at the N-terminus to l-plastin. Computer-assisted structural analysis revealed that p65 is a multidomain molecule involving at least three intriguing functional domains: two putative calcium-binding sites along the N-terminal 80 amino acid residues; a putative calmodulin-binding site following the calcium-binding region; and two tandem repeats of putative actin-binding domains in its middle and C-terminal parts, each containing approximately 240 amino acid residues. These results suggest that p65 belongs to actin-binding proteins.

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

    SciTech Connect

    Lerch, Thomas F.; Chapman, Michael S.

    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.

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

    SciTech Connect

    Lerch, Thomas F.; Chapman, Michael S.

    2012-05-24

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

  2. Protein binding site analysis for drug discovery using a computational fragment-based method.

    PubMed

    Ludington, Jennifer L

    2015-01-01

    One of the most powerful tools for designing drug molecules is an understanding of the target protein's binding site. Identifying key amino acids and understanding the electronic, steric, and solvation properties of the site enables the design of potent ligands. Of equal importance for the success of a drug discovery program is the evaluation of binding site druggability. Determining, a priori, if a particular binding site has the appropriate character to bind drug-like ligands saves research time and money.While there are a variety of experimental and computational techniques to identify and characterize binding sites, the focus of this chapter is on Binding Site Analysis (BSA) using virtual fragment simulations. The methodology of the technique is described, along with examples of successful application to drug discovery programs. BSA both indicates if a protein is a viable target for drug discovery and provides a roadmap for designing ligands. Using a computational fragment-based method is a effective means of understanding of a binding site.

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

    PubMed Central

    Si, Jingna; Zhao, Rui; Wu, Rongling

    2015-01-01

    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. PMID:25756377

  4. Localization of gonadotropin binding sites in human ovarian neoplasms

    SciTech Connect

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

    1989-10-01

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

  5. Binding sites associated with inhibition of photosystem II

    SciTech Connect

    Shipman, L.L.

    1981-01-01

    A variety of experimental and theoretical evidence has been integrated into coherent molecular mechanisms for the action of photosystem II herbicides. Photosystem II herbicides act by inhibiting electron transfers between the first and second plastoquinones on the reducing side of photosystem II. Each herbicide molecule must have a flat polar component with hydrophobic substituents to be active. The hydrophobic substituents serve to partition the molecule into lipid regions of the cell and to fit the hydrophobic region of the herbicide binding site. The flat polar portion of the herbicide is used for electrostatic binding to the polar region of the herbicide binding site. Theoretical calculations have been carried out to investigate the binding of herbicides to model proteinaceous binding sites.

  6. Autoradiographic localization of relaxin binding sites in rat brain

    SciTech Connect

    Osheroff, P.L.; Phillips, H.S. )

    1991-08-01

    Relaxin is a member of the insulin family of polypeptide hormones and exerts its best understood actions in the mammalian reproductive system. Using a biologically active 32P-labeled human relaxin, the authors have previously shown by in vitro autoradiography specific relaxin binding sites in rat uterus, cervix, and brain tissues. Using the same approach, they describe here a detailed localization of human relaxin binding sites in the rat brain. Displaceable relaxin binding sites are distributed in discrete regions of the olfactory system, neocortex, hypothalamus, hippocampus, thalamus, amygdala, midbrain, and medulla of the male and female rat brain. Characterization of the relaxin binding sites in the subfornical organ and neocortex reveals a single class of high-affinity sites (Kd = 1.4 nM) in both regions. The binding of relaxin to two of the circumventricular organs (subfornical organ and organum vasculosum of the lamina terminalis) and the neurosecretory magnocellular hypothalamic nuclei (i.e., paraventricular and supraoptic nuclei) provides the anatomical and biochemical basis for emerging physiological evidence suggesting a central role for relaxin in the control of blood pressure and hormone release. They conclude that specific, high-affinity relaxin binding sites are present in discrete regions of the rat brain and that the distribution of some of these sites may be consistent with a role for relaxin in control of vascular volume and blood pressure.

  7. Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2011-08-15

    Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.

  8. Mapping of SPARC/BM-40/Osteonectin-binding Sites on Fibrillar Collagens*S⃞

    PubMed Central

    Giudici, Camilla; Raynal, Nicolas; Wiedemann, Hanna; Cabral, Wayne A.; Marini, Joan C.; Timpl, Rupert; Bächinger, Hans Peter; Farndale, Richard W.; Sasaki, Takako; Tenni, Ruggero

    2008-01-01

    The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix αA of the extracellular calcium-binding domain of SPARC and is partially masked by helix αC. Previously, we found that the removal of helix αC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site ∼180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven α2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among α chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo. PMID:18487610

  9. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

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

    SciTech Connect

    Borden, L.A.; Gibbs, T.T.; Farb, D.H.

    1987-06-01

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

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

    PubMed Central

    Schroeder, Michael

    2013-01-01

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

  12. Toward an atomistic model for predicting transcription-factor binding sites.

    PubMed

    Endres, Robert G; Schulthess, Thomas C; Wingreen, Ned S

    2004-11-01

    Identifying the specific DNA-binding sites of transcription-factor proteins is essential to understanding the regulation of gene expression in the cell. Bioinformatics approaches are fast compared to experiments, but require prior knowledge of multiple binding sites for each protein. Here, we present an atomistic force-field method to predict binding sites based only on the X-ray structure of a related bound complex. Specific flexible contacts between the protein and DNA are modeled by a library of amino acid side-chain rotamers. Using the example of the mouse transcription factor, Zif268, a well-studied zinc-finger protein, we show that the protein sequence alone, without the detailed experimental structure, gives a strong bias toward the consensus binding site.

  13. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

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

    SciTech Connect

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

    1987-04-01

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

  15. Aminoglycoside antibiotics: A-site specific binding to 16S

    NASA Astrophysics Data System (ADS)

    Baker, Erin Shammel; Dupuis, Nicholas F.; Bowers, Michael T.

    2009-06-01

    The A-site of 16S rRNA, which is a part of the 30S ribosomal subunit involved in prokaryotic translation, is a well known aminoglycoside binding site. Full characterization of the conformational changes undergone at the A-site upon aminoglycoside binding is essential for development of future RNA/drug complexes; however, the massiveness of 16S makes this very difficult. Recently, studies have found that a 27 base RNA construct (16S27) that comprises the A-site subdomain of 16S behaves similarly to the whole A-site domain. ESI-MS, ion mobility and molecular dynamics methods were utilized in this study to analyze the A-site of 16S27 before and after the addition of ribostamycin (R), paromomycin (P) and lividomycin (L). The ESI mass spectrum for 16S27 alone illustrated both single-stranded 16S27 and double-stranded (16S27)2 complexes. Upon aminoglycoside addition, the mass spectra showed that only one aminoglycoside binds to 16S27, while either one or two bind to (16S27)2. Ion mobility measurements and molecular dynamics calculations were utilized in determining the solvent-free structures of the 16S27 and (16S27)2 complexes. These studies found 16S27 in a hairpin conformation while (16S27)2 existed as a cruciform. Only one aminoglycoside binds to the single A-site of the 16S27 hairpin and this attachment compresses the hairpin. Since two A-sites exist for the (16S27)2 cruciform, either one or two aminoglycosides may bind. The aminoglycosides compress the A-sites causing the cruciform with just one aminoglycoside bound to be larger than the cruciform with two bound. Non-specific binding was not observed in any of the aminoglycoside/16S27 complexes.

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

    SciTech Connect

    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. - 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.

  17. A graph kernel method for DNA-binding site prediction.

    PubMed

    Yan, Changhui; Wang, Yingfeng

    2014-01-01

    Protein-DNA interactions play important roles in many biological processes. Computational methods that can accurately predict DNA-binding sites on proteins will greatly expedite research on problems involving protein-DNA interactions. This paper presents a method for predicting DNA-binding sites on protein structures. The method represents protein surface patches using labeled graphs and uses a graph kernel method to calculate the similarities between graphs. A new surface patch is predicted to be interface or non-interface patch based on its similarities to known DNA-binding patches and non-DNA-binding patches. The proposed method achieved high accuracy when tested on a representative set of 146 protein-DNA complexes using leave-one-out cross-validation. Then, the method was applied to identify DNA-binding sites on 13 unbound structures of DNA-binding proteins. In each of the unbound structure, the top 1 patch predicted by the proposed method precisely indicated the location of the DNA-binding site. Comparisons with other methods showed that the proposed method was competitive in predicting DNA-binding sites on unbound proteins. The proposed method uses graphs to encode the feature's distribution in the 3-dimensional (3D) space. Thus, compared with other vector-based methods, it has the advantage of taking into account the spatial distribution of features on the proteins. Using an efficient kernel method to compare graphs the proposed method also avoids the demanding computations required for 3D objects comparison. It provides a competitive method for predicting DNA-binding sites without requiring structure alignment.

  18. 2( sup 125 I)Iodomelatonin binding sites in spleens of guinea pigs

    SciTech Connect

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

    1992-01-01

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

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

    PubMed Central

    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/. PMID:26987123

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

    PubMed

    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/.

  1. Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity.

    PubMed

    Rummel, Andreas; Bade, Steffen; Alves, Jürgen; Bigalke, Hans; Binz, Thomas

    2003-02-21

    Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.

  2. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.

    PubMed

    Dian, Cyril; Vitale, Sylvia; Leonard, Gordon A; Bahlawane, Christelle; Fauquant, Caroline; Leduc, Damien; Muller, Cécile; de Reuse, Hilde; Michaud-Soret, Isabelle; Terradot, Laurent

    2011-03-01

    Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS₄ site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

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

    PubMed

    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.

  4. Challenges of docking in large, flexible and promiscuous binding sites.

    PubMed

    Kotev, Martin; Soliva, Robert; Orozco, Modesto

    2016-10-15

    After decades of work, the correct determination of the binding mode of a small molecule into a target protein is still a challenging problem, whose difficulty depends on: (i) the sizes of the binding site and the ligand; (ii) the flexibility of both interacting partners, and (iii) the differential solvation of bound and unbound partners. We have evaluated the performance of standard rigid(receptor)/flexible(ligand) docking approaches with respect to last-generation fully flexible docking methods to obtain reasonable poses in a very challenging case: soluble Epoxide Hydrolase (sEH), a flexible protein showing different binding sites. We found that full description of the flexibility of both protein and ligand and accurate description of solvation leads to significant improvement in the ability of docking to reproduce well known binding modes, and at the same time capture the intrinsic binding promiscuity of the protein. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Binding sites for gonadotropins in human postmenopausal ovaries

    SciTech Connect

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

    1989-02-01

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

  6. Mg NMR in DNA solutions: Dominance of site binding effects.

    PubMed

    Rose, D M; Bleam, M L; Record, M T; Bryant, R G

    1980-11-01

    (25)Mg NMR spectroscopy is applied to a study of magnesium ion interactions with DNA, which is considered as a model for a linear polyelectrolyte. It is demonstrated that the magnesium ion spectrum is complicated by a non-Lorent-zian line shape and is dominated by the effects of chemical exchange with macromolecule binding sites. A distinction is made between specific-site interactions in which the magnesium ion loses a water molecule from the first coordination sphere on binding and those interactions, referred to as territorial binding, in which the ion maintains its first coordination sphere complement of solvent. The first type of site-binding interactions are shown to dominate the magnesium ion NMR spectrum, based on a consideration of the magnitudes of the observed (25)Mg relaxation rates compared with (23)Na relaxation rates, the clear contributions of chemical exchange-limited relaxation, and an ion displacement experiment employing sodium.

  7. Characterization of peripheral benzodiazepine binding sites in human term placenta.

    PubMed

    Fares, F; Gavish, M

    1986-01-15

    Peripheral benzodiazepine binding sites were characterized in human term placental membranes using [3H]PK 11195, which is a ligand specific for peripheral benzodiazepine binding sites. Binding of [3H]PK 11195 to human term placental membranes was found to be saturable. Scatchard analysis revealed a single population of binding sites (r = 0.98). Equilibrium dissociation constant (KD) was 2.1 +/- 0.3 nM, and density of binding sites (Bmax) was 920 +/- 105 fmol/mg protein. The KD value calculated from kinetic experiments was 3.6 +/- 0.2 nM. The ability of various drugs to displace [3H]PK 11195 from human term placental binding sites was tested: the inhibition constants (KI) for PK 11195, Ro 5-4864, and diazepam were 2.9, 11.8, and 177 nM, respectively, whereas clonazepam, methyl-beta-carboline-3-carboxylate, Ro 15-1788, chlordiazepoxide, atropine, and estradiol were inefficient in displacing [3H]PK 11195 (KI greater than 10(-5) M).

  8. Development of a protein-ligand-binding site prediction method based on interaction energy and sequence conservation.

    PubMed

    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.

  9. SiteLight: binding-site prediction using phage display libraries.

    PubMed

    Halperin, Inbal; Wolfson, Haim; Nussinov, Ruth

    2003-07-01

    Phage display enables the presentation of a large number of peptides on the surface of phage particles. Such libraries can be tested for binding to target molecules of interest by means of affinity selection. Here we present SiteLight, a novel computational tool for binding site prediction using phage display libraries. SiteLight is an algorithm that maps the 1D peptide library onto a three-dimensional (3D) protein surface. It is applicable to complexes made up of a protein Template and any type of molecule termed Target. Given the three-dimensional structure of a Template and a collection of sequences derived from biopanning against the Target, the Template interaction site with the Target is predicted. We have created a large diverse data set for assessing the ability of SiteLight to correctly predict binding sites. SiteLight predictive mapping enables discrimination between the binding and nonbinding parts of the surface. This prediction can be used to effectively reduce the surface by 75% without excluding the binding site. In 63% of the cases we have tested, there is at least one binding site prediction that overlaps the interface by at least 50%. These results suggest the applicability of phage display libraries for automated binding site prediction on three-dimensional structures. For most effective binding site prediction we propose using a random phage display library twice, to scan both binding partners of a given complex. The derived peptides are mapped to the other binding partner (now used as a Template). Here, the surface of each partner is reduced by 75%, focusing their relative positions with respect to each other significantly. Such information can be utilized to improve docking algorithms and scoring functions.

  10. Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

    PubMed Central

    Ballesta, J. J.; Garcia, A. G.; Gutierrez, L. M.; Hidalgo, M. J.; Palmero, M.; Reig, J. A.; Viniegra, S.

    1990-01-01

    1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown. PMID:1704272

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

    SciTech Connect

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. )

    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.

  12. Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites

    PubMed Central

    Smith, Amy Rhoden; Iverson, Brent L.

    2013-01-01

    The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound. We recently reported a threading tetraintercalator with a dissociation half-life of 16 days, the longest reported to date, from its preferred 14 bp binding site. Herein, three new tetraintercalator derivatives were synthesized with one, two, and three additional methylene units in the central major groove-binding linker. These molecules displayed dissociation half-lives of 57, 27, and 18 days, respectively, from the 14 bp site. The optimal major groove-binding linker was used in the design of an NDI hexaintercalator that was analyzed by gel-shift assays, DNase I footprinting, and UV-visible spectroscopy. The hexaintercalator bound its entire 22 bp binding site, the longest reported specific binding site for a synthetic, non-nucleic acid based DNA binding molecule, but with a significantly faster dissociation rate compared to the tetraintercalators. PMID:23919778

  13. Characterisation of imidazoline I2 binding sites in pig brain.

    PubMed

    Anderson, Neil J; Lupo, Patrick A; Nutt, David J; Hudson, Alan L; Robinson, Emma S J

    2005-09-05

    The imidazoline I2 binding sites in the central nervous system have previously been described in several different species including rat, mouse, rabbit and frog. The present study has investigated the imidazoline I2 binding site, and its relationship to the monoamine oxidase isoforms, in pig whole brain and compared the results obtained with data from other species. Results from saturation binding studies revealed that the imidazoline I2-selective ligand, [3H]2BFI (2-(2-benzofuranyl)-2-imidazoline) labelled a single saturable population of sites with a KD=6.6 nM and Bmax=771.7 fmol/mg protein. The pharmacological characterisation of the sites was similar to that previously reported with a rank order of potency for the imidazoline I2 ligands of 2BFI>BU224>Idazoxan>BU226. Displacement by the imidazoline I1 ligands was low affinity and the monoamine oxidase inhibitors displaced with micromolar affinity. The majority of compounds displaced the binding in a monophasic manner, however, displacement by the putative endogenous ligand, harmane was biphasic. The relative populations of the two monoamine oxidase isoforms revealed a 10 fold greater expression of monoamine oxidase B relative to monoamine oxidase A. These data confirm the presence of imidazoline I2 binding sites in pig brain and show that their pharmacology is characteristic of that seen in other species. The proportion of monoamine oxidase A and B expressed in the pig brain is similar to that seen in the human brain therefore, given the association between imidazoline I2 binding sites and monoamine oxidase, the pig may provide a more useful model for human imidazoline I2 binding sites than other species such as the rat.

  14. Characterization of Staphylococcus aureus SarA binding sites.

    PubMed

    Sterba, Kristen M; Mackintosh, Samuel G; Blevins, Jon S; Hurlburt, Barry K; Smeltzer, Mark S

    2003-08-01

    The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.

  15. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

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

    2013-03-01

    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 for actin 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. © 2012 Institute of Botany, Chinese Academy of Sciences.

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

    SciTech Connect

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

    1985-03-04

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

  17. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    PubMed

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  18. Liver Fatty Acid Binding Protein and Obesity

    PubMed Central

    Atshaves, B.P.; Martin, G.G.; Hostetler, H.A.; McIntosh, A.L.; Kier, A.B.; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15 member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP, or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age- and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair-fed a high fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age- or diet-induced obesity. PMID:20537520

  19. Models of metal binding structures in fulvic acid from the Suwannee River, Georgia

    SciTech Connect

    Leenheer, J.A.; Brown, G.K.; Cabaniss, S.E.; MacCarthy, P.

    1998-08-15

    Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca{sup 2+}, Cd{sup 2+}, Cu{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca{sup 2+} ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The metal binding fraction was characterized by quantitative {sup 13}C NMR, {sup 1}H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short-chain aliphatic dibasic acid structures. The Ca{sup 2+} binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.

  20. The binding sites for tRNA on eukaryotic ribosomes.

    PubMed Central

    Leader, D P; Machray, G C

    1975-01-01

    We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes. PMID:1098024

  1. The binding sites for tRNA on eukaryotic ribosomes.

    PubMed

    Leader, D P; Machray, G C

    1975-07-01

    We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.

  2. Transcription Factor Binding Sites Prediction Based on Modified Nucleosomes

    PubMed Central

    Talebzadeh, Mohammad; Zare-Mirakabad, Fatemeh

    2014-01-01

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

  3. Application of a continuous distribution model for proton binding by humic acids extracted from acidic lake sediments

    SciTech Connect

    Rhea, J.R.; Young, T.C. )

    1987-01-01

    The proton binding characteristics of humic acids extracted from the sediments of Cranberry Pond, an acidic water body located in the Adirondack Mountain region of New York State, were explored by the application of a nultiligand distribution model. The model characterizes a class of proton binding sites by mean log K values and the standard deviations of log K values and the mean. Mean log K values and their relative abundances were determined directly from experimental titration data. The model accurately predicts the binding of protons by the humic acids for pH values in the range 3.5 to 10.0.

  4. Application of a continuous distribution model for proton binding by humic acids extracted from acidic lake sediments

    NASA Astrophysics Data System (ADS)

    Rhea, James R.; Young, Thomas C.

    1987-10-01

    The proton binding characteristics of humic acids extracted from the sediments of Cranberry Pond, an acidic water body located in the Adirondack Mountain region of New York State, were explored by the application of a multiligand distribution model. The model characterizes a class of proton binding sites by mean log K values and the standard deviations of log K values about the mean. Mean log K values and their relative abundances were determined directly from experimental titration data. The model accurately predicts the binding of protons by the humic acids for pH values in the range 3.5 to 10.0.

  5. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: evidence for multiple estrogen binding sites

    SciTech Connect

    Lesch, K.P.; Schott, W.; Gross, S.

    1987-09-01

    The existence of estrogen receptors in human meningiomas has long been a controversial issue. This may be explained, in part, by apparent heterogeneity of estrogen binding sites in meningioma tissue. In this study, estrogen receptors were determined in 58 meningiomas with an enzyme immunoassay using monoclonal antibodies against human estrogen receptor protein (estrophilin) and with a sensitive radioligand binding assay using /sup 125/I-labeled estradiol (/sup 125/I-estradiol) as radioligand. Low levels of estrophilin immunoreactivity were found in tumors from 62% of patients, whereas radioligand binding activity was demonstrated in about 46% of the meningiomas examined. In eight (14%) tissue samples multiple binding sites for estradiol were observed. The immunoreactive binding sites correspond to the classical, high affinity estrogen receptors: the Kd for /sup 125/I-estradiol binding to the receptor was approximately 0.2 nM and the binding was specific for estrogens. The second, low affinity class of binding sites considerably influenced measurement of the classical receptor even at low ligand concentrations. The epidemiological and clinical data from patients with meningiomas, and the existence of specific estrogen receptors confirmed by immunochemical detection, may be important factors in a theory of oncogenesis.

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

    PubMed Central

    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

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

    PubMed

    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.

  8. Gaussian mapping of chemical fragments in ligand binding sites

    NASA Astrophysics Data System (ADS)

    Wang, Kun; Murcia, Marta; Constans, Pere; Pérez, Carlos; Ortiz, Angel R.

    2004-02-01

    We present a new approach to automatically define a quasi-optimal minimal set of pharmacophoric points mapping the interaction properties of a user-defined ligand binding site. The method is based on a fitting algorithm where a grid of sampled interaction energies of the target protein with small chemical fragments in the binding site is approximated by a linear expansion of Gaussian functions. A heuristic approximation selects from this expansion the smallest possible set of Gaussians required to describe the interaction properties of the binding site within a prespecified accuracy. We have evaluated the performance of the approach by comparing the computed Gaussians with the positions of aromatic sites found in experimental protein-ligand complexes. For a set of 53 complexes, good correspondence is found in general. At a 95% significance level, ˜65% of the predicted interaction points have an aromatic binding site within 1.5 Å. We then studied the utility of these points in docking using the program DOCK. Short docking times, with an average of ˜0.18 s per conformer, are obtained, while retaining, both for rigid and flexible docking, the ability to sample native-like binding modes for the ligand. An average 4-5-fold speed-up in docking times and a similar success rate is estimated with respect to the standard DOCK protocol. Abbreviations: RMSD - root mean square deviation; ASA - Atomic Shell Approximation; LSF - Least-Squares Fitting; 3D - three-dimensional; VDW - Van der Waals.

  9. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  10. Identification of an imidazoline binding protein: Creatine kinase and an imidazoline-2 binding site

    PubMed Central

    Kimura, Atsuko; Tyacke, Robin J.; Robinson, James J.; Husbands, Stephen M.; Minchin, Michael C.W.; Nutt, David J.; Hudson, Alan L.

    2009-01-01

    Drugs that bind to imidazoline binding proteins have major physiological actions. To date, three subtypes of such proteins, I1, I2 and I3, have been proposed, although characterisations of these binding proteins are lacking. I2 binding sites are found throughout the brain, particularly dense in the arcuate nucleus of the hypothalamus. Selective I2 ligands demonstrate antidepressant-like activity and the identity of the proteins that respond to such ligands remained unknown until now. Here we report the isolation of a ∼ 45 kDa imidazoline binding protein from rabbit and rat brain using a high affinity ligand for the I2 subtype, 2-BFI, to generate an affinity column. Following protein sequencing of the isolated ∼ 45 kDa imidazoline binding protein, we identified it to be brain creatine kinase (B-CK). B-CK shows high binding capacity to selective I2 ligands; [3H]-2-BFI (5 nM) specifically bound to B-CK (2330 ± 815 fmol mg protein− 1). We predicted an I2 binding pocket near the active site of B-CK using molecular modelling. Furthermore, B-CK activity was inhibited by a selective I2 irreversible ligand, where 20 μM BU99006 reduced the enzyme activity by 16%, confirming the interaction between B-CK and the I2 ligand. In summary, we have identified B-CK to be the ∼ 45 kDa imidazoline binding protein and we have demonstrated the existence of an I2 binding site within this enzyme. The importance of B-CK in regulating neuronal activity and neurotransmitter release may well explain the various actions of I2 ligands in brain and the alterations in densities of I2 binding sites in psychiatric disorders. PMID:19410564

  11. ConBind: motif-aware cross-species alignment for the identification of functional transcription factor binding sites.

    PubMed

    Lelieveld, Stefan H; Schütte, Judith; Dijkstra, Maurits J J; Bawono, Punto; Kinston, Sarah J; Göttgens, Berthold; Heringa, Jaap; Bonzanni, Nicola

    2016-05-05

    Eukaryotic gene expression is regulated by transcription factors (TFs) binding to promoter as well as distal enhancers. TFs recognize short, but specific binding sites (TFBSs) that are located within the promoter and enhancer regions. Functionally relevant TFBSs are often highly conserved during evolution leaving a strong phylogenetic signal. While multiple sequence alignment (MSA) is a potent tool to detect the phylogenetic signal, the current MSA implementations are optimized to align the maximum number of identical nucleotides. This approach might result in the omission of conserved motifs that contain interchangeable nucleotides such as the ETS motif (IUPAC code: GGAW). Here, we introduce ConBind, a novel method to enhance alignment of short motifs, even if their mutual sequence similarity is only partial. ConBind improves the identification of conserved TFBSs by improving the alignment accuracy of TFBS families within orthologous DNA sequences. Functional validation of the Gfi1b + 13 enhancer reveals that ConBind identifies additional functionally important ETS binding sites that were missed by all other tested alignment tools. In addition to the analysis of known regulatory regions, our web tool is useful for the analysis of TFBSs on so far unknown DNA regions identified through ChIP-sequencing. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. The nucleic acid-binding zinc finger protein of potato virus M is translated by internal initiation as well as by ribosomal frameshifting involving a shifty stop codon and a novel mechanism of P-site slippage.

    PubMed

    Gramstat, A; Prüfer, D; Rohde, W

    1994-09-25

    The genes for the capsid protein CP and the nucleic acid-binding 12K protein (pr12) of potato virus M (PVM) constitute the 3' terminal gene cluster of the PVM RNA genome. Both proteins are presumably translated from a single subgenomic RNA. We have identified two translational strategies operating in pr12 gene expression. Internal initiation at the first and the second AUG codon of the pr12 coding sequence results in the synthesis of the 12K protein. In addition the protein is produced as a CP/12K transframe protein by ribosomal frameshifting. For these studies parts of the CP and pr12 coding sequences including the putative frameshift region were introduced into an internal position of the beta-glucuronidase gene. Mutational analyses in conjunction with in vitro translation experiments identified a homopolymeric string of four adenosine nucleotides which together with a 3' flanking UGA stop codon were required for efficient frameshifting. The signal AAAAUGA is the first frameshift signal with a shifty stop codon to be analyzed in the eukaryotic system. Substitution of the four consecutive adenosine nucleotides by UUUU increased the efficiency of frameshifting, while substitution by GGGG or CCCC dramatically reduced the synthesis of the transframe protein. Also, UAA and UAG could replace the opal stop codon without effect on the frameshifting event, but mutation of UGA to the sense codon UGG inhibited transframe protein formation. These findings suggest that the mechanism of ribosomal frameshifting at the PVM signal is different from the one described by the 'simultaneous slippage' model in that only the string of four adenosine nucleotides represents the slippery sequence involved in a -1 P-site slippage.

  13. Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid

    SciTech Connect

    Giedroc, D.P.; Sinha, S.K.; Brew, K.; Puett, D.

    1985-11-05

    The CaS -dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing CaS by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by (TH)acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in CaS -saturated calmodulin. In the presence of CaS and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptides and proteins, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in CaS -mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the CaS -saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein.

  14. Exploring the orthosteric binding site of the γ-aminobutyric acid type A receptor using 4-(Piperidin-4-yl)-1-hydroxypyrazoles 3- or 5-imidazolyl substituted: design, synthesis, and pharmacological evaluation.

    PubMed

    Krall, Jacob; Jensen, Claus H; Sørensen, Troels E; Nielsen, Birgitte; Jensen, Anders A; Sander, Tommy; Balle, Thomas; Frølund, Bente

    2013-08-22

    A series of 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP) 3- or 5-imidazolyl substituted analogues have been designed, synthesized, and characterized pharmacologically. All analogues showed binding affinities in the low micro- to low nanomolar range at native rat GABAA receptors and were found to be antagonists at the human α1β2γ2s receptor. The structure-activity relationship of the compound series demonstrates distinct differences in size and architecture of previously discovered cavities in the vicinity of the 4-PHP scaffold in the orthosteric binding site.

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

    SciTech Connect

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

    1986-08-01

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

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

    SciTech Connect

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

    1983-07-01

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

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

    PubMed Central

    2010-01-01

    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, except for few specific

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

    PubMed

    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

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

    SciTech Connect

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

    2015-03-09

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

  20. Bordetella dermonecrotic toxin binds to target cells via the N-terminal 30 amino acids.

    PubMed

    Fukui-Miyazaki, Aya; Ohnishi, Shinya; Kamitani, Shigeki; Abe, Hiroyuki; Horiguchi, Yasuhiko

    2011-03-01

    Bordetella dermonecrotic toxin (DNT) affects the biological function of host cells by activating intracellular Rho GTPases. The toxin binds to unidentified receptor(s) via 54 N-terminal amino acids, undergoes intramolecular cleavage on the C-terminal side of Arg(44) by furin or furin-like protease, and eventually enters the cytoplasm where the Rho GTPases reside. The binding to the receptor(s) and intramolecular cleavage are essential for DNT to intoxicate cells, and the 54 amino-acid binding domain encompasses the cleavage site, however, it is unclear whether these two events are related. In this study, we could narrow down the cell-binding domain to the N-terminal amino acids 2-30. The region does not contain the furin-recognition site, indicating that the cell binding and the intramolecular cleavage are independent events.

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

    SciTech Connect

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

    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.

  2. ASCONA: Rapid Detection and Alignment of Protein Binding Site Conformations.

    PubMed

    Bietz, Stefan; Rarey, Matthias

    2015-08-24

    The usage of conformational ensembles constitutes a widespread technique for the consideration of protein flexibility in computational biology. When experimental structures are applied for this purpose, alignment techniques are usually required in dealing with structural deviations and annotation inconsistencies. Moreover, many application scenarios focus on protein ligand binding sites. Here, we introduce our new alignment algorithm ASCONA that has been specially geared to the problem of aligning multiple conformations of sequentially similar binding sites. Intense efforts have been directed to an accurate detection of highly flexible backbone deviations, multiple binding site matches within a single structure, and a reliable, but at the same time highly efficient, search algorithm. In contrast, most available alignment methods rather target other issues, e.g., the global alignment of distantly related proteins that share structurally conserved regions. For conformational ensembles, this might not only result in an overhead of computation time but could also affect the achieved accuracy, especially for more complicated cases as highly flexible proteins. ASCONA was evaluated on a test set containing 1107 structures of 65 diverse proteins. In all cases, ASCONA was able to correctly align the binding site at an average alignment computation time of 4 ms per target. Furthermore, no false positive matches were observed when searching the same query sites in the structures of other proteins. ASCONA proved to cope with highly deviating backbone structures and to tolerate structural gaps and moderate mutation rates. ASCONA is available free of charge for academic use at http://www.zbh.uni-hamburg.de/ascona .

  3. Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1985-01-01

    Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made. PMID:3977850

  4. Investigation of competitive binding of ibuprofen and salicylic acid with serum albumin by affinity capillary electrophoresis.

    PubMed

    Li, Zhu-Mei; Wei, Chuan-Wan; Zhang, Yintang; Wang, Di-Shan; Liu, You-Nian

    2011-07-01

    Ibuprofen and salicylic acid, two typical non-steroidal anti-inflammatory drugs, are used commonly as analgesic drug in clinical medicine and sometimes are co-administered. When the drugs are co-administered, the drug-drug interactions may occur, and can lead to alter the safety and efficacy of drugs, resulting in variations in drug response of the co-administered drugs. Affinity capillary electrophoresis (ACE) was employed to investigate the competitive binding of ibuprofen and salicylic acid on serum albumin. Mobility ratio, derivatives from mobility shift method, was used to deduce the binding constant (K(b)). The binding constants of ibuprofen with HSA are 2.97×10⁶ M⁻¹ and 7.07×10⁴ M⁻¹, respectively; while for salicylic acid, the binding constant is 5.99×10⁴ M⁻¹. The competitive binding of the two drugs was investigated by addition of excessive ibuprofen into the solutions containing constant concentrations of salicylic acid and serum albumin. The results confirmed that ibuprofen and salicylic acid have different high-affinity binding site, but share some low-affinity binding sites on the serum albumin; and ibuprofen is able to partially replace salicylic acid from the preformed binary complexes of serum albumin and salicylic acid. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  6. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

    PubMed

    Sage, Jay M; Cura, Anthony J; Lloyd, Kenneth P; Carruthers, Anthony

    2015-05-15

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

  7. Chemical modification of human albumin at cys34 by ethacrynic acid: structural characterisation and binding properties.

    PubMed

    Bertucci, C; Nanni, B; Raffaelli, A; Salvadori, P

    1998-10-01

    Derivatization of the free cys3,4 in human albumin, which is reported to occur under physiological conditions, has been performed in vitro by reaction of the protein with ethacrynic acid. This modification has been investigated by mass spectrometry and circular dichroism. Ethacrynic acid has been proven to bind human albumin either covalently and non-covalently. This post-translational modification does not determine significant changes in the secondary structure of the protein, as shown by the comparable circular dichroism spectra of the native and the modified proteins. Furthermore, the binding properties of the human albumin samples have been investigated by circular dichroism and equilibrium dialysis. The affinity to the higher affinity binding sites does not change either for drugs binding to site I, like phenylbutazone, or to site II, like diazepam, while a small but significant increase has been observed for bilirubin, known to bind to site III. Nevertheless significant decreases of the affinity at the lower affinity binding sites of the modified protein were observed for both drugs binding to site I or to site II.

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

    PubMed Central

    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

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

    PubMed

    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.

  10. Specific binding sites for muramyl peptides on murine macrophages

    SciTech Connect

    Silverman, D.H.S.; Krueger, J.M.; Karnovsky, M.L.

    1986-03-15

    Two radiolabeled (/sup 125/I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and K/sub D/ values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.

  11. Curcumin recognizes a unique binding site of tubulin.

    PubMed

    Chakraborti, Soumyananda; Das, Lalita; Kapoor, Neha; Das, Amlan; Dwivedi, Vishnu; Poddar, Asim; Chakraborti, Gopal; Janik, Mark; Basu, Gautam; Panda, Dulal; Chakrabarti, Pinak; Surolia, Avadhesha; Bhattacharyya, Bhabatarak

    2011-09-22

    Although curcumin is known for its anticarcinogenic properties, the exact mechanism of its action or the identity of the target receptor is not completely understood. Studies on a series of curcumin analogues, synthesized to investigate their tubulin binding affinities and tubulin self-assembly inhibition, showed that: (i) curcumin acts as a bifunctional ligand, (ii) analogues with substitution at the diketone and acetylation of the terminal phenolic groups of curcumin are less effective, (iii) a benzylidiene derivative, compound 7, is more effective than curcumin in inhibiting tubulin self-assembly. Cell-based studies also showed compound 7 to be more effective than curcumin. Using fluorescence spectroscopy we show that curcumin binds tubulin 32 Å away from the colchicine-binding site. Docking studies also suggests that the curcumin-binding site to be close to the vinblastine-binding site. Structure-activity studies suggest that the tridented nature of compound 7 is responsible for its higher affinity for tubulin compared to curcumin.

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

    PubMed Central

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

    2016-01-01

    ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

  13. Molecular simulations of multimodal ligand-protein binding: elucidation of binding sites and correlation with experiments.

    PubMed

    Freed, Alexander S; Garde, Shekhar; Cramer, Steven M

    2011-11-17

    Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

  14. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

    PubMed Central

    Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

    2015-01-01

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

  15. Quest for the binding mode of malachite green with humic acid

    NASA Astrophysics Data System (ADS)

    Zhang, Hongmei; Yin, Mingxing; Shi, Jinghua; Wang, Yanqing

    2015-02-01

    The association of malachite green (MG) with humic acid (HA) was investigated by using fluorescence, UV-vis spectroscopy and molecular Modelling method. The fluorescence spectral results indicated that the binding between MG and HA occurred by mainly hydrophobic and electrostatic forces with association constants of KA (298 K) = 6.24 × 105 L/mol and KA (310 K) = 10.20 × 105 L/mol. There were more than one binding sites on HA to bind with MG. The binding sites of MG with HA primarily located at the aromatic rings of HA. MG could enter into the hydrophobic cavities of HA to quench the fluorescence of HA. On the contrary, HA binding caused MG to a coplanar conformation with more extended π bond distribution by π-π stacking interactions. The experiment and calculation data both showed that the hydrophobic binding cavities in HA played a key role in its binding with MG.

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

    PubMed

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

    2015-07-01

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

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

    PubMed

    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.

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

    PubMed

    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.

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

    SciTech Connect

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

    PubMed

    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.

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

    PubMed Central

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

    1995-01-01

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

  2. Site-specific mutagenesis of the calcium-binding photoprotein aequorin

    PubMed Central

    Tsuji, Frederick I.; Inouye, Satoshi; Goto, Toshio; Sakaki, Yoshiyuki

    1986-01-01

    The luminescent protein aequorin from the jellyfish Aequoria victoria emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. In order to understand the mechanism of the reaction, a study of structure-function relationships was undertaken with respect to modifying certain of its amino acid residues. This was done by carrying out oligonucleotide-directed site-specific mutagenesis of apoaequorin cDNA and expressing the mutagenized cDNA in Escherichia coli. Amino acid substitutions were made at the three Ca2+-binding sites, the three cysteines, and a histidine in one of the hydrophobic regions. Subsequent assay of the modified aequorin showed that the Ca2+-binding sites, the cysteines, and probably the histidine all play a role in the bioluminescence reaction of aequorin. PMID:16593774

  3. Elucidating the Influence of Gold Nanoparticles on the Binding of Salvianolic Acid B and Rosmarinic Acid to Bovine Serum Albumin

    PubMed Central

    Peng, Xin; Qi, Wei; Huang, Renliang; Su, Rongxin; He, Zhimin

    2015-01-01

    Salvianolic acid B and rosmarinic acid are two main water-soluble active ingredients from Salvia miltiorrhiza with important pharmacological activities and clinical applications. The interactions between salvianolic acid B (or rosmarinic acid) and bovine serum albumin (BSA) in the presence and absence of gold nanoparticles (Au NPs) with three different sizes were investigated by using biophysical methods for the first time. Experimental results proved that two components quenched the fluorescence of BSA mainly through a static mechanism irrespective of the absence or presence of Au NPs. The presence of Au NPs decreased the binding constants of salvianolic acid B with BSA from 27.82% to 10.08%, while Au NPs increased the affinities of rosmarinic acid for BSA from 0.4% to 14.32%. The conformational change of BSA in the presence of Au NPs (caused by a noncompetitive binding between Au NPs and drugs at different albumin sites) induced changeable affinity and binding distance between drugs and BSA compared with no Au NPs. The competitive experiments revealed that the site I (subdomain IIA) of BSA was the primary binding site for salvianolic acid B and rosmarinic acid. Additionally, two compounds may induce conformational and micro-environmental changes of BSA. The results would provide valuable binding information between salvianolic acid B (or rosmarinic acid) and BSA, and also indicated that the Au NPs could alter the interaction mechanism and binding capability of drugs to BSA, which might be beneficial to understanding the pharmacokinetics and biological activities of the two drugs. PMID:25861047

  4. Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

    PubMed Central

    Akahane, K; Kimura, Y; Tsutomi, Y; Hayakawa, I

    1994-01-01

    The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA. PMID:7840564

  5. Characterization of a labile naloxone binding site (lambda site) in rat brain.

    PubMed

    Grevel, J; Yu, V; Sadée, W

    1985-05-01

    A high-affinity binding site selective for naloxone and other 4,5-epoxymorphinans (lambda site) has been previously described in rat brain. Following homogenization of freshly dissected brain, the lambda sites convert from a high-affinity to a low-affinity state. When measured with [3H]naloxone, the decay is very rapid at 20 degrees C (t 1/2 less than 2 min), whereas it is progressively slowed at lower temperatures. Proteinase inhibitors, antoxidants, and sulfhydryl group-protecting agents failed to prevent this conversion. Kinetic measurements of mu and lambda binding at varying temperatures demonstrated that the decrease in lambda binding does not coincide with the concurrent increase in mu binding and that the loss of high-affinity lambda binding at 20 degrees C can be partially restored when the temperature is lowered to 0 degrees C. The low-affinity state of the lambda site is rather stable in the Tris buffer homogenates and is susceptible to digestion by a protease. The (-)-isomer of WIN 44,441, a benzomorphan drug, binds to lambda sites with moderate affinity (dissociation constant, KD = 63 nM), whereas the (+)-isomer does not (KD greater than 10,000 nM), thus establishing stereoselectivity of the binding process. Neither the high-affinity nor the low-affinity state of lambda binding is significantly affected by the presence of 100 mM sodium chloride or 50 microM Gpp(NH)p, (a GTP analog), which is in contrast to the dramatic effect of these agents on the established opioid receptor system. Naltrexone, naloxone, nalorphine, and morphine (in this order of decreasing potency) bind to the lambda site in vivo in intact rat brain over dosage ranges that are commonly employed in pharmacological studies.

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

    PubMed Central

    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

  7. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

    PubMed Central

    Barchan, D; Kachalsky, S; Neumann, D; Vogel, Z; Ovadia, M; Kochva, E; Fuchs, S

    1992-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well. Images PMID:1380164

  8. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

    PubMed

    Barchan, D; Kachalsky, S; Neumann, D; Vogel, Z; Ovadia, M; Kochva, E; Fuchs, S

    1992-08-15

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

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

  11. Probing Molecular Docking in a Charged Model Binding Site

    PubMed Central

    Brenk, Ruth; Vetter, Stefan W.; Boyce, Sarah E.; Goodin, David B.; Shoichet, Brian K.

    2011-01-01

    A model binding site was used to investigate charge–charge interactions in molecular docking. This simple site, a small (180 Å3) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre”ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20 μM and 60 μM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the “naked” site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions

  12. Variable structure motifs for transcription factor binding sites.

    PubMed

    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

  13. Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression

    NASA Astrophysics Data System (ADS)

    Lengyel, Iván M.; Morelli, Luis G.

    2017-04-01

    Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

  14. Multiple binding sites for transcriptional repressors can produce regular bursting and enhance noise suppression.

    PubMed

    Lengyel, Iván M; Morelli, Luis G

    2017-04-01

    Cells may control fluctuations in protein levels by means of negative autoregulation, where transcription factors bind DNA sites to repress their own production. Theoretical studies have assumed a single binding site for the repressor, while in most species it is found that multiple binding sites are arranged in clusters. We study a stochastic description of negative autoregulation with multiple binding sites for the repressor. We find that increasing the number of binding sites induces regular bursting of gene products. By tuning the threshold for repression, we show that multiple binding sites can also suppress fluctuations. Our results highlight possible roles for the presence of multiple binding sites of negative autoregulators.

  15. E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

    PubMed Central

    Rabinovich, Alina; Jin, Victor X.; Rabinovich, Roman; Xu, Xiaoqin; Farnham, Peggy J.

    2008-01-01

    We have previously shown that most sites bound by E2F family members in vivo do not contain E2F consensus motifs. However, differences between in vivo target sites that contain or lack a consensus E2F motif have not been explored. To understand how E2F binding specificity is achieved in vivo, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) assays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are (1) the site must be in a core promoter and (2) the region must be utilized as a promoter in that cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including (1) indirect recruitment, (2) looping to the core promoter mediated by an E2F bound to a distal motif, and (3) assisted binding of E2F to a site that weakly resembles an E2F motif. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated fragments. Our findings suggest that in vivo (1) a consensus motif is not sufficient to recruit E2Fs, (2) E2Fs can bind to isolated regions that lack a consensus motif, and (3) binding can require regions other than the best match to the E2F motif. PMID:18836037

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

    PubMed

    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)

  17. Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

    PubMed

    Abou-Zied, Osama K

    2015-01-01

    Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena.

  18. Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites

    PubMed Central

    Angelini, Sandra; Boy, Diana; Schiltz, Emile; Koch, Hans-Georg

    2006-01-01

    Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRα subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRβ subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY–SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor. PMID:16923832

  19. Peanut lectin-binding sites in large bowel carcinoma.

    PubMed

    Cooper, H S

    1982-10-01

    Peanut lectin is known to bind to B-D-Gal-(1 leads to 3)-D-GalNac which provides antigenic determination for the T (TAg) blood group antigen. We examined 33 rectosigmoid carcinomas and 15 corresponding controls for their ability to express peanut lectin-binding sites. In controls one could localize TAg to the supranuclear portion of the cell, however, in cancers one noticed a cytostructural relocalization of TAg with the following two major patterns: localization to the region of the glycocalyx and localization intracytoplasmically in the apical portion of the cell. These two patterns were associated with glandular differentiation. Less frequently noted or in association with the above was a mucin glob-like pattern and/or a fine diffuse intracytoplasmic pattern associated with solid, nonglandular areas. The more poorly differentiated cancers less frequently expressed peanut lectin-binding sites. Benign (nontransitional zone) epithelium in those patients whose tumor expressed TAg was negative for peanut lectin-binding sites in 66 per cent of the cases. Reduced tumoral glycosyltransferases may explain this increased synthesis of TAg in cancers as compared with controls, if one considers TAg to be an incomplete glycoprotein of the MN blood group system.

  20. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

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

    2015-01-01

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

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

    DOE PAGES

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; ...

    2015-03-09

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

  2. Neuraminidase Receptor Binding Variants of Human Influenza A(H3N2) Viruses Resulting from Substitution of Aspartic Acid 151 in the Catalytic Site: a Role in Virus Attachment?▿

    PubMed Central

    Lin, Yi Pu; Gregory, Victoria; Collins, Patrick; Kloess, Johannes; Wharton, Stephen; Cattle, Nicholas; Lackenby, Angie; Daniels, Rodney; Hay, Alan

    2010-01-01

    Changes in the receptor binding characteristics of human H3N2 viruses have been evident from changes in the agglutination of different red blood cells (RBCs) and the reduced growth capacity of recently isolated viruses, particularly in embryonated eggs. An additional peculiarity of viruses circulating in 2005 to 2009 has been the poor inhibition of hemagglutination by postinfection ferret antisera for many viruses isolated in MDCK cells, including homologous reference viruses. This was shown not to be due to an antigenic change in hemagglutinin (HA) but was shown to be the result of a mutation in aspartic acid 151 of neuraminidase (NA) to glycine, asparagine, or alanine, which caused an oseltamivir-sensitive agglutination of RBCs. The D151G substitution was shown to cause a change in the specificity of NA such that it acquired the capacity to bind receptors, which were refractory to enzymatic cleavage, without altering its ability to remove receptors for HA. Thus, the inhibition of NA-dependent agglutination by the inclusion of oseltamivir carboxylate in the assay was effective in restoring the anti-HA specificity of the hemagglutination inhibition (HI) assay for monitoring antigenic changes in HA. Since the NA-dependent binding activity did not affect virus neutralization, and virus populations in clinical specimens possessed, at most, low levels of the “151 mutant,” the biological significance of this feature of NA in, for example, immune evasion is unclear. It is apparent, however, that an important role of aspartic acid 151 in the activity of NA may be to restrict the specificity of the NA interaction and its receptor-destroying activity to complement that of HA receptor binding. PMID:20410266

  3. Photoaffinity labeling in target- and binding-site identification

    PubMed Central

    Smith, Ewan; Collins, Ian

    2015-01-01

    Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

  4. Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site.

    PubMed

    Al-Zyoud, Walid A; Hynson, Robert M G; Ganuelas, Lorraine A; Coster, Adelle C F; Duff, Anthony P; Baker, Matthew A B; Stewart, Alastair G; Giannoulatou, Eleni; Ho, Joshua W K; Gaus, Katharina; Liu, Dali; Lee, Lawrence K; Böcking, Till

    2016-02-18

    Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR-DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR-DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter

    NASA Astrophysics Data System (ADS)

    Ritchie, Jason D.; Perdue, E. Michael

    2003-01-01

    The acid-base properties of 14 standard and reference materials from the International Humic Substances Society (IHSS) were investigated by potentiometric titration. Titrations were conducted in 0.1 M NaCl under a nitrogen atmosphere, averaging 30 min from start to finish. Concentrations of carboxyl groups and phenolic groups were estimated directly from titration curves. Titration data were also fit to a modified Henderson-Hasselbalch model for two classes of proton-binding sites to obtain "best fit" parameters that describe proton-binding curves for the samples. The model was chosen for its simplicity, its ease of implementation in computer spreadsheets, and its excellent ability to describe the shapes of the titration curves. The carboxyl contents of the IHSS samples are in the general order: terrestrial fulvic acids > aquatic fulvic acids > Suwannee River natural organic matter (NOM) > aquatic humic acids > terrestrial humic acids. Overall, fulvic acids and humic acids have similar phenolic contents; however, all of the aquatically derived samples have higher phenolic contents than the terrestrially derived samples. The acid-base properties of reference Suwannee River NOM are surprisingly similar to those of standard Suwannee River humic acid. Results from titrations in this study were compared with other published results from both direct and indirect titrations. Typically, carboxyl contents for the IHSS samples were in agreement with the results from both methods of titration. Phenolic contents for the IHSS samples were comparable to those determined by direct titrations, but were significantly less than estimates of phenolic content that were based on indirect titrations with Ba(OH) 2 and Ca(OAc) 2. The average phenolic-to-carboxylic ratio of the IHSS samples is approximately 1:4. Models that assume a 1:2 ratio of phenolic-to-carboxylic groups may overestimate the relative contribution of phenolic groups to the acid-base chemistry of humic substances.

  6. [Kinetics of ligand binding to nucleic acids at random fillings].

    PubMed

    Arakelian, V B; Babaian, S Iu; Tairian, V I; Arakelian, A V; Parsadanian, M A; Vardevanian, P O

    2006-01-01

    Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.

  7. Comparison of SARS and NL63 papain-like protease binding sites and binding site dynamics: inhibitor design implications.

    PubMed

    Chaudhuri, Rima; Tang, Sishi; Zhao, Guijun; Lu, Hui; Case, David A; Johnson, Michael E

    2011-11-25

    The human severe acute respiratory syndrome coronavirus (SARS-CoV) and the NL63 coronaviruses are human respiratory pathogens for which no effective antiviral treatment exists. The papain-like cysteine proteases encoded by the coronavirus (SARS-CoV: PLpro; NL63: PLP1 and PLP2) represent potential targets for antiviral drug development. Three recent inhibitor-bound PLpro structures highlight the role of an extremely flexible six-residue loop in inhibitor binding. The high binding site plasticity is a major challenge in computational drug discovery/design efforts. From conventional molecular dynamics and accelerated molecular dynamics (aMD) simulations, we find that with conventional molecular dynamics simulation, PLpro translationally samples the open and closed conformation of BL2 loop on a picosecond-nanosecond timescale but does not reproduce the peptide bond inversion between loop residues Tyr269 and Gln270 that is observed on inhibitor GRL0617 binding. Only aMD simulation, starting from the closed loop conformation, reproduced the 180° ϕ-ψ dihedral rotation back to the open loop state. The Tyr-Gln peptide bond inversion appears to involve a progressive conformational change of the full loop, starting at one side, and progressing to the other. We used the SARS-CoV apo X-ray structure to develop a model of the NL63-PLP2 catalytic site. Superimposition of the PLP2 model on the PLpro X-ray structure identifies binding site residues in PLP2 that contribute to the distinct substrate cleavage site specificities between the two proteases. The topological and electrostatic differences between the two protease binding sites also help explain the selectivity of non-covalent PLpro inhibitors.

  8. Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

    DOE PAGES

    Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; ...

    2015-07-13

    Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. In thismore » paper, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. Finally, H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.« less

  9. Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

    SciTech Connect

    Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; Sapparapu, Gopal; Crowe, James. E.; Spiller, Benjamin W.

    2015-07-13

    Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. In this paper, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. Finally, H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.

  10. Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

    PubMed Central

    Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; Sapparapu, Gopal; Crowe, James. E.; Spiller, Benjamin W.

    2015-01-01

    Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. Here, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility. PMID:26170302

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

    SciTech Connect

    Fiorica, E.; Spector, S.

    1988-01-01

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

  12. N2 Binding to an Iron-Sulfur-Carbon Site

    PubMed Central

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

    2015-01-01

    Nitrogenases are found in some microorganisms, and these enzymes convert atmospheric N2 to ammonia, thereby providing essential nitrogen atoms for higher organisms. Some nitrogenases reduce atmospheric N2 at the FeMoco, a sulfur-rich iron-molybdenum cluster1–5. The iron centers that are coordinated to sulfur and carbon atoms in FeMoco have been proposed as the substrate binding sites, based on kinetic and spectroscopic studies5,6. Studies on the enzyme indicate that iron atom Fe6 and possibly also adjacent belt iron sites are involved.5–8 In the resting state, the central Fe sites (including Fe6) have identical environments consisting of three sulfides and a carbide. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown5. In this manuscript, we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe–N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site. PMID:26416755

  13. Assembly of a π-π stack of ligands in the binding site of an acetylcholine-binding protein.

    PubMed

    Stornaiuolo, Mariano; De Kloe, Gerdien E; Rucktooa, Prakash; Fish, Alexander; van Elk, René; Edink, Ewald S; Bertrand, Daniel; Smit, August B; de Esch, Iwan J P; Sixma, Titia K

    2013-01-01

    Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π-π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design.

  14. Noble gas binding to human serum albumin using docking simulation: nonimmobilizers and anesthetics bind to different sites.

    PubMed

    Seto, Tomoyoshi; Isogai, Hideto; Ozaki, Masayuki; Nosaka, Shuichi

    2008-10-01

    Nonimmobilizers are structurally similar to anesthetics, but do not produce anesthesia at clinically relevant concentrations. Xenon, krypton, and argon are anesthetics, whereas neon and helium are nonimmobilizers. The structures of noble gases with anesthetics or nonimmobilizers are similar and their interactions are simple. Whether the binding site of anesthetics differs from that of nonimmobilizers has long been a question in molecular anesthesiology. We investigated the binding sites and energies of anesthetic and nonimmobilizer noble gases in human serum albumin (HSA) because the 3D structure of HSA is well known and it has an anesthetic binding site. The computational docking simulation we used searches for binding sites and calculates the binding energy for small molecules and a template molecule. Xenon, krypton, and argon were found to bind to the enflurane binding site of HSA, whereas neon and helium were found to bind to sites different from the xenon binding site. Rare gas anesthetic binding was dominated by van der Waals energy, while nonimmobilizer binding was dominated by solvent-effect energy. Binding site preference was determined by the ratios of local binding energy (van der Waals energy) and nonspecific binding energy (solvent-effect energy) to the total binding energy. van der Waals energy dominance is necessary for anesthetic binding. This analysis of binding energy components provides a rationale for the binding site difference of anesthetics and nonimmobilizers, reveals the differences between the binding interactions of anesthetics and nonimmobilizers, may explain pharmacological differences between anesthetics and nonimmobilizers, and provide an understanding of anesthetic action at the atomic level.

  15. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  16. Neuropeptide Y binding sites in rat brain identified with purified neuropeptide Y-I125

    SciTech Connect

    Walker, M.W.; Miller, R.J.

    1986-03-05

    Neuropeptide Y (NPY) is a widely distributed neuronally localized peptide with 36 amino acids, 5 of which are tyrosines. The authors wished to investigate the properties of specific receptors for NPY. They therefore labeled the tyrosines with I125 using chloramine T and then purified the peptide using HPLC. A single mono-iodinated species of NPY which yielded > 85% specific binding in rat forebrain synaptosomes was selected as the ligand for all subsequent experiments. A time course of binding showed that equilibrium conditions were reached in 60 minutes at 21/sup 0/C. Scatchard plots revealed a single class of binding sites with a Kd and a Bmax of 3 x 10-10 M and 28 pmol/mg, respectively. Competition binding with unlabeled NPY showed 50% displacement of bound ligand at 1 x 10-10 M NPY. Competition binding with rat pancreatic polypeptide (RPP), a homologous peptide possessing little NPY-like activity, showed 50% displacement of bound ligand at 2 x 10/sup -7/ M RPP. No binding was observed on F-11 or PC12 neuronal cell lines, or on HSWP fibroblast cells. They conclude that NPY-I125 purified to homogeneity with HPLC is a highly selective ligand for NPY receptor sites. They are currently investigating such sites in brain, gut, and other tissues.

  17. Positive cooperative regulation of double binding sites for human acetylcholinesterase.

    PubMed

    Liu, Hao; Ye, Wei; Chen, Hai-Feng

    2016-10-25

    Acetylcholinesterase is a potent enzyme that regulates neurotransmission by rapidly hydrolyzing the neurotransmitter acetylcholine in synapses of the nervous system. As drug target of anti-AD, it has catalytic and peripheral anionic sites. However, the regulation relation between these two sites is unclear. Therefore, we constructed dynamics fluctuation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in double-site system (hAChE/TZ5) is distinctly different from that in the free state and single-site systems (hAChE/huprine and hAChE/1YL). The community network analysis indicates that the information freely transfers from the peripheral anionic site to the catalytic active site in hAChE/TZ5. Furthermore, the binding free energy between the inhibitor and hAChE for hAChE/TZ5 is significantly lower than of either hAChE/huprine or hAChE/1YL. Thus, a hypothesis of 'positive cooperative regulation' is proposed for the regulation of double binding sites and further confirmed by the weakening and mutation community analyses. Finally, one possible cooperative regulation pathway of W86-TZ5-W286 was identified based on the shortest path algorithm and was confirmed by the network perturbation analysis. Interestingly, the regulation pathway for single-site systems is significantly different from that of dual-site system. The process targeting on the shortest pathway can better regulate the hydrolyzing the neurotransmitter acetylcholine and significantly inhibit the aggregation of Aβ amyloid.

  18. Thermodynamics of Molybdate Binding to Humic Acid

    NASA Astrophysics Data System (ADS)

    Thalhammer, K.; Gilbert, B.

    2016-12-01

    Molybdenum is an essential nutrient for diazotrophic bacteria that use nitrogenase I to fix atmospheric nitrogen in soils into bioavailable forms such as ammonia. This metalloid is released during rock weathering processes and at neutral pH it exists primarily as the soluble oxyanion molybdate, MoO42-. It has been established that molybdate mobility and bioavailability in soils is influenced by sorption to mineral surfaces and complexation by natural organic matter (NOM). The molybdate ion is readily bound by ortho dihydroxybenzene molecules such as catechol and catechol groups in siderophores. Humic acids (HA) found in NOM contain abundant phenolic groups and extended X-ray absorption fine structure (EXAFS) spectroscopy demonstrated that molybdate is bound by catechol-containing molecules in soil organic matter1. However, to our knowledge no quantitative determination of the affinity of molybdate to HA has been reported. We studied the interactions of molybdate with Suwannee River HA using ultraviolet-visible (UV-vis) absorption spectroscopy and isothermal titration calorimetry (ITC) to determine the conditional equilibrium constant for complexation at neutral pH. We further used ITC to investigate the thermodynamic contributions to complexation and the interaction kinetics. Addition of molybdate to HA caused the formation of complexes with UV-vis absorption spectra in good agreement with molybdate-catechol species indicating catechol groups to be the primary ligands in HA. ITC data revealed that binding enthalpies and kinetics were strongly influenced by ionic strength, suggesting a role for macromolecular reorganization driven by metalloid addition. 1. Wichard et al., Nature Geoscience 2, 625 - 629 (2009).

  19. Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

    PubMed Central

    Liaw, S. H.; Kuo, I.; Eisenberg, D.

    1995-01-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS

  20. Components of the macrolide binding site on the ribosome.

    PubMed

    Tejedor, F; Ballesta, J P

    1985-07-01

    Carbomycin A, niddamycin and tylosin, macrolide antibiotics that inhibit ribosomal activity, have alpha-beta unsaturated ketone groups in their structure that make them photoreactive. These drugs can also take part in thermic reactions, probably through an addition mechanism to the double bond. Given of the photoactivity and thermic reactivity of their molecules, affinity labeling of the macrolide binding site on the ribosome has been performed using radioactive derivatives of these drugs. After either irradiating or incubating samples containing antibiotics and ribosomal particles, radioactivity appears covalent associated to the proteins. Ribosomal protein L27 is the major labeled component, indicating that this polypeptide, which seems to be part of the peptidyl transferase centre of the ribosome, also plays an important role on the macrolide binding site.

  1. Different sites of acivicin binding and inactivation of gamma-glutamyl transpeptidases.

    PubMed Central

    Smith, T K; Ikeda, Y; Fujii, J; Taniguchi, N; Meister, A

    1995-01-01

    Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site. PMID:7892271

  2. Theoretical investigation on nevirapine and HIV-1 reverse transcriptase binding site interaction, based on ONIOM method

    NASA Astrophysics Data System (ADS)

    Kuno, Mayuso; Hannongbua, Supa; Morokuma, Keiji

    2003-10-01

    The ONIOM method was applied to the interaction of nevirapine with the HIV-1 reverse transcriptase binding site. The isolated complex of pyridine (part of nevirapine) and methyl phenol (part of Tyr181) was found at the MP2/6-31+G(d) level to have stacking interaction with 8.8 kcal/mol binding energy. Optimization of nevirapine and Tyr181 geometry in the pocket of 16 amino acid residues at the ONIOM3(MP2/6-31G(d):HF/3-21G:PM3) level gave the complex structure with weak hydrogen bonding but without stacking interaction. The binding energy of 8.9 kcal/mol comes almost entirely from the interaction of nevirapine with amino acid residues other than Tyr181.

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

    SciTech Connect

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

    1986-10-01

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

  4. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

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

    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.

  5. Minimal Zn2+ Binding Site of Amyloid-β

    PubMed Central

    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

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

    PubMed

    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.

  7. Cation binding sites on the projected structure of bacteriorhodopsin.

    PubMed

    Katre, N V; Kimura, Y; Stroud, R M

    1986-08-01

    Divalent cations are involved in the function of bacteriorhodopsin (bR) as a light-driven proton pump. If cations are removed from purple membranes they become blue. Divalent cations such as Ca2+ or Pb2+ or trivalent ions, can be stoichiometrically titrated back on to these deionized membranes. The color transitions as a function of ion concentration for Ca2+ or Pb2+ are precisely comparable and indicate that approximately three stoichiometric equivalents of cations are required to effect the color transition (Kimura et al., 1984). We found four main partially occupied binding sites for cations at a stoichiometric ratio of 3 Pb2+/bR. We localized the binding sites for Pb2+ using x-ray diffraction of membranes reconstituted with 1, 2, and 3 equivalents of Pb2+ per bR. The site of highest affinity is located on helix 7. At 2 Pb2+/bR, sites on helix 6 and between helix 2 and 3 are occupied. At 3 Pb2+/bR a fourth site above helix 3 is occupied.

  8. Analysis of zinc binding sites in protein crystal structures.

    PubMed Central

    Alberts, I. L.; Nadassy, K.; Wodak, S. J.

    1998-01-01

    The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. PMID:10082367

  9. Molecular modeling of a putative antagonist binding site on helix III of the β-adrenoceptor

    NASA Astrophysics Data System (ADS)

    van Vlijmen, H. W. Th.; Ijzerman, A. P.

    1989-06-01

    In recent biochemical studies it was demonstrated that residue Asp113 of theβ-adrenoceptor (β-AR) is an indispensable amino acid for the binding of β-AR antagonists. Earlier fluorescence studies showed that a tryptophan-rich region of the β-AR is involved in the binding of propranolol, the prototype β-AR antagonist. Bearing these two biochemical findings in mind, we explored the β-AR part containing Asp113, for an energetically favorable antagonist binding site. This was done by performing molecular docking studies with the antagonist propranolol and a specific β-AR peptide which included, besides Asp113, two possibly relevant tryptophan residues. In the docking calculations, the propranolol molecule was allowed to vary all its internal torsional angles. The receptor peptide was kept in an α-helix conformation, while side chains relevant to ligand binding were flexible to enable optimal adaptations to the ligand's binding conformation. By means of force-field calculations the total energy was minimized, consisting of the intramolecular energies of both ligand and receptor peptide, and the intermolecular energy. We found an antagonist binding site, consisting of amino acids Asp113 and Trp109, which enabled energetically favorable interactions with the receptor-binding groups of propranolol. According to these results, binding involves three main interaction points: (i) a reinforced ionic bond; (ii) a hydrogen bond; and (iii) a hydrophobic/charge transfer interaction. The deduced binding site shows a difference in affinity between the levo- and dextrorotatory isomers of propranolol caused by a difference in ability to form a hydrogen bond, which is in conformity with the experimentally observed stereoselectivity. Moreover, it also provides an explanation for the β 1-selectivity of p-phenyl substituted phenoxypropanolamines like betaxolol. The p-phenyl substituent of betaxolol was shown to be sterically hindered upon binding to the β 2-AR peptide, whereas

  10. A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

    PubMed Central

    Rademacher, Christoph; Bru, Thierry; McBride, Ryan; Robison, Elizabeth; Nycholat, Corwin M; Kremer, Eric J; Paulson, James C

    2012-01-01

    Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid. PMID:22522600

  11. The synergistic binding of anions and Fe3+ by transferrin. Implications for the interlocking sites hypothesis.

    PubMed

    Schlabach, M R; Bates, G W

    1975-03-25

    The finding that transferrin does not bind Fe3+ at the specific metal binding sites in the absence of carbonate and synergistic anions emphasizes the fundamental importance of the anion binding site to the chemistry of Fe3+-transferrin-CO32-. An important question regards the chemical and structural requirements for carbonate substitution. This has been, however, an area of some dispute in the literature. We have utilized four synthetic routes for the preparation of Fe3+-transferrin-anion complexes. The products have been examined with regard to spectral properties, and reaction with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) sodium citrate under CO2-free conditions. The results provide information as to which anions are synergistic, and the basic properties of the Fe3+-transferrin-anion complexes that are formed. The 6 inorganic anions that were tested were all found to be nonsynergistic. Dihydroxyacetone and glyceraldehyde were also nonsynergistic. Dicarboxylic acids were found to form stable Fe3+-transferrin-anion complexes which were only slowly displaced by carbonate. Several monocarboxylic acids with proximal aldehyde, ketone, alcohol, amino, or thiol functional groups proved to be synergistic. CPK molecular model studies suggested the functional group and the carboxylic acid must be able to fit within a site between 6.3 and 7.0 A in maximal length. One large substituent could be accommodated by the site, however, two methylgroups on the alpha carbon to a carboxylate group could not be accommodated. Chloroacetate and monocarboxylic acids were nonsynergistic. The results are interpreted in terms of an interlocking sites hypothesis which envisions the synergistic anion as interacting with the protein via its its carboxyl group and bonding with the Fe3+ via its proximal functional group.

  12. A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor

    PubMed Central

    Kaji, Mark D; Kwaka, Ariel; Callanan, Micah K; Nusrat, Humza; Desaulniers, Jean-Paul; Forrester, Sean G

    2015-01-01

    Background and Purpose Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. Experimental Approach We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. Key Results The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(−)-4-amino-3-hydroxybutyric acid [R(−)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > β-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(−)- and S(+)-GABOB. Conclusions and Implications The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes. PMID:25850584

  13. Cloud Computing for Protein-Ligand Binding Site Comparison

    PubMed Central

    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

  14. Two mechanisms of ion selectivity in protein binding sites.

    PubMed

    Yu, Haibo; Noskov, Sergei Yu; Roux, Benoît

    2010-11-23

    A theoretical framework is presented to clarify the molecular determinants of ion selectivity in protein binding sites. The relative free energy of a bound ion is expressed in terms of the main coordinating ligands coupled to an effective potential of mean force representing the influence of the rest of the protein. The latter is separated into two main contributions. The first includes all the forces keeping the ion and the coordinating ligands confined to a microscopic subvolume but does not prevent the ligands from adapting to a smaller or larger ion. The second regroups all the remaining forces that control the precise geometry of the coordinating ligands best adapted to a given ion. The theoretical framework makes it possible to delineate two important limiting cases. In the limit where the geometric forces are dominant (rigid binding site), ion selectivity is controlled by the ion-ligand interactions within the matching cavity size according to the familiar "snug-fit" mechanism of host-guest chemistry. In the limit where the geometric forces are negligible, the ion and ligands behave as a "confined microdroplet" that is free to fluctuate and adapt to ions of different sizes. In this case, ion selectivity is set by the interplay between ion-ligand and ligand-ligand interactions and is controlled by the number and the chemical type of ion-coordinating ligands. The framework is illustrated by considering the ion-selective binding sites in the KcsA channel and the LeuT transporter.

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

    PubMed

    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.

  16. Elucidation of Novel Structural Scaffold in Rohu TLR2 and Its Binding Site Analysis with Peptidoglycan, Lipoteichoic Acid and Zymosan Ligands, and Downstream MyD88 Adaptor Protein

    PubMed Central

    Sahoo, Bikash Ranjan; Basu, Madhubanti; Swain, Banikalyan; Dikhit, Manas Ranjan; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-01-01

    Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)–ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16–19, LRR12–14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)—protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, αB-helix, αC-helix, and CD loop in TLR2-TIR and BB loop, αB-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling. PMID:23956969

  17. Spectrofluorimetric study of the binding of codeine to nucleic acids

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Huang, Wei; Su, Liang; Dong, Zijia; Zhang, Shuai

    2009-06-01

    The characteristics of the interaction between codeine (CD) and nucleic acids were studied by ultraviolet-visible spectra and fluorescent spectra. It shows that there is a powerful ability in nucleic acids to quench the fluorescence intensity of codeine. The fluorescence quenching data were analyzed according to Stern-Volmer equation and Förster's nonradiative energy transfer mechanism. Thus the binding constant and the thermodynamic parameters between codeine and nucleic acids were obtained. The results show that codeine interacts with nucleic acids in a mode of groove binding and -OCH 3 of the codeine molecular combines with the groove of nucleic acids through hydrogen bond or van der Waals force.

  18. The Next Generation of Transcription Factor Binding Site Prediction

    PubMed Central

    Mathelier, Anthony; Wasserman, Wyeth W.

    2013-01-01

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

  19. The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin.

    PubMed

    Barchan, D; Ovadia, M; Kochva, E; Fuchs, S

    1995-07-18

    The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Direct GR Binding Sites Potentiate Clusters of TF Binding across the Human Genome.

    PubMed

    Vockley, Christopher M; D'Ippolito, Anthony M; McDowell, Ian C; Majoros, William H; Safi, Alexias; Song, Lingyun; Crawford, Gregory E; Reddy, Timothy E

    2016-08-25

    The glucocorticoid receptor (GR) binds the human genome at >10,000 sites but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter-gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady-state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs and may therefore play a major role in driving gene activation in response to GCs.

  1. Capture and release of acid-gasses with acid-gas binding organic compounds

    DOEpatents

    Heldebrant, David J; Yonker, Clement R; Koech, Phillip K

    2015-03-17

    A system and method for acid-gas capture wherein organic acid-gas capture materials form hetero-atom analogs of alkyl-carbonate when contacted with an acid gas. These organic-acid gas capture materials include combinations of a weak acid and a base, or zwitterionic liquids. This invention allows for reversible acid-gas binding to these organic binding materials thus allowing for the capture and release of one or more acid gases. These acid-gas binding organic compounds can be regenerated to release the captured acid gasses and enable these organic acid-gas binding materials to be reused. This enables transport of the liquid capture compounds and the release of the acid gases from the organic liquid with significant energy savings compared to current aqueous systems.

  2. Sec12 Binds to Sec16 at Transitional ER Sites

    PubMed Central

    Montegna, Elisabeth A.; Bhave, Madhura; Liu, Yang; Bhattacharyya, Dibyendu; Glick, Benjamin S.

    2012-01-01

    COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export. PMID:22347445

  3. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

    PubMed

    Chia, Ju-Chen; Yang, Chien-Chih; Sui, Yu-Ting; Lin, Shin-Yu; Juang, Rong-Huay

    2013-01-01

    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-Cys)2-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.

  4. Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding.

    PubMed

    Fleury, F; Ianoul, A; Berjot, M; Feofanov, A; Alix, A J; Nabiev, I

    1997-07-14

    Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)-binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3'-azido-3'-deoxythymidine (a ligand occupying IIIA structural sub-domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA-bound CPT may be replaced by warfarin, occupying IIA structural sub-domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT-binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT-induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp-residue to hydrophilic environment. These data suggest that CPT is bound in the region of interdomain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

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

    PubMed

    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.

  6. Expression of messenger ribonucleic acid and presence of immunoreactive proteins for epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and EGF/TGF alpha receptors and 125I-EGF binding sites in human fallopian tube.

    PubMed

    Chegini, N; Zhao, Y; McLean, F W

    1994-05-01

    Reverse transcription polymerase chain reaction (RT-PCR) revealed that the Fallopian tubes express epidermal growth factor (EGF), transforming growth factor (TGF alpha), and EGF receptor (EGF-R) mRNA. The RT-PCR product was verified by restriction enzyme digestion analysis. Immunohistochemically, EGF, TGF alpha, and EGF-R were localized in Fallopian tubes by use of specific antibodies to human EGF, mature fragments of human TGF alpha, and monoclonal antibodies to the extracellular binding domain of EGF-R. The tubal epithelial cells were the primary site of immunoreactive EGF, TGF alpha, and EGF-R, which were present to a lesser extent in the stromal cells, smooth muscle cell layers, fibroblasts of serosal tissue, and arterial endothelial and smooth muscle cells. Using antibodies generated against the amino and carboxy termini of TGF alpha precursor produced a similar cellular distribution to that observed for mature TGF alpha. The intensity of immunoreactive TGF alpha with these antibodies was similar to that seen with EGF. The ciliated and nonciliated epithelial cells in the ampullary and isthmus regions immunostained with similar intensity for EGF, TGF alpha, and EGF-R. The immunostaining for EGF, TGF alpha, and EGF-R was cycle-dependent, was considerably higher during late proliferative and early-to-mid-secretory phases than during early proliferative and late secretory phases of the menstrual cycle, and was reduced during the postmenopausal period. Specimens obtained 5-12 yr after tubal ligation immunostained for EGF, TGF alpha, and EGF-R similarly to sections from unligated tubes taken during the same phase of the cycle. Quantitative autoradiography of 125I-EGF binding generated a pattern similar to that of immunostaining for EGF-R binding. Net grain density/100 microns 2 calculated for different cell types indicated that the epithelial cells had a significantly higher grain density than did other tubal cell types (p < 0.05) without the cycle dependency seen

  7. The elusive permeability barriers and binding sites for proflavine in Escherichia coli.

    PubMed

    Gravelle, M J; Mehta, B M; Kushner, D J

    1972-06-01

    Cells of proflavine-sensitive and -resistant Escherichia coli strains were altered in different ways, and the proflavine binding of the changed material was studied. Spheroplasts prepared from sensitive and resistant cells bound similar amounts of proflavine at saturation, whether or not they were osmotically protected by 10% sucrose. Intact cells bound approximately the same amounts of proflavine as spheroplasts. On addition of glucose, osmotically protected resistant but not sensitive spheroplasts released proflavine; unprotected spheroplasts did not release bound proflavine. Thus, osmotically protected membranes are not required for proflavine binding (a passive process) but are required for proflavine release (an active process). The presence of sucrose reduced proflavine binding by resistant cells. Adding glucose to cells in 20% sucrose did not cause a release of residual proflavine, though glucose caused a release of proflavine from cells suspended in 0 or 10% sucrose. On treatment of heated cells or ruptured spheroplasts with nucleases and Pronase, practically all nucleic acids were removed. Proflavine-binding ability of such preparations fell by only 30 to 50%. Washing heated cells with ethanol did not reduce their proflavine-binding ability. There appear to be important binding sites in cells aside from nucleic acids.

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

    PubMed Central

    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. PMID:26600856

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

    PubMed

    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.

  10. Defining the binding site of homotetrameric R67 dihydrofolate reductase and correlating binding enthalpy with catalysis.

    PubMed

    Strader, Michael Brad; Chopra, Shaileja; Jackson, Michael; Smiley, R Derike; Stinnett, Lori; Wu, Jun; Howell, Elizabeth E

    2004-06-15

    R67 dihydrofolate reductase (DHFR) is a novel protein that possesses 222 symmetry. A single active site pore traverses the length of the homotetramer. Although the 222 symmetry implies that four symmetry-related binding sites should exist for each substrate as well as each cofactor, isothermal titration calorimetry (ITC) studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate with one cofactor. The latter is the productive ternary complex. To evaluate the roles of A36, Y46, T51, G64, and V66 residues in binding and catalysis, a site-directed mutagenesis approach was employed. One mutation per gene produces four mutations per active site pore, which often result in large cumulative effects. Conservative mutations at these positions either eliminate the ability of the gene to confer trimethoprim resistance or have no effect on catalysis. This result, in conjunction with previous mutagenesis studies on K32, K33, S65, Q67, I68, and Y69 [Strader, M. B., et al. (2001) Biochemistry 40, 11344-11352; Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Park, H., et al. (1997) Protein Eng. 10, 1415-1424], allows mapping of the active site surface. Residues for which conservative mutations have large effects on binding and catalysis include K32, Q67, I68, and Y69. These residues form a stripe that establishes the ligand binding surface. Residues that accommodate conservative mutations that do not greatly affect catalysis include K33, Y46, T51, S65, and V66. Isothermal titration calorimetry studies were also conducted on many of the mutants described above to determine the enthalpy of folate binding to the R67 DHFR.NADPH complex. A linear correlation between this DeltaH value and log k(cat)/K(m) is observed. Since structural tightness appears to be correlated with the exothermicity of the binding interaction, this leads to the hypothesis that enthalpy-driven formation of the ternary

  11. Characterization of the proton binding sites of extracellular polymeric substances in an anaerobic membrane bioreactor.

    PubMed

    Liu, Yi; Chang, Sheng; Defersha, Fantahun M

    2015-07-01

    This paper focuses on the characterization of the chemical compositions and acidic constants of the extracellular polymeric substances (EPSs) in an anaerobic membrane bioreactor treating synthetic brewery wastewater by using chemical analysis, linear programming analysis (LPA) of titration data, and FT-IR analysis. The linear programming analysis of titration data revealed that the EPSs have proton binding sites with pKa values from pKa ≤ 6, between 6 and 7, and approximately 9.8. The strong acidic sites (pKa ≤ 6) and some weak acidic sites (7.5 < pKa < 9.0) were found to be readily removed by 0.45-μm membrane filtration. In addition, the FT-IR analysis confirmed the presence of proteins, carbohydrates, nucleic acids, and lipids in the EPS samples. Based on the FT-IR analysis and the main chemical functional groups at the bacterial cell surfaces, the identified proton binding sites were related to carboxyl, phosphate, and hydroxyl/amine groups with pKa values of 4.6 ± 0.7, 6.6 ± 0.01, and 9.7 ± 0.1, respectively, with the corresponding respective intensities of 0.31 ± 0.05, 0.96 ± 0.3, and 1.53 ± 0.3 mmole/g-EPS. The pKa values and intensities of the proton binding sites are the fundamental molecular properties of EPSs that affect the EPS charge, molecular interactions, and metal complexation characteristics. Determination of such properties can advance Derjaguin-Landau-Verwey-Overbeek (DLVO)-based concentration polarization modeling, facilitate the estimation of the osmotic pressure of the EPS concentration polarization layers, and lead to a deeper understanding of the role of metal complexation in membrane fouling.

  12. Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish

    PubMed Central

    1984-01-01

    Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also

  13. The genomic binding sites of a noncoding RNA

    PubMed Central

    Simon, Matthew D.; Wang, Charlotte I.; Kharchenko, Peter V.; West, Jason A.; Chapman, Brad A.; Alekseyenko, Artyom A.; Borowsky, Mark L.; Kuroda, Mitzi I.; Kingston, Robert E.

    2011-01-01

    Long noncoding RNAs (lncRNAs) have important regulatory roles and can functio