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Sample records for acetylcholine binding site

  1. 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.

  2. 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.

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

    PubMed Central

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

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  4. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  5. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.

  6. 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

  7. 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.

  8. Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

    PubMed

    Iturriaga-Vásquez, Patricio; Alzate-Morales, Jans; Bermudez, Isabel; Varas, Rodrigo; Reyes-Parada, Miguel

    2015-11-01

    For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.

  9. 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)

  10. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    PubMed Central

    Martinez-Archundia, Marlet; Cordomi, Arnau; Garriga, Pere; Perez, Juan J.

    2012-01-01

    The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand. PMID:22500107

  11. Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

    NASA Technical Reports Server (NTRS)

    Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

    1987-01-01

    A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

  12. A mutational analysis of the acetylcholine receptor channel transmitter binding site.

    PubMed Central

    Akk, G; Zhou, M; Auerbach, A

    1999-01-01

    Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR. PMID:9876135

  13. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  14. The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

    PubMed Central

    Williams, Dustin K.; Stokes, Clare; Horenstein, Nicole A.

    2011-01-01

    We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists. PMID:21444659

  15. Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Gershoni, J M; Fridkin, M; Fuchs, S

    1985-01-01

    Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site. Images PMID:2582416

  16. Quantitative autoradiography of brain binding sites for the vesicular acetylcholine transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH5183)

    SciTech Connect

    Marien, M.R.; Parsons, S.M.; Altar, C.A.

    1987-02-01

    2-(4-Phenylpiperidino)cyclohexanol (AH5183) is a noncompetitive and potent inhibitor of high-affinity acetylcholine transport into cholinergic vesicles. It is reported here that (/sup 3/H)AH5183 binds specifically and saturably to slide-mounted sections of the rat forebrain (Kd = 1.1 to 2.2 X 10(-8) M; Bmax = 286 to 399 fmol/mg of protein). The association and dissociation rate constants for (/sup 3/H)AH5183 binding are 8.6 X 10(6) M-1 X min-1 and 0.18 min-1, respectively. Bound (/sup 3/H)AH5183 can be displaced by nonradioactive AH5183 and by the structural analog (2 alpha,3 beta,4A beta,8A alpha)-decahydro-3-(4-phenyl-1-piperidinyl)-2- naphthalenol but not by 10 microM concentrations of the cholinergic drugs acetylcholine, choline, atropine, hexamethonium, eserine, or hemicholinium-3 or by the structurally related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, (+/-)-N-allylnormetazocine (SKF 10,047), levoxadrol, or dexoxadrol. Quantitative autoradiography reveals that (/sup 3/H)AH5183 binding sites are distributed heterogenously throughout the rat forebrain and are highly localized to cholinergic nerve terminal regions. At the level of the caudate nucleus-putamen, the highest concentrations of saturable (/sup 3/H)AH5183 binding (713-751 fmol/mg of protein) are found in the vertical limb of the diagonal band and the olfactory tubercle, with lesser amounts (334-516 fmol/mg of protein) in the caudate-putamen, nucleus accumbens, superficial layers of the cerebral cortex, and the primary olfactory cortex. At day 7 after transsection of the left fimbria, (/sup 3/H)AH5183 binding and choline acetyltransferase activity in the left hippocampus were reduced by 33 +/- 6% and 61 +/- 7%, respectively. These findings indicate that (/sup 3/H)AH5183 binds to a unique recognition site in rat brain that is topographically associated with cholinergic nerve terminals.

  17. [Sites of synthesis of acetylcholine receptors in denervated muscles].

    PubMed

    Giacobini Robecchi, M G; Garelli, M; Filogamo, G

    1980-09-01

    Muscle fibres binding with 125I alpha-bungarotoxine from Bungarus Multicinctus, after treatment with saponine, shows (in electron microscope autoradiography) intracellular binding sites identifying sites of acetylcholine receptor synthesis. In innervated muscle, the acetylcholine receptor is located only at the neuromuscular junction. In denervated muscle the receptor is distributed along the whole sarcolemma; in this situation the acetylcholine receptor is synthesized "ex novo" in the membrane system over the whole length of the muscle fibre.

  18. Binding of rabies virus to purified Torpedo acetylcholine receptor.

    PubMed

    Lentz, T L; Benson, R J; Klimowicz, D; Wilson, P T; Hawrot, E

    1986-12-01

    The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

  19. The α-bungarotoxin binding site on the nicotinic acetylcholine receptor: Analysis using a phage–epitope library

    PubMed Central

    Balass, Moshe; Katchalski-Katzir, Ephraim; Fuchs, Sara

    1997-01-01

    The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. α-Neurotoxins, in particular α-bungarotoxin (α-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with α-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to α-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the α-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind α-BTX. The library-derived peptide and the corresponding peptides containing residues 187–199 of the Torpedo AcChoR α-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human α7; ERFYECCKEPYPD) that binds α-BTX, inhibit the binding of α-BTX to the intact Torpedo AcChoR with IC50 values of 10−6 M. A synthetic peptide from a neuronal AcChoR that does not bind α-BTX (e.g., human α2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the α-BTX-binding protein (α7)—i.e., Lys in α2 and Tyr in α7—does not inhibit the binding of α-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to α-BTX. PMID:9177167

  20. Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.

    PubMed Central

    Gershoni, J M

    1987-01-01

    Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots. Images PMID:3295881

  1. 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.

  2. Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile.

    PubMed

    Möller-Acuña, Patricia; Contreras-Riquelme, J Sebastián; Rojas-Fuentes, Cecilia; Nuñez-Vivanco, Gabriel; Alzate-Morales, Jans; Iturriaga-Vásquez, Patricio; Arias, Hugo R; Reyes-Parada, Miguel

    2015-01-01

    Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets.

  3. Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile

    PubMed Central

    Möller-Acuña, Patricia; Contreras-Riquelme, J. Sebastián; Rojas-Fuentes, Cecilia; Nuñez-Vivanco, Gabriel; Alzate-Morales, Jans; Iturriaga-Vásquez, Patricio; Arias, Hugo R.; Reyes-Parada, Miguel

    2015-01-01

    Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets. PMID:26244344

  4. Comparison of (/sup 3/H)nicotine and (/sup 3/H)acetylcholine binding in mouse brain: regional distribution

    SciTech Connect

    Sershen, H.; Reith, M.E.; Hashim, A.; Lajtha, A.

    1985-06-01

    In a continuing study of nicotine binding sites, the authors determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both (/sup 3/H)nicotine and (/sup 3/H)acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of (/sup 3/H)nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-(/sup 3/H)nicotine and that of (/sup 3/H)acetylcholine.

  5. Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site.

    PubMed

    Shimomura, Masaru; Yokota, Maiko; Ihara, Makoto; Akamatsu, Miki; Sattelle, David B; Matsuda, Kazuhiko

    2006-10-01

    The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha4beta2 nAChR and Drosophila melanogaster Dalpha2/chicken beta2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha4beta2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the Dalpha2beta2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha4beta2 and Dalpha2beta2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

  6. Binding sites for. alpha. -bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the. alpha. -subunit of the nicotinic acetylcholine receptor

    SciTech Connect

    Donnelly-Roberts, D.L.; Lentz, T.L. )

    1991-07-30

    The binding of the competitive antagonist {alpha}-bungarotoxin ({alpha}-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the {alpha}-subunit of the Torpedo acetylcholine receptor has been characterized. {sup 125}I-{alpha}-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by {alpha}-Btx d-tubocurarine and NaCl. In the presence of 0.02% sodium dodecyl sulfate, {sup 125}I-{alpha}-Btx bound to the 56-residue peptide with a K{sub D} of 3.5 nM, as determined by equilibrium saturation binding studies. Because {alpha}Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, ({sup 3}H)PCP was bound to the 172-227 peptide. ({sup 3}H)PCP binding was inhibited by chlorpromazine, tetracaine, and dibucaine. It is concluded that a high-affinity binding site for PCP is located between residues 205 and 227, which includes the first 18 residues of transmembrane segment M1, and that a low-affinity site is located in the competitive antagonist binding site between residues 173 and 204. These results show that a synthetic peptide comprising residues 172-227 of the {alpha} subunit contains three binding sites, one for {alpha}-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel.

  7. Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.

    PubMed Central

    Balass, M; Heldman, Y; Cabilly, S; Givol, D; Katchalski-Katzir, E; Fuchs, S

    1993-01-01

    Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable. Images Fig. 5 PMID:7504273

  8. Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms*

    PubMed Central

    Lucero, Linda M.; Weltzin, Maegan M.; Eaton, J. Brek; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.; Whiteaker, Paul

    2016-01-01

    Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect. PMID:26644472

  9. Differential α4(+)/(-)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

    PubMed

    Lucero, Linda M; Weltzin, Maegan M; Eaton, J Brek; Cooper, John F; Lindstrom, Jon M; Lukas, Ronald J; Whiteaker, Paul

    2016-01-29

    Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

  10. Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

    PubMed Central

    Hamouda, Ayman K.; Wang, Ze-Jun; Stewart, Deirdre S.; Jain, Atul D.; Glennon, Richard A.

    2015-01-01

    Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([3H]phencyclidine; IC50 = 4 μM) than in the resting state ([3H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([3H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [3H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [3H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [3H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity. PMID:25870334

  11. Insights into a highly conserved network of hydrogen bonds in the agonist binding site of nicotinic acetylcholine receptors: a structural and theoretical study.

    PubMed

    Atkinson, Alexandre; Graton, Jérôme; Le Questel, Jean-Yves

    2014-10-01

    Structural and theoretical studies on the geometrical features of a hydrogen-bond network occurring in the binding site of nicotinic acetylcholine receptors (nAChRs) and composed of interconnected WxPD (Trp-x-Pro-Asp) and SWyz (Ser-Trp-yz) sequences from loops A and B, respectively, have been carried out. Multiple sequence alignments using as template the sequence of the apoform of Aplysia californica acetylcholine binding protein (Ac-AChBP) show the strict conservation of serine and tryptophan residues of the loop B SWyz sequence. Considering a sample of 19 high resolution AChBP structures, the strong conformational preferences of the key tryptophan residue has been pointing out, whatever the form, free or bounded, of AChBP. The geometry of the motif hydrogen-bond network has been characterized through the analyses of seven distances. The robustness of the various hydrogen-bond interactions is pointed out, the one involving the aspartate carboxylate group and the serine residue being the shortest of the network. The role of a cooperative effect involving a NH(His145)…OH (Ser142) hydrogen bond is highlighted. Density functional theory calculations on several simplified models based on the motif hydrogen-bond network allow probing the importance of the various hydrogen-bond interactions. The removal of the Ser142 hydroxyl group induces strong structural rearrangements, in agreement with the structural observations. Molecular electrostatic potential calculations on model systems highlight the importance of a cooperative effect in the whole hydrogen-bond network. More precisely, the key role of the Ser142 hydroxyl group, involved in several hydrogen bonds, is underlined.

  12. Increased Nicotinic Acetylcholine Receptor Protein Underlies Chronic Nicotine-Induced Up-Regulation of Nicotinic Agonist Binding Sites in Mouse Brain

    PubMed Central

    McClure-Begley, Tristan D.; Whiteaker, Paul; Salminen, Outi; Brown, Robert W. B.; Cooper, John; Collins, Allan C.; Lindstrom, Jon M.

    2011-01-01

    Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of α4β2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion. Tissue sections were prepared, and binding of [125I]3-((2S)-azetidinylmethoxy)-5-iodo-pyridine (A85380) to β2*-nAChR sites, [125I]monoclonal antibody (mAb) 299 to α4 nAChR subunits, and [125I]mAb 270 to β2 nAChR subunits was determined by quantitative autoradiography. Chronic nicotine treatment dose-dependently increased binding of all three ligands. In regions that express α4β2-nAChR almost exclusively, binding of all three ligands increased coordinately. However, in brain regions containing significant β2*-nAChR without α4 subunits, relatively less increase in mAb 270 binding to β2 subunits was observed. Signal intensity measured with the mAbs was lower than that with [125I]A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [125I]epibatidine binding sites with mAb 270 in select regions of nicotine-treated mice was nearly quantitative, although somewhat less so with mAb 299, confirming that the mAbs effectively recognize their targets. The patterns of change measured using immunoprecipitation were comparable with those determined autoradiographically. Thus, increases in α4β2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein. PMID:21228066

  13. Binding of Alpha-Bungarotoxin to Single Identified Neurons of ’Aplysia’ which have Different Ionic Responses to Acetylcholine,

    DTIC Science & Technology

    1976-09-01

    Identifiable Aplysia neurons have one or more of three different ionic responses to acetylcholine, due to Na, Cl, and K conductance increases... Aplysia acetylcholine receptors. Thus the inhibition of the Na response by hexamethonium may be a result of the binding to a site which prevent the conductance change rather than preventing acetylcholine from binding to its receptor.

  14. Bupropion Binds to Two Sites in the Torpedo Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analog [125I]-SADU-3-72

    PubMed Central

    Pandhare, Akash; Hamouda, Ayman K.; Staggs, Brandon; Aggarwal, Shaili; Duddempudi, Phaneendra K.; Lever, John R.; Lapinsky, David J.; Jansen, Michaela; Cohen, Jonathan B.; Blanton, Michael P.

    2012-01-01

    Bupropion, a clinically-used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analog, (±)-2-(N-tert-butylamino)-3′-[125I]-iodo-4′-azidopropiophenone (SADU-3-72). Based upon inhibition of [125I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC50 = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with three-fold higher affinity in the desensitized (IC50 = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [125I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [125I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu265, βLeu257) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP, but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr213 in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket. PMID:22394379

  15. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  16. Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation [corrected and republished article originally printed in J Cell Biol 1988 May;106(5):1723-34

    PubMed Central

    1988-01-01

    Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation. PMID:3170634

  17. The conformation of acetylcholine at its target site in the membrane-embedded nicotinic acetylcholine receptor

    PubMed Central

    Williamson, P. T. F.; Verhoeven, A.; Miller, K. W.; Meier, B. H.; Watts, A.

    2007-01-01

    The conformation of the neurotransmitter acetylcholine bound to the fully functional nicotinic acetylcholine receptor embedded in its native membrane environment has been characterized by using frequency-selective recoupling solid-state NMR. Six dipolar couplings among five resolved 13C-labeled atoms of acetylcholine were measured. Bound acetylcholine adopts a bent conformation characterized with a quaternary ammonium-to-carbonyl distance of 5.1 Å. In this conformation, and with its orientation constrained to that previously determined by us, the acetylcholine could be docked satisfactorily in the agonist pocket of the agonist-bound, but not the agonist-free, crystal structure of a soluble acetylcholine-binding protein from Lymnaea stagnali. The quaternary ammonium group of the acetylcholine was determined to be within 3.9 Å of five aromatic residues and its acetyl group close to residues C187/188 of the principle and residue L112 of the complementary subunit. The observed >CO chemical shift is consistent with H bonding to the nicotinic acetylcholine receptor residues γY116 and δT119 that are homologous to L112 in the soluble acetylcholine-binding protein. PMID:17989232

  18. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    PubMed Central

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands. PMID:28091608

  19. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

  20. Anesthetics Target Interfacial Transmembrane Sites in Nicotinic Acetylcholine Receptors

    PubMed Central

    Forman, Stuart A.; Chiara, David C.; Miller, Keith W.

    2014-01-01

    General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. PMID:25316107

  1. Acetylcholine Promotes Binding of α-Conotoxin MII for α3β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Sambasivarao, Somisetti V.; Roberts, Jessica; Bharadwaj, Vivek S.; Slingsby, Jason G.; Rohleder, Conrad; Mallory, Chris; Groome, James R.

    2014-01-01

    α-Conotoxin MII (α-CTxMII) is a 16 amino acid peptide with the sequence GCCSNPVCHLEHSNLC containing disulfide bonds between Cys2-Cys8 and Cys3-Cys16. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER using crystal structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3 and β2 subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with commensurate binding energies to previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, which was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs. PMID:24420650

  2. The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype

    PubMed Central

    Eaton, J. Brek; Lucero, Linda M.; Stratton, Harrison; Chang, Yongchang; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.

    2014-01-01

    Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb+ efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(−)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit–specific [125I]mAb295 labeling. However, maximum function is approximately five times greater on a “per-receptor” basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(−)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(−)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely

  3. The ligand binding domain of the nicotinic acetylcholine receptor. Immunological analysis.

    PubMed

    Kachalsky, S G; Aladjem, M; Barchan, D; Fuchs, S

    1993-03-08

    The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with alpha-bungarotoxin (alpha-BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR alpha-subunit binds alpha-BTX, whereas the mongoose-expressed domain is not recognized by alpha-BTX. On the other hand, both the mouse and mongoose domains bind to the site-specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of alpha-BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.

  4. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-01-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  5. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-12-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  6. Substituted 2-Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins.

    PubMed

    Kaczanowska, Katarzyna; Camacho Hernandez, Gisela Andrea; Bendiks, Larissa; Kohs, Larissa; Cornejo-Bravo, Jose Manuel; Harel, Michal; Finn, M G; Taylor, Palmer

    2017-03-15

    Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4β2-nAChR, and a serotonin receptor (5-HT3AR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 μM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC50 values of 70 nM and Kd values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4β2-nAChR or 5-HT3AR at concentrations up to 10 μM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.

  7. Docking of 6-chloropyridazin-3-yl derivatives active on nicotinic acetylcholine receptors into molluscan acetylcholine binding protein (AChBP).

    PubMed

    Artali, Roberto; Bombieri, Gabriella; Meneghetti, Fiorella

    2005-04-01

    The crystal structure of Acetylcholine Binding Protein (AChBP), homolog of the ligand binding domain of nAChR, has been used as model for computational investigations on the ligand-receptor interactions of derivatives of 6-chloropyridazine substituted at C3 with 3,8-diazabicyclo[3.2.1]octane, 2,5-diazabicyclo[2.2.1]heptane and with piperazine and homopiperazine, substituted or not at N4. The ligand-receptor complexes have been analyzed by docking techniques using the binding site of HEPES complexed with AChBP as template. The good relationship between the observed binding affinity and the calculated docking energy confirms that this model provides a good starting point for understanding the binding domain of neuronal nicotinic receptors. An analysis of the possible factors significant for the ligand recognition has evidenced, besides the cation-pi interaction, the distance between the chlorine atom of the pyridazinyl group and the carbonylic oxygen of Leu B112 as an important parameter in the modulation of the binding energy.

  8. Exploration of the molecular architecture of the orthosteric binding site in the α4β2 nicotinic acetylcholine receptor with analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridin-3-yl)-1,4-diazepane.

    PubMed

    Bach, Tinna B; Jensen, Anders A; Petersen, Jette G; Sørensen, Troels E; Della Volpe, Serena; Liu, Jun; Blaazer, Antoni R; van Muijlwijk-Koezen, Jacqueline E; Balle, Thomas; Frølund, Bente

    2015-09-18

    X-ray crystal structures of acetylcholine binding proteins (AChBPs) have revealed two different possible extensions to the classical ligand binding pocket known to accommodate various nicotinic agonists. One of the pockets is limited in size while the other is of considerable dimensions and protrudes along the interfacial cleft between subunits. To probe these putative extensions in functional nicotinic acetylcholine receptors (nAChRs), elongated analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridine-3-yl)-1,4-diazepane were prepared and characterized pharmacologically at neuronal heteromeric nAChRs. Although the new analogs, relative to parent compounds, displayed lower binding affinities, functional characterization of selected compounds revealed that they had retained partial α4β2 nAChR agonist activity. The structure-activity relationship data did not indicate an upper limit to the size of substituents as would have been expected if the ligand was bound in the smaller pocket. The data were better in agreement with a binding mode in which substituents protrude along the interfacial cleft of the receptor. This was further supported by docking into a homology model of the α4-β2 nAChR interface and by surface plasmon resonance biosensor analysis of binding of the compounds to acetylcholine-binding proteins, where they exhibit preference for Lymnaea stagnalis ACh binding protein (Ls-AChBP) over the Aplysia california ACh binding protein (Ac-AChBP). These results suggest new opportunities for expanding chemical space in the development of partial agonist and may be of interest in relation to development of novel smoking cessation aids.

  9. Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein.

    PubMed

    Kaczanowska, Katarzyna; Harel, Michal; Radić, Zoran; Changeux, Jean-Pierre; Finn, M G; Taylor, Palmer

    2014-07-22

    The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are pentameric oligomers in which binding sites for nicotinic agonists and competitive antagonists are found at selected subunit interfaces. The nAChR spontaneously exists in multiple conformations associated with its activation and desensitization steps, and conformations are selectively stabilized by binding of agonists and antagonists. In the nAChR, agonist binding and the associated conformational changes accompanying activation and desensitization are cooperative. AChBP, which lacks the transmembrane spanning and cytoplasmic domains, serves as a homology model of the extracellular domain of the nAChRs. We identified unique cooperative binding behavior of a number of 4,6-disubstituted 2-aminopyrimidines to Lymnaea AChBP, with different molecular variants exhibiting positive, nH > 1.0, and negative cooperativity, nH < 1.0. Therefore, for a distinctive set of ligands, the extracellular domain of a nAChR surrogate suffices to accommodate cooperative interactions. X-ray crystal structures of AChBP complexes with examples of each allowed the identification of structural features in the ligands that confer differences in cooperative behavior. Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epibatidine. An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, noncooperative, nicotinic ligands. Positively and negatively cooperative ligands exhibited unique features in the detailed binding determinants and poses of the complexes.

  10. Neurotoxin Binding Site on the Acetylcholine Receptor

    DTIC Science & Technology

    1987-03-01

    8794. 40. Neumann,D., D. Barchan , A. Safran, J.M. Gershoni, and S. Fuchs (1986) Proc. Nat. Acad. Sci. USA 83:3008-3011. 41. Juillerat, M.A., B... Barchan , M. Fridkin, and S. Fuchs (1986) Proc. Nat. Acad. Sci. USA 83:9250-9253. 57. Burl ey, S.K. and G.A. Petsko (19855’Science 229:23-28. 58 ;a

  11. The M1 muscarinic receptor allosteric agonists AC-42 and 1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one bind to a unique site distinct from the acetylcholine orthosteric site.

    PubMed

    Jacobson, Marlene A; Kreatsoulas, Constantine; Pascarella, Danette M; O'Brien, Julie A; Sur, Cyrille

    2010-10-01

    Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one), and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297-1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.

  12. Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

    SciTech Connect

    Shimohama, S.; Tsukahara, T.; Taniguchi, T.; Fujiwara, M.

    1985-11-18

    Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.

  13. Acetylcholine Receptor and Ion Conductance Modulator Sites at the Murine Neuromuscular Junction: Evidence from Specific Toxin Reactions

    PubMed Central

    Albuquerque, Edson X.; Barnard, Eric A.; Chiu, Tieh H.; Lapa, Antonio J.; Dolly, J. Oliver; Jansson, Sten-Erik; Daly, John; Witkop, Bernhard

    1973-01-01

    The perhydro derivative of histrionicotoxin reversibly blocks the excitatory ionic transduction system in the synaptic and sarcolemmal membranes of mammalian skeletal muscle cells. The efficacy of perhydrohistrionicotoxin as an antagonist at the post-synaptic membrane is increased by the transient presence of acetylcholine in the endplate of innervated muscles and at extrajunctional receptors in denervated muscles. α-Bungarotoxin and [3H]monoacetyl-α-bungarotoxin block the endplate acetylcholine receptors, each binding to the same extent. The effect of bungarotoxin is partially reversible. These electrophysiological results, together with the effects of perhydrohistrionicotoxin and/or d-tubocurarine on the binding of [3H]monoacetyl-α-bungarotoxin at endplates of murine diaphragm muscle and on the bungarotoxin-elicited irreversible blockade of neuromuscular transmission, suggest that at least two types of sites participate in the synaptic excitation by acetylcholine. One site, competitively blocked by bungarotoxin and by curare, is presumably the acetylcholine receptor. Binding of bungarotoxin at this site is responsible for an irreversible blockade of neuromuscular transmission. The second site, competitively blocked by bungarotoxin and perhydrohistrionicotoxin, is proposed to be part of the cholinergic ion conductance modulator. Binding of bungarotoxin to this site does not result in an irreversible blockade. Images PMID:4351811

  14. Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

    PubMed Central

    Mulac-Jericevic, B; Atassi, M Z

    1987-01-01

    The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites. PMID:3435488

  15. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  16. Rabies virus binding to an acetylcholine receptor alpha-subunit peptide.

    PubMed

    Lentz, T L

    1990-04-01

    The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.

  17. Reductions in (/sup 3/H)nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study

    SciTech Connect

    Whitehouse, P.J.; Martino, A.M.; Wagster, M.V.; Price, D.L.; Mayeux, R.; Atack, J.R.; Kellar, K.J.

    1988-05-01

    In Alzheimer's disease (AD) and Parkinson's disease (PD), dysfunction in the basal forebrain cholinergic system is accompanied by a consistent loss of presynaptic cholinergic markers in cortex, but changes in cholinergic receptor binding sites are poorly understood. In the present study, we used receptor autoradiography to map the distribution of nicotinic (/sup 3/H)acetylcholine binding sites in cortices of individuals with AD and PD and matched control subjects. In both diseases, a profound loss of nicotinic receptors occurs in all cortical layers, particularly the deepest layers.

  18. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: (/sup 3/H)chlorpromazine labels homologous residues in the. beta. and delta chains

    SciTech Connect

    Giraudat, J.; Dennis, M.; Heidmann, T.; Haumont, P.Y.; Lederer, F.; Changeux, J.P.

    1987-05-05

    The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker (/sup 3/H)chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled ..beta.. chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by (/sup 3/H)chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the ..beta.. chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

  19. Cation-pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine.

    PubMed

    Beene, Darren L; Brandt, Gabriel S; Zhong, Wenge; Zacharias, Niki M; Lester, Henry A; Dougherty, Dennis A

    2002-08-13

    A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

  20. Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

    PubMed

    Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

    1994-09-01

    Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

  1. Ligand-binding properties of an unusual nicotinic acetylcholine receptor subtype on isolated outer hair cells from guinea pig cochlea.

    PubMed

    Lawoko, G; Järlebark, L; Heilbronn, E

    1995-07-28

    Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [125I]alpha-bungarotoxin revealed a KD of 62 +/- 2 nM, Bmax = 7.2 +/- 1.8 x 10(7) binding sites/OHC, and a slowly reversible dissociation rate constant, kappa-1 = 2.2 +/- 0.01 x 10(-4) min-1. L-[3H]Nicotine bound reversibly (estimated KD approximately 230 nM and Bmax approximately 5 x 10(7)) with kinetic rate constants of association kappa-1 = 6.2 +/- 0.06 x 10(4) min-1 nM-1 and dissociation kappa-1 = 0.23 +/- 0.003 min-1. [3H]Strychnine bound to OHC with a KD of 35 +/- 6 nM and Bmax = 2.6 +/- 0.5 x 10(7), and binding increased 3-4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > nM concentrations, of [3H]3-quinuclidinyl benzilate (KD = 11.5 +/- 5 nM; Bmax = 2.5 +/- 0.6 x 10(6)) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 +/- 0.5 and 6.5 +/- 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.

  2. Ligand binding to nicotinic acetylcholine receptor investigated by surface plasmon resonance.

    PubMed

    Kröger, D; Hucho, F; Vogel, H

    1999-08-01

    Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A K(D) = 3.5 × 10(-)(6) M for the detergent-solubilized receptor and a K(D) = 1.4 × 10(-)(5) M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a K(D) = 4.5 × 10(-)(5) M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligand-receptor interactions in case of small ligand molecules by mass-sensitive techniques.

  3. Acetylcholine-binding protein in the hemolymph of the planorbid snail Biomphalaria glabrata is a pentagonal dodecahedron (60 subunits).

    PubMed

    Saur, Michael; Moeller, Vanessa; Kapetanopoulos, Katharina; Braukmann, Sandra; Gebauer, Wolfgang; Tenzer, Stefan; Markl, Jürgen

    2012-01-01

    Nicotinic acetylcholine receptors (nAChR) play important neurophysiological roles and are of considerable medical relevance. They have been studied extensively, greatly facilitated by the gastropod acetylcholine-binding proteins (AChBP) which represent soluble structural and functional homologues of the ligand-binding domain of nAChR. All these proteins are ring-like pentamers. Here we report that AChBP exists in the hemolymph of the planorbid snail Biomphalaria glabrata (vector of the schistosomiasis parasite) as a regular pentagonal dodecahedron, 22 nm in diameter (12 pentamers, 60 active sites). We sequenced and recombinantly expressed two ∼25 kDa polypeptides (BgAChBP1 and BgAChBP2) with a specific active site, N-glycan site and disulfide bridge variation. We also provide the exon/intron structures. Recombinant BgAChBP1 formed pentamers and dodecahedra, recombinant BgAChBP2 formed pentamers and probably disulfide-bridged di-pentamers, but not dodecahedra. Three-dimensional electron cryo-microscopy (3D-EM) yielded a 3D reconstruction of the dodecahedron with a resolution of 6 Å. Homology models of the pentamers docked to the 6 Å structure revealed opportunities for chemical bonding at the inter-pentamer interfaces. Definition of the ligand-binding pocket and the gating C-loop in the 6 Å structure suggests that 3D-EM might lead to the identification of functional states in the BgAChBP dodecahedron.

  4. Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor.

    PubMed

    Lentz, T L; Hawrot, E; Wilson, P T

    1987-01-01

    Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha

  5. Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor.

    PubMed

    Bracci, L; Antoni, G; Cusi, M G; Lozzi, L; Niccolai, N; Petreni, S; Rustici, M; Santucci, A; Soldani, P; Valensin, P E

    1988-09-01

    It has been reported that binding to muscle nicotinic acetylcholine receptor at the post-synaptic membrane is an important event of the rabies virus neurotropism. The binding site can be located within the 190-203 region of the virus glycoprotein sharing a high degree of homology with the "toxic loop" of the curare-mimetic snake neurotoxins. We have synthesized a tetradecapeptide corresponding to this glycoprotein region and used it, following conjugation with an immunogenic carrier to raise MAbs. We found that some MAbs raised against the peptide were able to recognize both the virus glycoprotein and the snake neurotoxin alpha-bungarotoxin; moreover, they can inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor extracted from the electric organs of Torpedo marmorata. On the basis of this cross-reactivity, we suggest that rabies virus glycoprotein and curare-mimetic snake neurotoxins share three-dimensionally similar structures in order to bind to the nicotinic cholinergic receptor. The potential use of the immunogenic properties of the peptide for the rational design of a synthetic vaccine against rabies is proposed.

  6. Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.

    PubMed

    Young, Gareth T; Zwart, Ruud; Walker, Alison S; Sher, Emanuele; Millar, Neil S

    2008-09-23

    Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

  7. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-11-07

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding.

  8. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed Central

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479887

  9. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  10. Postischemic alteration of muscarinic acetylcholine and adenosine A1 binding sites in gerbil brain. Protective effects of a novel vinca alkaloid derivative, vinconate, and pentobarbital using an autoradiographic study.

    PubMed

    Araki, T; Kato, H; Kogure, K

    1992-01-01

    We studied the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors following transient cerebral ischemia in Mongolian gerbils and examined the effects of the novel vinca alkaloid derivative vinconate and pentobarbital against the alterations in the binding of these receptors. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Quinuclidinyl benzilate (QNB) and [3H]cyclohexyladenosine (CHA) were used to label muscarinic cholinergic and adenosine A1 receptors, respectively. The [3H]QNB and [3H]CHA bindings showed no significant alteration in the gerbil brain 5 h after ischemia. However, these bindings in the striatum, the hippocampal CA1 sector, and the hippocampal CA3 sector revealed a significant reduction 7 days after ischemia. The [3H]CHA binding also showed a significant decline in the dentate molecular layer 7 days after ischemia. Intraperitoneal application of vinconate (100 and 300 mg/kg) 10 min and pentobarbital (40 mg/kg) 30 min before ischemia showed a mild reduction in the [3H]CHA binding in the brain 5 h after ischemia. Especially, the reduction was found in the hippocampal CA1 sector and the dentate molecular layer. However, the [3H]QNB binding revealed no significant alteration in the brain 5 h after ischemia. Seven days after ischemia, both drugs prevented a marked reduction in the [3H]CHA binding in the striatum, but not in the hippocampal CA1 sector, the hippocampal CA3 sector, and the dentate molecular layer. By contrast, vinconate and pentobarbital failed to prevent the reduction in the [3H]QNB binding in the striatum. Morphological study indicated that vinconate and pentobarbital ameliorated the neuronal damage to the striatum, but not the hippocampal damage 7 days after ischemia. This histological finding was relatively consistent with the alteration in the [3H]CHA binding. These receptor autoradiographic and histological

  11. The nicotinic acetylcholine receptor: Binding of nitroxide analogs of a local anesthetic and a photoactivatable analog of phosphatidylserine

    SciTech Connect

    Blanton, M.P.

    1989-01-01

    Electron spin resonance was used to contrast the accessibility of tertiary and quaternary amine local anesthetics to their high affinity binding site in the desensitized Torpedo californica acetylcholine receptor (AchR). Preincubation of AchR-rich membranes with agonist resulted in a substantial reduction in the initial association of the quaternary amine local anesthetic C6SLMEI with the receptor. The time-dependent reduction in association follows a biphasic exponential function having rate constants of 0.19 min{sup {minus}1} and 0.03 min{sup {minus}1}. In contrast, agonist preincubation did not produce a comparable decrease in the association of C6SL, a tertiary amine analog, with the AchR. The results are modeled in two ways: (1) A charge gate near the channel mouth in the desensitized receptor limits access of the permanently charged cationic local anesthetic (C6SLMEI), but not for the uncharged form of the tertiary amine anesthetic C6SL. (2) A hydrophobic pathway, possibly through a corridor in the annular lipid surrounding receptor subunits, allows the uncharged form of C6SL to reach the high affinity binding site in the AchR. A photoactivatable analog of phosphatidylserine {sup 125}I 4-azido salicylic acid-phosphatidylserine ({sup 125}I ASA-PS) was use to label both Torpedo californica acetylcholine receptor-rich membranes and reconstituted AchR membranes. All four subunits of the AchR were found to incorporate label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporate {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. Eighty-one per cent of the incorporated label was localized to 11.7 and 10.1 kdal V8 cleavage fragments.

  12. Computational determination of the binding mode of α-conotoxin to nicotinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Tabassum, Nargis; Yu, Rilei; Jiang, Tao

    2016-12-01

    Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The α-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, α-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of α-conotoxins in complex with acetylcholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the α1 and α9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of α-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of α-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between α-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of α-conotoxins on AChRs allows rational design of α-conotoxin analogues with improved potency or selectivity to nAChRs.

  13. Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

    PubMed Central

    Schloss, P; Hermans-Borgmeyer, I; Betz, H; Gundelfinger, E D

    1988-01-01

    The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system. PMID:3141150

  14. Anticonvulsants Based on the α-Substituted Amide Group Pharmacophore Bind to and Inhibit Function of Neuronal Nicotinic Acetylcholine Receptors.

    PubMed

    Krivoshein, Arcadius V

    2016-03-16

    Although the antiepileptic properties of α-substituted lactams, acetamides, and cyclic imides have been known for over 60 years, the mechanism by which they act remains unclear. I report here that these compounds bind to the nicotinic acetylcholine receptor (nAChR) and inhibit its function. Using transient kinetic measurements with functionally active, nondesensitized receptors, I have discovered that (i) α-substituted lactams and cyclic imides are noncompetitive inhibitors of heteromeric subtypes (such as α4β2 and α3β4) of neuronal nAChRs and (ii) the binding affinity of these compounds toward the nAChR correlates with their potency in preventing maximal electroshock (MES)-induced convulsions in mice. Based on the hypothesis that α-substituted amide group is the essential pharmacophore of these drugs, I found and tested a simple compound, 2-phenylbutyramide. This compound indeed inhibits nAChR and shows good anticonvulsant activity in mice. Molecular docking simulations suggest that α-substituted lactams, acetamides, and cyclic imides bind to the same sites on the extracellular domain of the receptor. These new findings indicate that inhibition of brain nAChRs may play an important role in the action of these antiepileptic drugs, a role that has not been previously recognized.

  15. Insight into the Binding Mode of Agonists of the Nicotinic Acetylcholine Receptor from Calculated Electron Densities

    PubMed Central

    Beck, Michael E; Gutbrod, Oliver; Matthiesen, Svend

    2015-01-01

    Insect nicotinic acetylcholine receptors (nAChRs) are among the most prominent and most economically important insecticide targets. Thus, an understanding of the modes of binding of respective agonists is important for the design of specific compounds with favorable vertebrate profiles. In the case of nAChRs, the lack of available high-resolution X-ray structures leaves theoretical considerations as the only viable option. Starting from classical homology and docking approaches, binding mode hypotheses are created for five agonists of the nAChR, covering insecticides in the main group 4 of the Insecticide Resistance Action Committee (IRAC) mode of action (MoA) classification, namely, neonicotinoids, nicotine, sulfoxaflor, and butenolides. To better understand these binding modes, the topologies of calculated electron densities of small-model systems are analyzed in the framework of the quantum theory of atoms in molecules. The theoretically obtained modes of binding are very much in line with the biology-driven IRAC MoA classification of the investigated ligands. PMID:26175091

  16. Insight into the Binding Mode of Agonists of the Nicotinic Acetylcholine Receptor from Calculated Electron Densities.

    PubMed

    Beck, Michael E; Gutbrod, Oliver; Matthiesen, Svend

    2015-07-15

    Insect nicotinic acetylcholine receptors (nAChRs) are among the most prominent and most economically important insecticide targets. Thus, an understanding of the modes of binding of respective agonists is important for the design of specific compounds with favorable vertebrate profiles. In the case of nAChRs, the lack of available high-resolution X-ray structures leaves theoretical considerations as the only viable option. Starting from classical homology and docking approaches, binding mode hypotheses are created for five agonists of the nAChR, covering insecticides in the main group 4 of the Insecticide Resistance Action Committee (IRAC) mode of action (MoA) classification, namely, neonicotinoids, nicotine, sulfoxaflor, and butenolides. To better understand these binding modes, the topologies of calculated electron densities of small-model systems are analyzed in the framework of the quantum theory of atoms in molecules. The theoretically obtained modes of binding are very much in line with the biology-driven IRAC MoA classification of the investigated ligands.

  17. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  18. 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.

  19. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  20. Structural Characterization of the Putative Cholinergic Binding Region alpha(179-201) of the Nicotinic Acetylcholine Receptor. Part 1. Review and Experimental Design.

    DTIC Science & Technology

    1993-04-01

    alpha-subunit of the acetylcholine receptor. Proc. Natl. Acad. Sci USA, vol. 82, pp. 3490-3493, 1985 Neumann, D, Barchan , D., Safran, A., GershoniJ., and...pp. 3008-3011, 1986 MAY. Neumann, D., Barchan , D., Fuchs, S., Analysis of ligand binding to the synthetic dodeca-peptide 185-1% of the acetylcholine

  1. 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.

  2. Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides.

    PubMed Central

    Marinou, Martha; Tzartos, Socrates J

    2003-01-01

    The neuronal alpha7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin alpha-bungarotoxin (alpha-Bgt). Fine mapping of the alpha-Bgt-binding site on the human alpha7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human alpha7 subunit (residues 1-206). Screening of these peptides for (125)I-alpha-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186-197, which exhibited the best (125)I-alpha-Bgt binding, and one at residues 159-165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the alpha7(186-197) peptide was almost completely inhibited by unlabelled alpha-Bgt or d -tubocurarine. Alanine substitutions within the sequence 186-198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the alpha-Bgt binding site of the alpha1 subunit (residues 188-200 in Torpedo AChR). In competition experiments, the soluble peptides alpha7(186-197) and Torpedo alpha1(184-200) inhibited the binding of (125)I-alpha-Bgt to the immobilized alpha7(186-197) peptide, to native Torpedo AChR, and to the extracellular domain of the human alpha1 subunit. These results suggest that the toxin-binding sites of the neuronal alpha7 and muscle-type AChRs bind to identical or overlapping sites on the alpha-Bgt molecule. In support of this, when synthetic alpha-Bgt peptides were tested for binding to the recombinant extracellular domains of the human alpha7 and alpha1 subunits, and to native Torpedo and alpha7 AChR, the results indicated that alpha-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail. PMID:12614199

  3. Efficient expression of acetylcholine-binding protein from Aplysia californica in Bac-to-Bac system.

    PubMed

    Lin, Bo; Meng, Hailing; Bing, Hui; Zhangsun, Dongting; Luo, Sulan

    2014-01-01

    The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 10(6) cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

  4. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  5. Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface.

    PubMed

    Ahring, Philip K; Olsen, Jeppe A; Nielsen, Elsebet Ø; Peters, Dan; Pedersen, Martin H F; Rohde, Line A; Kastrup, Jette S; Shahsavar, Azadeh; Indurthi, Dinesh C; Chebib, Mary; Gajhede, Michael; Balle, Thomas

    2015-05-01

    The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-β2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2(HQT)), which converts the β(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(β2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-β2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors.

  6. 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)

  7. Identification of a Negative Allosteric Site on Human α4β2 and α3β4 Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Pavlovicz, Ryan E.; Henderson, Brandon J.; Bonnell, Andrew B.; Boyd, R. Thomas; McKay, Dennis B.; Li, Chenglong

    2011-01-01

    Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and

  8. Discovery, synthesis, biological evaluation and structure-based optimization of novel piperidine derivatives as acetylcholine-binding protein ligands

    PubMed Central

    Shen, Jian; Yang, Xi-cheng; Yu, Ming-cheng; Xiao, Li; Zhang, Xun-jie; Sun, Hui-jiao; Chen, Hao; Pan, Guan-xin; Yan, Yu-rong; Wang, Si-chen; Li, Wei; Zhou, Lu; Xie, Qiong; Yu, Lin-qian; Wang, Yong-hui; Shao, Li-ming

    2017-01-01

    The homomeric α7 nicotinic receptor (α7 nAChR) is widely expressed in the human brain that could be activated to suppress neuroinflammation, oxidative stress and neuropathic pain. Consequently, a number of α7 nAChR agonists have entered clinical trials as anti-Alzheimer's or anti-psychotic therapies. However, high-resolution crystal structure of the full-length α7 receptor is thus far unavailable. Since acetylcholine-binding protein (AChBP) from Lymnaea stagnalis is most closely related to the α-subunit of nAChRs, it has been used as a template for the N-terminal domain of α-subunit of nAChR to study the molecular recognition process of nAChR-ligand interactions, and to identify ligands with potential nAChR-like activities. Here we report the discovery and optimization of novel acetylcholine-binding protein ligands through screening, structure-activity relationships and structure-based design. We manually screened in-house CNS-biased compound library in vitro and identified compound 1, a piperidine derivative, as an initial hit with moderate binding affinity against AChBP (17.2% inhibition at 100 nmol/L). During the 1st round of optimization, with compound 2 (21.5% inhibition at 100 nmol/L) as the starting point, 13 piperidine derivatives with different aryl substitutions were synthesized and assayed in vitro. No apparent correlation was demonstrated between the binding affinities and the steric or electrostatic effects of aryl substitutions for most compounds, but compound 14 showed a higher affinity (Ki=105.6 nmol/L) than nicotine (Ki=777 nmol/L). During the 2nd round of optimization, we performed molecular modeling of the putative complex of compound 14 with AChBP, and compared it with the epibatidine-AChBP complex. The results suggested that a different piperidinyl substitution might confer a better fit for epibatidine as the reference compound. Thus, compound 15 was designed and identified as a highly affinitive acetylcholine-binding protein ligand. In

  9. Synthesis, Nicotinic Acetylcholine Receptor Binding, Antinociceptive and Seizure Properties of Methyllycaconitine Analogs

    PubMed Central

    Carroll, F. Ivy; Ma, Wei; Navarro, Hernán A.; Abraham, Philip; Wolckenhauer, Scott A.; Damaj, M. I.; Martin, Billy R.

    2007-01-01

    A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl- and 2-cyclohexylsuccinimidobenzoyl (1b–f) group. The analogs 1b–f were evaluated for their inhibition of [125I]iodo MLA binding at rat brain α7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b–f were evaluated for inhibition of binding to rat brain α, β nAChR using [3H]epibatidine. At the α7 nAChR, MLA showed a Ki value of 0.87 nM, analogs 1b–e possessed Ki values of 1.68–2.16 nM, and 1f showed a Ki value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (Ki = 1.68 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for α, β nAChRs. MLA antagonized nicotine-induced seizures with an AD50 = 2mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine’s antinociceptive effects in the tail-flick assay. Compound 1c (Ki = 1.78 nM at α7 nAChR) with an AD50 value of 1.8 mg/kg was 6.7 times more potent than MLA (AD50 = 12 mg/kg) in antagonizing nicotine’s antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against β-amyloid1–42, these new analogs which have high α7 nAChR affinity and good selectivity relative to α, β nAChRs will be useful biological tools for studying the effects of α7 nAChR antagonist and neuroprotection. PMID:17098430

  10. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    SciTech Connect

    Bencherif, M.; Lukas, R.J. )

    1991-06-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

  11. (/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.

  12. Acetylcholine receptor binding antibody-associated myasthenia gravis and rhabdomyolysis induced by nivolumab in a patient with melanoma.

    PubMed

    Shirai, Takushi; Sano, Tasuku; Kamijo, Fuminao; Saito, Nana; Miyake, Tomomi; Kodaira, Minori; Katoh, Nagaaki; Nishie, Kenichi; Okuyama, Ryuhei; Uhara, Hisashi

    2016-01-01

    We reported an 81-year-old woman with metastatic melanoma, in whom myasthenia gravis and rhabdomyolysis developed after nivolumab monotherapy. The first symptom of myasthenia gravis was dyspnea. Ultrasonography detected hypokinesis of the bilateral diaphragm suggesting myasthenia gravis, although there was no abnormal finding of the lungs in computed tomography images. Acetylcholine receptor binding antibodies were low-titer positive in the preserved serum before administration of nivolumab, strongly suggesting that the myasthenia gravis was a nivolumab-related immune adverse event. Despite the remarkable clinical benefits of immune checkpoint inhibitors for patients with advanced melanoma, it is important to recognize unexpected immune-related adverse events.

  13. Nicotinic binding in rat brain: autoradiographic comparison of (/sup 3/H)acetylcholine, (/sup 3/H)nicotine, and (/sup 125/I)-alpha-bungarotoxin

    SciTech Connect

    Clarke, P.B.; Schwartz, R.D.; Paul, S.M.; Pert, C.B.; Pert, A.

    1985-05-01

    Three radioligands have been commonly used to label putative nicotinic cholinoceptors in the mammalian central nervous system: the agonists (/sup 3/H)nicotine and (/sup 3/H)acetylcholine ((/sup 3/H)ACh--in the presence of atropine to block muscarinic receptors), and the snake venom extract, (/sup 125/I)-alpha-bungarotoxin((/sup 125/I)BTX), which acts as a nicotinic antagonist at the neuromuscular junction. Binding studies employing brain homogenates indicate that the regional distributions of both (/sup 3/H)nicotine and (/sup 3/H)ACh differ from that of (/sup 125/I)BTX. The possible relationship between brain sites bound by (/sup 3/H)nicotine and (/sup 3/H)ACh has not been examined directly. The authors have used the technique of autoradiography to produce detailed maps of (/sup 3/H)nicotine, (/sup 3/H)ACh, and (/sup 125/I)BTX labeling; near-adjacent tissue sections were compared at many levels of the rat brain. The maps of high affinity agonist labeling are strikingly concordant, with highest densities in the interpeduncular nucleus, most thalamic nuclei, superior colliculus, medial habenula, presubiculum, cerebral cortex (layers I and III/IV), and the substantia nigra pars compacta/ventral tegmental area. The pattern of (/sup 125/I)BTX binding is strikingly different, the only notable overlap with agonist binding being the cerebral cortex (layer I) and superior colliculus. (/sup 125/I)BTX binding is also dense in the inferior colliculus, cerebral cortex (layer VI), hypothalamus, and hippocampus, but is virtually absent in thalamus. Various lines of evidence suggest that the high affinity agonist-binding sites in brain correspond to nicotinic cholinergic receptors similar to those found at autonomic ganglia; BTX-binding sites may also serve as receptors for nicotine and are possibly related to neuromuscular nicotinic cholinoceptors.

  14. 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.

  15. Identification and Functional Characterization of a Novel Acetylcholine-binding Protein from the Marine Annelid Capitella teleta

    SciTech Connect

    McCormack, T.; Petrovich,; Mercier, K; DeRose, E; Cuneo, M; Williams, J; Johnson, K; Lamb, P; London, R; Yakel, J

    2010-01-01

    We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) {alpha} subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and {alpha}-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 {micro}M, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K{sub D} = 163 {micro}M). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.

  16. Identification and Functional Characterization of a Novel Acetylcholine-Binding Protein from the Marine Annelid Capitella teleta†

    PubMed Central

    McCormack, Thomas; Petrovich, Robert M.; Mercier, Kelly A.; DeRose, Eugene F.; Cuneo, Matthew J.; Williams, Jason; Johnson, Katina L.; Lamb, Patricia W.; London, Robert E.; Yakel, Jerrel L.

    2016-01-01

    We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21–30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) α subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and α-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 μM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (KD = 163 μM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca. PMID:20136097

  17. Kinetic analysis of 3-quinuclidinyl 4-( sup 125 I)iodobenzilate transport and specific binding to muscarinic acetylcholine receptor in rat brain in vivo: Implications for human studies

    SciTech Connect

    Sawada, Y.; Hiraga, S.; Francis, B.; Patlak, C.; Pettigrew, K.; Ito, K.; Owens, E.; Gibson, R.; Reba, R.; Eckelman, W. )

    1990-11-01

    Radioiodinated R- and S-Quinuclidinyl derivatives of RS-benzilate (R- and S-125IQNB) have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor binding in vivo. Two sets of experiments were performed in rats. The first involved determining the metabolite-corrected blood concentration and tissue distribution of tracer R-IQNB (active enantiomer) and S-IQNB (inactive enantiomer) in brain 1 min to 26 h after intravenous injection. The second involved the measurement of brain tissue washout over a 2-min period after loading the brain by an intracarotid artery injection of the ligands. Various pharmacokinetic models were tested, which included transport across the blood-brain barrier (BBB), nonspecific binding, low-affinity binding, and high-affinity binding. Our analysis demonstrated that the assumptions of rapid equilibrium across the BBB and rapid nonspecific binding are incorrect and result in erroneous estimates of the forward rate constant for binding at the high-affinity receptor sites (k3). The estimated values for influx across the BBB (K1), the steady-state accumulation rate in cerebrum (K), and the dissociation rate constant at the high-affinity site (k4) of R-IQNB were independent of the specific compartmental model used to analyze these data (K1 approximately 0.23 ml/min/g, K approximately 0.13 ml/min/g, and k4 approximately 0.0019 min-1 for caudate). In contrast, the estimated values of k3 and the efflux rate constant (k2) varied over a 10-fold range between different compartmental models (k3 approximately 2.3-22 min-1 and k2 approximately 1.6-16 min-1 in caudate), but their ratios were constant (k3/k2 approximately 1.4).

  18. 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.

  19. Imidacloprid and thiacloprid neonicotinoids bind more favourably to cockroach than to honeybee α6 nicotinic acetylcholine receptor: insights from computational studies.

    PubMed

    Selvam, Balaji; Graton, Jérôme; Laurent, Adèle D; Alamiddine, Zakaria; Mathé-Allainmat, Monique; Lebreton, Jacques; Coqueret, Olivier; Olivier, Christophe; Thany, Steeve H; Le Questel, Jean-Yves

    2015-02-01

    The binding interactions of two neonicotinoids, imidacloprid (IMI) and thiacloprid (THI) with the extracellular domains of cockroach and honeybee α6 nicotinic acetylcholine receptor (nAChR) subunits in an homomeric receptor have been studied through docking and molecular dynamics (MD) simulations. The binding mode predicted for the two neonicotinoids is validated through the good agreement observed between the theoretical results with the crystal structures of the corresponding complexes with Ac-AChBP, the recognized structural surrogate for insects nAChR extracellular ligand binding domain. The binding site of the two insect α6 receptors differs by only one residue of loop D, a serine residue (Ser83) in cockroach being replaced by a lysine residue (Lys108) in honeybee. The docking results show very close interactions for the two neonicotinoids with both α6 nAChR models, in correspondence to the trends observed in the experimental neonicotinoid-Ac-AChBP complexes. However, the docking parameters (scores and energies) are not significantly different between the two insect α6 nAChRs to draw clear conclusions. The MD results bring distinct trends. The analysis of the average interaction energies in the two insects α6 nAChRs shows indeed better affinity of neonicotinoids bound to α6 cockroach compared to honeybee nAChR. This preference is explained by tighter contacts with aromatic residues (Trp and Tyr) of the binding pocket. Interestingly, the non-conserved residue Lys108 of loop D of α6 honeybee nAChR interacts through van der Waals contacts with neonicotinoids, which appear more favourable than the direct or water mediated hydrogen-bond interaction between the OH group of Ser83 of α6 cockroach nAChR and the electronegative terminal group of the two neonicotinoids (nitro in IMI and cyano in THI). Finally, in both insects nAChRs, THI is consistently found to bind more favourably than IMI.

  20. 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.

  1. 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.

  2. Computational analysis of the binding ability of heterocyclic and conformationally constrained epibatidine analogs in the neuronal nicotinic acetylcholine receptor.

    PubMed

    Soriano, Elena; Marco-Contelles, José; Colmena, Inés; Gandía, Luis

    2010-05-01

    One of the most critical issues on the study of ligand-receptor interactions in drug design is the knowledge of the bioactive conformation of the ligand. In this study, we describe a computational approach aimed at estimating the binding ability of epibatidine analogs to interact with the neuronal nicotinic acetylcholine receptor (nAChR) and get insights into the bioactive conformation. The protocol followed consists of a docking analysis and evaluation of pharmacophore parameters of the docked structures. On the basis of the biological data, the results have revealed that the docking analysis is able to predict active ligands, whereas further efforts are needed to develop a suitable and solid pharmacophore model.

  3. Lipophilicity as a determinant of binding of procaine analogs to rat α3β4 nicotinic acetylcholine receptor.

    PubMed

    Cheffer, Arquimedes; Mustafa, Elba Vieira; T-do Amaral, Antonia; Ulrich, Henning

    2012-08-01

    Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structure-activity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Affinity values (log[1/IC50]) of these compounds for the α(3)β(4) nAChR were determined by their competition with [(3)H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structure-activity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity.

  4. Evidence of mineralization activity and supramolecular assembly by the N-terminal sequence of ACCBP, a biomineralization protein that is homologous to the acetylcholine binding protein family.

    PubMed

    Amos, Fairland F; Ndao, Moise; Evans, John Spencer

    2009-12-14

    Several biomineralization proteins that exhibit intrinsic disorder also possess sequence regions that are homologous to nonmineral associated folded proteins. One such protein is the amorphous calcium carbonate binding protein (ACCBP), one of several proteins that regulate the formation of the oyster shell and exhibit 30% conserved sequence identity to the acetylcholine binding protein sequences. To gain a better understanding of the ACCBP protein, we utilized bioinformatic approaches to identify the location of disordered and folded regions within this protein. In addition, we synthesized a 50 AA polypeptide, ACCN, representing the N-terminal domain of the mature processed ACCBP protein. We then utilized this polypeptide to determine the mineralization activity and qualitative structure of the N-terminal region of ACCBP. Our bioinformatic studies indicate that ACCBP consists of a ten-stranded beta-sandwich structure that includes short disordered sequence blocks, two of which reside within the primarily helical and surface-accessible ACCN sequence. Circular dichroism studies reveal that ACCN is partially disordered in solution; however, ACCN can be induced to fold into an alpha helix in the presence of TFE. Furthermore, we confirm that the ACCN sequence is multifunctional; this sequence promotes radial calcite polycrystal growth on Kevlar threads and forms supramolecular assemblies in solution that contain amorphous-appearing deposits. We conclude that the partially disordered ACCN sequence is a putative site for mineralization activity within the ACCBP protein and that the presence of short disordered sequence regions within the ACCBP fold are essential for function.

  5. Novel Triazole-Quinoline Derivatives as Selective Dual Binding Site Acetylcholinesterase Inhibitors.

    PubMed

    Mantoani, Susimaire P; Chierrito, Talita P C; Vilela, Adriana F L; Cardoso, Carmen L; Martínez, Ana; Carvalho, Ivone

    2016-02-05

    Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as hAChE and hBChE inhibitors, and some derivatives showed IC50 values in the micro-molar range and were remarkably selective towards hAChE. Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.

  6. 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.

  7. 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

  8. Marine Natural Products Acting on the Acetylcholine-Binding Protein and Nicotinic Receptors: From Computer Modeling to Binding Studies and Electrophysiology

    PubMed Central

    Kudryavtsev, Denis; Makarieva, Tatyana; Utkina, Natalia; Santalova, Elena; Kryukova, Elena; Methfessel, Christoph; Tsetlin, Victor; Stonik, Valentin; Kasheverov, Igor

    2014-01-01

    For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources. PMID:24686559

  9. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    SciTech Connect

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M. . E-mail: laurie.chan@mcgill.ca

    2005-05-15

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

  10. Loop 2 of Ophiophagus hannah toxin b binds with neuronal nicotinic acetylcholine receptors and enhances intracranial drug delivery.

    PubMed

    Zhan, Changyou; Yan, Zhiqiang; Xie, Cao; Lu, Weiyue

    2010-12-06

    Three-finger snake neurotoxins have been widely investigated for their high binding affinities with nicotinic acetylcholine receptors (nAChRs), which are widely expressed in the central nervous system including the blood-brain barrier and thus mediate intracranial drug delivery. The loop 2 segments of three-finger snake neurotoxins are considered as the binding domain with nAChRs, and thus, they may have the potential to enhance drug or drug delivery system intracranial transport. In the present work, binding of the synthetic peptides to the neuronal nAChRs was assessed by measuring their ability to inhibit the binding of (125)I-α-bungarotoxin to the receptor. The loop 2 segment of Ophiophagus hannah toxin b (KC2S) showed high binding affinity, and the competitive binding IC(50) value was 32.51 nM. Furthermore, the brain targeting efficiency of KC2S had been investigated in vitro and in vivo. The specific uptake by brain capillary endothelial cells (BCECs) demonstrated that KC2S could be endocytosized after binding with nAChRs. In vivo, the qualitative and quantitative biodistribution results of fluorescent dyes (DiR or coumarin-6) indicated that KC2S modified poly(ethylene glycol)-poly(lactic acid) micelles (KC2S-PEG-PLA micelles) could enhance intracranial drug delivery. Furthermore, intravenous treatment with paclitaxel-encapsulated KC2S-PEG-PLA micelles (KC2S-PEG-PLA-PTX micelles) afforded robust inhibition of intracranial glioblastoma. The median survival time of KC2S-PEG-PLA-PTX-micelle-treated mice (47.5 days) was significantly longer than that of mice treated by mPEG-PLA-PTX micelles (41.5 days), Taxol (38.5 days), or saline (34 days). Compared with the short peptide derived from rabies virus glycoprotein (RVG29) that has been previously reported as an excellent brain targeting ligand, KC2S has a similar binding affinity with neuronal nAChRs but fewer amino acid residues. Thus, we concluded that the loop 2 segment of Ophiophagus hannah toxin b could bind

  11. Progesterone Modulates a Neuronal Nicotinic Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Valera, S.; Ballivet, M.; Bertrand, D.

    1992-10-01

    The major brain nicotinic acetylcholine receptor is assembled from two subunits termed α 4 and nα 1. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroid is not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.

  12. Is the acetylcholine receptor a rabies virus receptor?

    PubMed

    Lentz, T L; Burrage, T G; Smith, A L; Crick, J; Tignor, G H

    1982-01-08

    Rabies virus was found on mouse diaphragms and on cultured chick myotubes in a distribution coinciding with that of the acetylcholine receptor. Treatment of the myotubes with alpha-bungarotoxin and d-tubocurarine before the addition of the virus reduced the number of myotubes that became infected with rabies virus. These findings together suggest that acetylcholine receptors may serve as receptors for rabies virus. The binding of virus to acetylcholine receptors, which are present in high density at the neuromuscular junction, would provide a mechanism whereby the virus could be locally concentrated at sites in proximity to peripheral nerves facilitating subsequent uptake and transfer to the central nervous system.

  13. Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands.

    PubMed

    Eldefrawi, M E; Eldefrawi, A T; Wilson, D B

    1975-09-23

    Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.

  14. The mechanism of acetylcholine receptor in binding MuSK in myasthenia gravis and the role of HSP90 molecular chaperone.

    PubMed

    Chen, Rongbo; Chen, Siqia; Liao, Juan; Chen, Xiaopu; Xu, Xiaoling

    2016-01-01

    As an autoimmune disease, myasthenia gravis is caused by the dysfunction of neural transmission. Acetylcholine is known to exert its function after entering into synaptic cleft through binding onto postsynaptic membrane. The role of acetylcholine in binding MuSK in myasthenia gravis, however, remains unknown. A total of 38 myasthenia gravis patients and 27 healthy controls were included in this study for the detection of the expression of MuSK using immunofluorescent method. Expression of both MuSK and interleukin-6 (IL-6) were measured by Western blot, followed by the correlation analysis between heat shock protein 90 (HSP90) and IL-6 which were measured by enzyme-linked immunosorbent assay (ELISA). In myasthenia gravis patients, MuSK was co-localized with acetylcholine at the postsynaptic membrane. Such accumulation of MuSK, however, did not occur in normal people. Meanwhile we also observed elevated expression of IL-6 in myasthenia gravis patients (p<0.05). ELISA assay showed higher expression of HSP90 in patients. Further signaling pathway screening revealed the activation of IL-6-mediated pathways including STAT3 and SPH2. In conclusion, MuSK was co-localized with acetylcholine in myasthenia gravis patients, with elevated expression. HSP90 in disease people can activate IL-6 mediated signaling pathways.

  15. The reaction site of a non-competitive antagonist in the delta-subunit of the nicotinic acetylcholine receptor.

    PubMed Central

    Oberthür, W; Muhn, P; Baumann, H; Lottspeich, F; Wittmann-Liebold, B; Hucho, F

    1986-01-01

    A site in the primary structure of the nicotinic acetylcholine receptor from Torpedo marmorata covalently labeled with the non-competitive antagonist [3H]triphenylmethylphosphonium (TPMP+) was localized. The label was found in position 262 of the delta-polypeptide chain. This site is specifically labeled in the presence of the agonist carbamoylcholine. Labeling is prevented by the non-competitive antagonist histrionicotoxin. Position 262, probably a serine, is located in the highly conserved membrane-spanning helix M2 (according to the predicted folding scheme of Finer-Moore and Stroud (1984). The relationship of this site to the receptor's ion channel and its regulation is discussed. Images Fig. 2. PMID:3758027

  16. Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography

    SciTech Connect

    Rosier, A.M.; Vandesande, F.; Orban, G.A. )

    1991-03-08

    The distribution of galanin (GAL) binding sites in the visual cortex of cat and monkey was determined by autoradiographic visualization of ({sup 125}I)-GAL binding to tissue sections. Binding conditions were optimized and, as a result, the binding was saturable and specific. In cat visual cortex, GAL binding sites were concentrated in layers I, IVc, V, and VI. Areas 17, 18, and 19 exhibited a similar distribution pattern. In monkey primary visual cortex, the highest density of GAL binding sites was observed in layers II/III, lower IVc, and upper V. Layers IVA and VI contained moderate numbers of GAL binding sites, while layer I and the remaining parts of layer IV displayed the lowest density. In monkey secondary visual cortex, GAL binding sites were mainly concentrated in layers V-VI. Layer IV exhibited a moderate density, while the supragranular layers contained the lowest proportion of GAL binding sites. In both cat and monkey, we found little difference between regions subserving central and those subserving peripheral vision. Similarities in the distribution of GAL and acetylcholine binding sites are discussed.

  17. 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

  18. α-Conotoxin OmIA Is a Potent Ligand for the Acetylcholine-binding Protein as Well as α3β2 and α7 Nicotinic Acetylcholine Receptors*

    PubMed Central

    Talley, Todd T.; Olivera, Baldomero M.; Han, Kyou-Hoon; Christensen, Sean B.; Dowell, Cheryl; Tsigelny, Igor; Ho, Kwok-Yiu; Taylor, Palmer; McIntosh, J. Michael

    2016-01-01

    The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the α-subunit of nicotinic acetylcholine receptors and in particular the homomeric α7 nicotinic receptor. We report the isolation and characterization of an α-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the α7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the α3β2 nAChR indicating that α-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of α3β2 nAChRs. α-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first α-conotoxin with higher affinity for the closely related receptor subtypes, α3β2 versus α6β2, and selectively blocks these two subtypes when compared with α2β2, α4β2, and α1β1δε nAChRs. PMID:16803900

  19. 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

  20. 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.

  1. Single-Channel Current Through Nicotinic Receptor Produced by Closure of Binding Site C-Loop

    SciTech Connect

    Wang, Hailong; Cheng, Xiaolin; McCammon, Jonathan

    2009-01-01

    We investigated the initial coupling of agonist binding to channel gating of the nicotinic acetylcholine receptor using targeted molecular-dynamics (TMD) simulation. After TMD simulation to accelerate closure of the C-loops at the agonist binding sites, the region of the pore that passes through the cell membrane expands. To determine whether the structural changes in the pore result in ion conduction, we used a coarse-grained ion conduction simulator, Biology Boltzmann transport Monte Carlo, and applied it to two structural frames taken before and after TMD simulation. The structural model before TMD simulation represents the channel in the proposed resting state, whereas the model after TMD simulation represents the channel in the proposed active state. Under external voltage biases, the channel in the active state was permeable to cations. Our simulated ion conductance approaches that obtained experimentally and recapitulates several functional properties characteristic of the nicotinic acetylcholine receptor. Thus, closure of the C-loop triggers a structural change in the channel sufficient to account for the open channel current. This approach of applying Biology Boltzmann transport Monte Carlo simulation can be used to further investigate the binding to gating transduction mechanism and the structural bases for ion selection and translocation.

  2. High density of benzodiazepine binding sites in the substantia innominata of the rat

    SciTech Connect

    Sarter, M.; Schneider, H.H.

    1988-07-01

    In order to study the neuronal basis of the pharmacological interactions between benzodiazepine receptor ligands and cortical cholinergic turnover, we examined the regional distribution of specific benzodiazepine binding sites using in vitro autoradiography. In the basal forebrain, the substantia innominata contained a high density of (/sup 3/H)lormetazepam (LMZ) binding sites (Bmax = 277 fmol/mg tissue; Kd = 0.55 nM). The label could be displaced by diazepam (IC50 = 100 nM), the benzodiazepine receptor antagonist beta-carboline ZK 93426 (45 nM) and the partial inverse agonist beta-carboline FG 7142 (540 nM). It is hypothesized that the amnesic effects of benzodiazepine receptor agonists are exerted through benzodiazepine receptors which are situated on cholinergic neurons in the substantia innominata and are involved in a tonic inhibition of cortical acetylcholine release. The benzodiazepine receptor antagonist ZK 93426 may exert its nootropic effects via benzodiazepine receptors in the substantia innominata and, consequently, by disinhibiting cortical acetylcholine release.

  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. Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

    PubMed Central

    Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

    1994-01-01

    Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

  5. 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

  6. 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.

  7. Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

    USGS Publications Warehouse

    Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

    2014-01-01

    Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

  8. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor. alpha. subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    SciTech Connect

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M. )

    1991-05-21

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) {alpha}subunit forms a binding site for {alpha}-bungarotoxin ({alpha}-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR {alpha}1 subunits were tested for their ability to bind {sup 125}I-{alpha}-BTX, and differences in {alpha}-BTX affinity were determined by using solution (IC{sub 50}s) and solid-phase (K{sub d}s) assays. Panels of overlapping peptides corresponding to the complete {alpha}1 subunit of mouse and human were also tested for {alpha}-BTX binding, but other sequence segments forming the {alpha}-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased {alpha}-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/{alpha}1-subunit interface a vicinal disulfide bound was not required for {alpha}-BTX binding.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

    Thomas, Trayder; McLean, Kimberley C; McRobb, Fiona M; Manallack, David T; Chalmers, David K; Yuriev, Elizabeth

    2014-01-27

    We have developed homology models of the acetylcholine muscarinic receptors M₁R-M₅R, based on the β₂-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M₂R model, using the M₃R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M₁R-M₅R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

  17. 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

  18. Acetylcholine inhibits Ca2+ current by acting exclusively at a site proximal to adenylyl cyclase in frog cardiac myocytes.

    PubMed

    Jurevicius, J; Fischmeister, R

    1996-03-15

    1. The effects of acetylcholine (ACh) on the L-type Ca2+ current (ICa) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 microM Iso produced a half-maximal increase in ICa since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 microM ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only approximately 50%. 3. When 10 microM Iso or 30 microM Fsk were applied to a Ca(2+)-free solution on one half of the cell, ICa was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3-10 microM) to the remote part had no effect on ICa, while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates ICa by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of ICa does not involve any additional cAMP-independent mechanisms occurring downstream from cAMP generation.

  19. Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

    PubMed Central

    Nys, Mieke; Wijckmans, Eveline; Farinha, Ana; Yoluk, Özge; Andersson, Magnus; Brams, Marijke; Spurny, Radovan; Peigneur, Steve; Tytgat, Jan; Lindahl, Erik; Ulens, Chris

    2016-01-01

    Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8–β9 loop in the extracellular ligand-binding domain. The β8–β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8–β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors. PMID:27791038

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Ligand Binding at the α4-α4 Agonist-Binding Site of the α4β2 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State

    PubMed Central

    Indurthi, Dinesh C.; Lewis, Trevor M.; Ahring, Philip K.; Balle, Thomas; Chebib, Mary; Absalom, Nathan L.

    2016-01-01

    The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors. PMID:27552221

  5. 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

  6. 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.

  7. 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.

  8. Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

    PubMed Central

    Schwimmbeck, P L; Dyrberg, T; Drachman, D B; Oldstone, M B

    1989-01-01

    The large majority of patients with the autoimmune disease myasthenia gravis characteristically have detectable antibodies against the acetylcholine receptor (AChR). We used synthetic peptides to identify antibodies in sera of myasthenia gravis patients reactive with the human acetylcholine receptor (HuAChR) alpha-subunit, residues 160-167. Affinity purification of these antibodies, using the HuAChR alpha-subunit 157-170 peptide immobilized on thiopropyl-Sepharose, yielded IgG antibodies that bound to the native AChR and inhibited the binding of alpha-bungarotoxin to the receptor. The HuAChR alpha-subunit 160-167 peptide demonstrated specific immunological cross-reactivity with a shared homologous domain on herpes simplex virus glycoprotein D, residues 286-293, by both binding and inhibition studies. Thus, HuAChR alpha-subunit, residues 160-167, elicits antibodies in myasthenic patients that binds to the native AChR protein and is capable of eliciting a biologic effect. Immunologic cross-reactivity of this "self" epitope with herpes simplex virus suggest that this virus may be associated with the initiation of some cases of myasthenia. Images PMID:2551924

  9. Identification of a high-affinity binding site for dinotefuran in the nerve cord of the American cockroach.

    PubMed

    Miyagi, Satoshi; Komaki, Iori; Ozoe, Yoshihisa

    2006-04-01

    The binding of the neonicotinoid insecticide dinotefuran to insect nicotinic acetylcholine receptors (nAChRs) was examined by a centrifugation method using the nerve cord membranes of American cockroaches and [3H]dinotefuran (78 Ci mmol-1). The Kd and Bmax values of [3H]dinotefuran binding were estimated to be 13.7 nM and 14.8 fmol 40 microg-1 protein respectively by Scatchard analysis. Epibatidine, an nAChR agonist, showed a rather lower affinity to the dinotefuran binding site (IC50=991 nM) than dinotefuran (IC50=5.02 nM). Imidacloprid and nereistoxin displayed lower potencies than dinotefuran but higher potencies than epibatidine. The potencies of five dinotefuran analogues in inhibiting the specific binding of [3H]dinotefuran to nerve cord membranes were determined. A good correlation (r2=0.970) was observed between the -log IC50 values of the tested compounds and their piperonyl butoxide-synergised insecticidal activities (-log LD50 values) against German cockroaches. The results indicate that a high-affinity binding site for dinotefuran is present in the nerve cord of the American cockroach and that the binding of ligands to the site leads to the manifestation of insecticidal activity.

  10. 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.

  11. 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.

  12. 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

  13. Spatial Relationships between Drug Binding Sites on the Surface of the Acetylcholine Receptor

    DTIC Science & Technology

    1989-02-13

    Proteins, 2, 298-307. 43. Dufton, M.J. and Hider, R.C. (1988’ CRC Crit. Rev. Biochem. 14, 113-171. 44. Garcia -Borron, 1-C., Bieber, A.L. a,.J Mart inez... Carrion , M. (1987) Biochemistry, 26, 4295-4303. 45. Martin, B.M., Chibber, B.A., and Maelicke, A. (1983) J. Biol. Chem., 258, 8714-8722. 46

  14. 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

  15. Non-charged amino acids from three different domains contribute to link agonist binding to channel gating in alpha7 nicotinic acetylcholine receptors.

    PubMed

    Aldea, Marcos; Mulet, José; Sala, Salvador; Sala, Francisco; Criado, Manuel

    2007-10-01

    Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.

  16. 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.

  17. 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.

  18. 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.

  19. Correlation of the vesicular acetylcholine transporter densities in the striata to the clinical abilities of women with Rett syndrome.

    PubMed

    Brašić, James Robert; Bibat, Genila; Kumar, Anil; Zhou, Yun; Hilton, John; Yablonski, Marybeth E; Dogan, Ahmet Semih; Guevara, Maria Rita; Stephane, Massoud; Johnston, Michael; Wong, Dean Foster; Naidu, Sakkubai

    2012-06-01

    Rett syndrome (RTT) is a neurodevelopmental disability characterized by mutations in the X-linked methyl-CpG-binding protein 2 located at the Xq28 region. The severity is modified in part by X chromosomal inactivation resulting in wide clinical variability. We hypothesized that the ability to perform the activities of daily living (ADL) is correlated with the density of vesicular acetylcholine transporters in the striata of women with RTT. The density of the vesicular acetylcholine transporters in the living human brain can be estimated by single-photon emission-computed tomography (SPECT) after the administration of (-)-5-[¹²³I]iodobenzovesamicol ([¹²³I]IBVM). Twenty-four hours following the intravenous injection of ∼333 MBq (9 mCi) [¹²³ I]IBVM, four women with RTT and nine healthy adult volunteer control participants underwent SPECT brain scans for 60 min. The Vesicular Acetylcholine Transporter Binding Site Index (Kuhl et al., 1994), a measurement of the density of vesicular acetylcholine transporters, was estimated in the striatum and the reference structure, the cerebellum. The women with RTT were assessed for certain ADL. Although the striatal Vesicular Acetylcholine Transporter Binding Site Index was not significantly lower in RTT (5.2 ± 0.9) than in healthy adults (5.7 ± 1.6), RTT striatal Vesicular Acetylcholine Transporter Binding Site Indices and ADL scores were linearly associated (ADL = 0.89*(Vesicular Acetylcholine Transporter Binding Site Index) + 4.5; R² = 0.93; P < 0.01), suggesting a correlation between the ability to perform ADL and the density of vesicular acetylcholine transporters in the striata of women with RTT. [¹²³I]IBVM is a promising tool to characterize the pathophysiological mechanisms of RTT and other neurodevelopmental disabilities.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors.

    PubMed Central

    Peralta, E G; Ashkenazi, A; Winslow, J W; Smith, D H; Ramachandran, J; Capon, D J

    1987-01-01

    To investigate the molecular basis for the diversity in muscarinic cholinergic function, we have isolated the genes encoding the human M1 and M2 muscarinic receptors (mAChR) as well as two previously undiscovered mAChR subtypes, designated HM3 and HM4. The amino acid sequence of each subtype reflects a structure consisting of seven, highly conserved transmembrane segments and a large intracellular region unique to each subtype, which may constitute the ligand-binding and effector-coupling domains respectively. Significant differences in affinity for muscarinic ligands were detected in individual mAChR subtypes produced by transfection of mammalian cells. Each subtype exhibited multiple affinity states for agonists; differences among subtypes in the affinities and proportions of such sites suggest the capacity of mAChR subtypes to interact differentially with the cellular effector-coupling apparatus. Subtype-specific mRNA expression was observed in the heart, pancreas and a neuronal cell line, indicating that the regulation of mAChR gene expression contributes to the differentiation of cholinergic activity. Images Fig. 3. PMID:3443095

  5. The E Loop of the Transmitter Binding Site Is a Key Determinant of the Modulatory Effects of Physostigmine on Neuronal Nicotinic α4β2 Receptors.

    PubMed

    Jin, Xiaochun; McCollum, Megan M; Germann, Allison L; Akk, Gustav; Steinbach, Joe Henry

    2017-02-01

    Physostigmine is a well known inhibitor of acetylcholinesterase, which can also activate, potentiate, and inhibit acetylcholine receptors, including neuronal nicotinic receptors comprising α4 and β2 subunits. We have found that the two stoichiometric forms of this receptor differ in the effects of physostigmine. The form containing three copies of α4 and two of β2 was potentiated at low concentrations of acetylcholine chloride (ACh) and physostigmine, whereas the form containing two copies of α4 and three of β2 was inhibited. Chimeric constructs of subunits indicated that the presence of inhibition or potentiation depended on the source of the extracellular ligand binding domain of the subunit. Further sets of chimeric constructs demonstrated that a portion of the ACh binding domain, the E loop, is a key determinant. Transferring the E loop from the β2 subunit to the α4 subunit resulted in strong inhibition, whereas the reciprocal transfer reduced inhibition. To control the number and position of the incorporated chimeric subunits, we expressed chimeric constructs with subunit dimers. Surprisingly, incorporation of a subunit with an altered E loop had similar effects whether it contributed either to an intersubunit interface containing a canonical ACh binding site or to an alternative interface. The observation that the α4 E loop is involved suggests that physostigmine interacts with regions of subunits that contribute to the ACh binding site, whereas the lack of interface specificity indicates that interaction with a particular ACh binding site is not the critical factor.

  6. 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/.

  7. 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

  8. Use of Monoclonal Antibodies to Study the Structure and Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

    DTIC Science & Technology

    1988-03-16

    observed when the sections were coincubated in 400 nrL cold mAb 270. Adjacent Nissl -stained sections were used to identify labeled structures...affinity reagent for the acetylcholine receptor binding site. J Biol Chem 259:11662-11665. 7. Whiting PJ, JM Lindstrom. 1986 . Purification and...characterization of a nicotinic acetylcholine receptor from chick brain. Biochemistry 2502082-2093. 8. Whiting PJ, JM Lindstrom. 1986 . Pharmacological

  9. 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.

  10. 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.

  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. 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.

  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. 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.

  16. 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.

  17. Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail.

    PubMed

    Hayashi, M K; Haga, T

    1997-04-15

    A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H] palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein-coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein beta gamma subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate Gi2 and G(o). However, the rate of [35S] GTP gamma S binding to Gi2 was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42-42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

  18. 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

  19. 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.

  20. 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.

  1. A Multi-Route Model of Nicotine-Cotinine Pharmacokinetics, Pharmacodynamics and Brain Nicotinic Acetylcholine Receptor Binding in Humans

    SciTech Connect

    Teeguarden, Justin G.; Housand, Conrad; Smith, Jordan N.; Hinderliter, Paul M.; Gunawan, Rudy; Timchalk, Charles

    2013-02-01

    The pharmacokinetics of nicotine, the pharmacologically active alkaloid in tobacco responsible for addiction, are well characterized in humans. We developed a physiologically based pharmacokinetic/pharmacodynamic model of nicotine pharmacokinetics, brain dosimetry and brain nicotinic acetylcholine receptor (nAChRs) occupancy. A Bayesian framework was applied to optimize model parameters against multiple human data sets. The resulting model was consistent with both calibration and test data sets, but in general underestimated variability. A pharmacodynamic model relating nicotine levels to increases in heart rate as a proxy for the pharmacological effects of nicotine accurately described the nicotine related changes in heart rate and the development and decay of tolerance to nicotine. The PBPK model was utilized to quantitatively capture the combined impact of variation in physiological and metabolic parameters, nicotine availability and smoking compensation on the change in number of cigarettes smoked and toxicant exposure in a population of 10,000 people presented with a reduced toxicant (50%), reduced nicotine (50%) cigarette Across the population, toxicant exposure is reduced in some but not all smokers. Reductions are not in proportion to reductions in toxicant yields, largely due to partial compensation in response to reduced nicotine yields. This framework can be used as a key element of a dosimetry-driven risk assessment strategy for cigarette smoke constituents.

  2. A model of the human M2 muscarinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Jöhren, Kirstin; Höltje, Hans-Dieter

    2002-11-01

    The M2 muscarinic acetylcholine receptor belongs to the family of rhodopsin like G-Protein Coupled Receptors. This subtype of muscarinic receptors is of special interest because it bears, aside from an orthosteric binding site, also an allosteric binding site. Based on the X-ray structure of bovine rhodopsin a complete homology model of the human M2 receptor was developed. For the orthosteric binding site point mutations and binding studies with different agonists and antagonists are available. This knowledge was utilized for an initial verification of the M2 model. Allosteric modulation of activity is mediated by structurally different ligands such as gallamine, caracurine V salts or W84 (a hexamethonium-derivative). Caracurine V derivatives with different affinities to M2 were docked using GRID-fields. Subsequent molecular dynamics simulations yielded different binding energies based on diverse electrostatic and lipophilic interactions. The calculated affinities are in good agreement to experimentally determined affinities.

  3. Acetylcholine receptors in the human retina

    SciTech Connect

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Synthetic peptides in the study of the interaction of rabies virus and the acetylcholine receptor.

    PubMed

    Lentz, T L; Hawrot, E; Donnelly-Roberts, D; Wilson, P T

    1988-01-01

    The neurotropism of some viruses may be explained in part by the attachment of these viruses to host cell receptors that are present on or even largely restricted to neurons. Rabies virus is an RNA virus that, after a period of replication in muscle, gains access to the central nervous system, where it selectively infects certain neuronal populations. The nicotinic acetylcholine receptor occurs in high density at the neuromuscular junction and is present in the central nervous system. Although several different cell surface constituents may act as attachment determinants for rabies, direct binding of radioactively labeled virus to affinity-purified acetylcholine receptor has been demonstrated. Binding of virus to the receptor was saturable and inhibited by up to 50% by alpha-bungarotoxin, a snake venom neurotoxin that binds at or near the acetylcholine binding site on the receptor. The molecular basis for the virus-receptor interaction may lie in an amino acid sequence similarity between the snake venom neurotoxins and a segment of the rabies virus glycoprotein. Two peptides (10 and 13 residues) of the rabies virus glycoprotein and homologous bungarotoxin peptides were synthesized and tested for ability to compete with labeled alpha-bungarotoxin for binding to the acetylcholine receptor. The peptides were found to compete with toxin binding with affinities comparable to those of the cholinergic ligands d-tubocurarine and nicotine. These findings indicate that a segment of the rabies virus glycoprotein interacts with the acetylcholine receptor at or near the acetylcholine binding site of the receptor. The similarity between the virus glycoprotein and the neurotoxin was further evidenced by the cross reaction of antibody raised against the virus 10-mer with the bungarotoxin 10-mer. Binding of rabies virus to the acetylcholine receptor or to other neuronal bungarotoxin-binding proteins may be related to the neurotropism of this virus. In addition, knowledge of both

  9. 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

  10. 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

  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. 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.

  13. Agonists binding nicotinic receptors elicit specific channel-opening patterns at αγ and αδ sites

    PubMed Central

    Stock, Patrick; Ljaschenko, Dmitrij; Heckmann, Manfred; Dudel, Josef

    2014-01-01

    ‘Embryonic’ muscle-type nicotinic acetylcholine receptor channels (nAChRs) bind ligands at interfaces of α- and γ- or δ-subunits. αγ and αδ sites differ in affinity, but their contributions to opening the channel have remained elusive. We compared high-resolution patch clamp currents evoked by epibatidine (Ebd), carbamylcholine (CCh) and acetylcholine (ACh). Ebd binds with 75-fold higher affinity at αγ than at αδ sites, whereas CCh and ACh prefer αδ sites. Similar short (τO1), intermediate (τO2) and long (τO3) types of opening were observed with all three agonists. τO2 openings were maximally prevalent at low Ebd concentrations, binding at αγ sites. By contrast, τO1 openings appear to be generated at αδ sites. In addition, two types of burst appeared: short bursts of an average of 0.75 ms (τB1) that should arise from the αγ site, and long bursts of 12–25 ms (τB2) in duration arising from double liganded receptors. Limited by the temporal resolution, the closings within bursts were invariant at 3 μs. Corrected for missed closings, in the case of ACh the openings within long bursts lasted 170 μs and those in short bursts about 30 μs. Blocking αδ sites with α-conotoxin M1 (CTx) eliminated both τO1 and τB2 and left only τO2 and the short τB1 bursts, as expected. Furthermore we found desensitization when the receptors bound ACh only at the αγ site. When CTx was applied to ‘embryonic’ mouse endplates, monoquantal current rise times were increased, and amplitude and decay time constants were reduced, as expected. Thus the αγ and αδ sites of nAChRs elicit specific channel-opening patterns. PMID:24665094

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Online magnetic bead based dynamic protein affinity selection coupled to LC-MS for the screening of acetylcholine binding protein ligands.

    PubMed

    Pochet, Lionel; Heus, Ferry; Jonker, Niels; Lingeman, Henk; Smit, August B; Niessen, Wilfried M A; Kool, Jeroen

    2011-06-15

    A magnetic beads based affinity-selection methodology towards the screening of acetylcholine binding protein (AChBP) binders in mixtures and pure compound libraries was developed. The methodology works as follows: after in solution incubation of His-tagged AChBP with potential ligands, and subsequent addition of cobalt (II)-coated paramagnetic beads, the formed bead-AChBP-ligand complexes are fetched out of solution by injection and trapping in LC tubing with an external adjustable magnet. Non binders are then washed to the waste followed by elution of ligands to a SPE cartridge by flushing with denaturing solution. Finally, SPE-LC-MS analysis is performed to identify the ligands. The advantage of the current methodology is the in solution incubation followed by immobilized AChBP ligand trapping and the capability of using the magnetic beads system as mobile/online transportable affinity SPE material. The system was optimized and then successfully demonstrated for the identification of AChBP ligands injected as pure compounds and for the fishing of ligands in mixtures. The results obtained with AChBP as target protein demonstrated reliable discrimination between binders with pK(i) values ranging from at least 6.26 to 8.46 and non-binders.

  19. 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.

  20. 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

  1. Acetylcholine Receptor Binding Characteristics of Snake and Cone Snail Venom Postsynaptic Neurotoxins: Further Studies with a Non-Radioactive Assay

    DTIC Science & Technology

    1993-01-01

    binding) results (BURDEN et at.. 1975: PETERSON, 1989: BARCHAN et al.. 1992). Obviously, the most reliable correlation between an in vivo and in vitro...SCHIMIDT "as particularly appreciated REFERENCES BARCHAN . D.. KACHALSKY. S.. NEUMANNv. D_. VOGFL.ý Z.. OVADIA. NI.. KOCHIXA. E. and [--.(itis. S. 1 192...Peptides isolated from the venom of’ Coitus xi’ographus block neuromuscular transmission. Neurosca, Lett. 24, 57-62. NEUMANN. D., BARCHAN . D.. FRIDKIN

  2. 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

  3. 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.

  4. 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

  5. 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.

  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. 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.

  8. Binding of HIV-1 gp120 to the nicotinic receptor.

    PubMed

    Bracci, L; Lozzi, L; Rustici, M; Neri, P

    1992-10-19

    We previously described a significant sequence homology between HIV-1 gp120 and the functional sites responsible for the specific binding of snake curare-mimetic neurotoxins and rabies virus glycoprotein to the nicotinic acetylcholine receptor. Here we report findings about the existence of a mechanism of functional molecular mimicry which could enable the binding of HIV-1 gp120 to nicotinic acetylcholine receptors in muscle cells and neurons.

  9. 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

  10. Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3β4 nicotinic acetylcholine receptor (nAChR).

    PubMed

    Kombo, David C; Hauser, Terry A; Grinevich, Vladimir P; Melvin, Matthew S; Strachan, Jon-Paul; Sidach, Serguei S; Chewning, Joseph; Fedorov, Nikolai; Tallapragada, Kartik; Breining, Scott R; Miller, Craig H

    2013-03-01

    We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3β4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3β4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC(50) values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3β4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3β4 receptor subtype. Docking into the orthosteric binding site of a α3β4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Spatial orientation of the antagonist granisetron in the ligand-binding site of the 5-HT3 receptor.

    PubMed

    Yan, Dong; White, Michael M

    2005-08-01

    The serotonin type 3 receptor (5-HT(3)R) is a member of the cys-loop ligand-gated ion channel (LGIC) superfamily. Like almost all membrane proteins, high-resolution structural data are unavailable for this class of receptors. We have taken advantage of the high degree of homology between LGICs and the acetylcholine binding protein (AChBP) from the freshwater snail Lymnea stagnalis, for which high-resolution structural data are available, to create a structural model for the extracellular (i.e., ligand-binding) domain of the 5-HT(3)R and to perform a series of ligand docking experiments to delineate the architecture of the ligand-binding site. Structural models were created using homology modeling with the AChBP as a template. Docking of the antagonist granisetron was carried out using a Lamarckian genetic algorithm to produce models of ligand-receptor complexes. Two energetically similar conformations of granisetron in the binding site were obtained from the docking simulations. In one model, the indazole ring of granisetron is near Trp90 and the tropane ring is near Arg92; in the other, the orientation is reversed. We used double-mutant cycle analysis to determine which of the two orientations is consistent with experimental data and found that the data are consistent with the model in which the indazole ring of granisetron interacts with Arg92 and the tropane ring interacts with Trp90. The combination of molecular modeling with double-mutant cycle analysis offers a powerful approach for the delineation of the architecture of the ligand-binding site.

  18. 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.

  19. 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.

  20. 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.

  1. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

    PubMed

    Haga, Kazuko; Kruse, Andrew C; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I; Okada, Tetsuji; Kobilka, Brian K; Haga, Tatsuya; Kobayashi, Takuya

    2012-01-25

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  2. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    SciTech Connect

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  3. 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.

  4. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  5. 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.

  6. 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.

  7. 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

  8. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    PubMed

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

  9. 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

  10. Pharmacological characterization of cis-nitromethylene neonicotinoids in relation to imidacloprid binding sites in the brown planthopper, Nilaparvata lugens.

    PubMed

    Xu, X; Bao, H; Shao, X; Zhang, Y; Yao, X; Liu, Z; Li, Z

    2010-02-01

    Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) and extensively used in areas of crop protection and animal health to control a variety of insect pest species. Here we describe that two cis-nitromethylene neonicotinoids (IPPA152002 and IPPA152004), recently synthesized in our laboratory, discriminated between the high and low affinity imidacloprid binding sites in the brown planthopper, Nilaparvata lugens, a major insect pest of rice crops in many parts of Asia. [(3)H]imidacloprid has two binding sites with different affinities (Kd value of 0.0035 +/- 0.0006 nM for the high-affinity site and 1.47 +/- 0.22 nM for the low-affinity site). Although the cis-nitromethylene neonicotinoids showed low displacement ability (Ki values of 0.15 +/- 0.03 microM and 0.42 +/- 0.07 microM for IPPA152002 and IPPA152004, respectively) against [(3)H]imidacloprid binding, low concentrations (0.01 microM) of IPPA152002 completely inhibited [(3)H]imidacloprid binding at its high-affinity site. In Xenopus oocytes co-injected with cRNA encoding Nlalpha1 and rat beta2 subunits, obvious inward currents were detected in response to applications of IPPA152002 and IPPA152004, although the agonist potency is reduced to that of imidacloprid. The previously identified Y151S mutation in Nlalpha1 showed significant effects on the agonist potency of IPPA152002 and IPPA152004, such as a 75.8% and 70.6% reduction in Imax, and a 2.4- and 2.1-fold increase in EC(50). This data clearly shows that the two newly described cis-nitromethylene neonicotinoids act on insect nAChRs and like imidacloprid, discriminated between high and low affinity binding sites in N. lugens native nAChRs. These compounds may be useful tools to further elucidate the pharmacology and nature of neonicotinoid binding sites.

  11. 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.

  12. Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.

    PubMed

    Kurup, C K; Sanadi, D R

    1977-02-01

    3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.

  13. A Unitary Anesthetic Binding Site at High Resolution

    SciTech Connect

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

    2011-12-31

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

  14. A Unitary Anesthetic-Binding Site at High Resolution

    SciTech Connect

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

    2009-01-01

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

  15. A Unitary Anesthetic Binding Site at High Resolution

    SciTech Connect

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

    2009-10-21

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

  16. 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.

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

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

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

  18. Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development.

    PubMed

    Kruse, Andrew C; Hu, Jianxin; Kobilka, Brian K; Wess, Jürgen

    2014-06-01

    Muscarinic acetylcholine receptor antagonists are widely used as bronchodilating drugs in pulmonary medicine. The therapeutic efficacy of these agents depends on the blockade of M3 muscarinic receptors expressed on airway smooth muscle cells. All muscarinic antagonists currently used as bronchodilating agents show high affinity for all five muscarinic receptor subtypes, thus increasing the likelihood of unwanted side effects. Recent X-ray crystallographic studies have provided detailed structural information about the nature of the orthosteric muscarinic binding site (the conventional acetylcholine binding site) and an 'outer' receptor cavity that can bind allosteric (non-orthosteric) drugs. These new findings should guide the development of selective M3 receptor blockers that have little or no effect on other muscarinic receptor subtypes.

  19. Genetic deletion of the adenosine A(2A) receptor prevents nicotine-induced upregulation of α7, but not α4β2* nicotinic acetylcholine receptor binding in the brain.

    PubMed

    Metaxas, Athanasios; Al-Hasani, Ream; Farshim, Pamela; Tubby, Kristina; Berwick, Amy; Ledent, Catherine; Hourani, Susanna; Kitchen, Ian; Bailey, Alexis

    2013-08-01

    Considerable evidence indicates that adenosine A(2A) receptors (A(2A)Rs) modulate cholinergic neurotransmission, nicotinic acetylcholine receptor (nAChR) function, and nicotine-induced behavioural effects. To explore the interaction between A(2A) and nAChRs, we examined if the complete genetic deletion of adenosine A(2A)Rs in mice induces compensatory alterations in the binding of different nAChR subtypes, and whether the long-term effects of nicotine on nAChR regulation are altered in the absence of the A(2A)R gene. Quantitative autoradiography was used to measure cytisine-sensitive [¹²⁵I]epibatidine and [¹²⁵I]α-bungarotoxin binding to α4β2* and α7 nAChRs, respectively, in brain sections of drug-naïve (n = 6) or nicotine treated (n = 5-7), wild-type and adenosine A(2A)R knockout mice. Saline or nicotine (7.8 mg/kg/day; free-base weight) were administered to male CD1 mice via subcutaneous osmotic minipumps for a period of 14 days. Blood plasma levels of nicotine and cotinine were measured at the end of treatment. There were no compensatory developmental alterations in nAChR subtype distribution or density in drug-naïve A(2A)R knockout mice. In nicotine treated wild-type mice, both α4β2* and α7 nAChR binding sites were increased compared with saline treated controls. The genetic ablation of adenosine A(2A)Rs prevented nicotine-induced upregulation of α7 nAChRs, without affecting α4β2* receptor upregulation. This selective effect was observed at plasma levels of nicotine that were within the range reported for smokers (10-50 ng ml⁻¹). Our data highlight the involvement of adenosine A(2A)Rs in the mechanisms of nicotine-induced α7 nAChR upregulation, and identify A(2A)Rs as novel pharmacological targets for modulating the long-term effects of nicotine on α7 receptors.

  20. 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.

  1. Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.

    PubMed

    Kasheverov, Igor E; Utkin, Yuri N; Tsetlin, Victor I

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

  2. 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.

  3. Spatial Relationships between Drug Binding Sites on the Surface of the Acetylcholine Receptor: Preparation and Interaction of Fluorescent Alpha-Toxins and Decidium with the Acetylcholine Receptor

    DTIC Science & Technology

    1985-10-15

    SC R " V. r "A C C S III O G Mo 3. n, c a• ’ ltt , T ’ C A• T A LO G wu umc ’ L . "TugL (.* s.oo**) L Tr.t" or RaiPo a PC-mo coveano SPATIAL ELATONS...Ti L I *u AwTo4•. S. COGITCACT 05OAA •U•T14UU•OI(.3 David A. Johnson DAM4,-17-84-4187 5. Pgmft O tO u 060 *AD AOCCU W PNOGRAN ILi9iq7r...phencyclidine, Bc3H- l cello, decidium, carbachol, fluorescence probes. ~ T~a report des"crfbe’s IM34 ~e~rpffent 5f 7T~steat Probes of the nico- tinic

  4. 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

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

    SciTech Connect

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

    2004-10-28

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

  7. 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.

  8. Conserved properties of individual Ca2+-binding sites in calmodulin

    PubMed Central

    Halling, D. Brent; Liebeskind, Benjamin J.; Hall, Amelia W.; Aldrich, Richard W.

    2016-01-01

    Calmodulin (CaM) is a Ca2+-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca2+ concentration into cellular signals by binding to a wide range of target proteins in a Ca2+-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca2+-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca2+ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease. PMID:26884197

  9. Imidazoline binding sites and receptors in cardiovascular tissue.

    PubMed

    Molderings, G J; Göthert, M

    1999-01-01

    1. Imidazoline binding sites and receptors and their endogenous ligands have been identified in cardiovascular tissue of various species including human beings. 2. I2- (but only exceptionally I1-)imidazoline binding sites have been shown to exist on cardiac myocytes and vascular smooth muscle cells; at present, their functional role is unknown. 3. The sympathetic nerves supplying the cardiovascular system are endowed with presynaptic inhibitory imidazoline receptors that may become of therapeutic relevance as targets of drugs. 4. ATP-sensitive K+ channels present in heart and blood vessels can be blocked by several imidazolines and guanidines; hence, those drugs can interfere with the cardioprotective effects resulting from K(ATP) channel activation by a decrease in the endogenous ligand ATP or by drugs. 5. Imidazoline derivatives exhibit antiarrhythmic properties that are due to a reduction of sympathetic tone by central and peripheral mechanisms and to blockade of postsynaptic alpha2-adrenoceptors in the heart and coronary arteries. 6. Agmatine and clonidine-displacing substance, which are endogenous ligands at imidazoline and alpha2-receptors, are present in the blood serum and appear to participate in vascular smooth muscle proliferation and blood pressure regulation.

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

    SciTech Connect

    Bianchi, C.; Thibault, G.; Wrobel-Konrad, E.; De Lean, A.; Genest, J.; Cantin, M. )

    1989-10-01

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

  11. The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.

    PubMed

    Moore, D D; Marks, A R; Buckley, D I; Kapler, G; Payvar, F; Goodman, H M

    1985-02-01

    Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins.

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

    PubMed Central

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

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein–DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD–DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein–DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw. PMID:22693214

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

    PubMed

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

    2007-08-23

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

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

    SciTech Connect

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

    2007-01-01

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

  15. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

    SciTech Connect

    Harada, Y.; Li, H.; Li, Hua; Lennarz, W. J.

    2009-04-28

    Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.

  16. GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.

    PubMed

    Zhang, Yu; Degen, David; Ho, Mary X; Sineva, Elena; Ebright, Katherine Y; Ebright, Yon W; Mekler, Vladimir; Vahedian-Movahed, Hanif; Feng, Yu; Yin, Ruiheng; Tuske, Steve; Irschik, Herbert; Jansen, Rolf; Maffioli, Sonia; Donadio, Stefano; Arnold, Eddy; Ebright, Richard H

    2014-04-22

    Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001.

  17. 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).

  18. A model of the rabies virus glycoprotein active site.

    PubMed

    Rustici, M; Bracci, L; Lozzi, L; Neri, P; Santucci, A; Soldani, P; Spreafico, A; Niccolai, N

    1993-06-01

    The glycoprotein from the neurotropic rabies virus shows a significant homology with the alpha neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the alpha neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side-chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194-Ser195-Arg196-Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection.

  19. Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA.

    PubMed

    Gourves, A S; Defais, M; Johnson, N P

    2001-03-30

    The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence anisotropy. The RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to measure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10(5) m(-1) (37 degrees C, 150 mm NaCl, pH 7.4). The reaction was enthalpy-driven and entropy-opposed. It depended on salt concentration and was sensitive to the type of monovalent anion, suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.

  20. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

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

  1. 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

  2. Activation of α7 Nicotinic Acetylcholine Receptor Decreases On-site Mortality in Crush Syndrome through Insulin Signaling-Na/K-ATPase Pathway

    PubMed Central

    Fan, Bo-Shi; Zhang, En-Hui; Wu, Miao; Guo, Jin-Min; Su, Ding-Feng; Liu, Xia; Yu, Jian-Guang

    2016-01-01

    On-site mortality in crush syndrome remains high due to lack of effective drugs based on definite diagnosis. Anisodamine (Ani) is widely used in China for treatment of shock, and activation of α7 nicotinic acetylcholine receptor (α7nAChR) mediates such antishock effect. The present work was designed to test whether activation of α7nAChR with Ani decreased mortality in crush syndrome shortly after decompression. Sprague-Dawley rats and C57BL/6 mice with crush syndrome were injected with Ani (20 mg/kg and 28 mg/kg respectively, i.p.) 30 min before decompression. Survival time, serum potassium, insulin, and glucose levels were observed shortly after decompression. Involvement of α7nAChR was verified with methyllycaconitine (selective α7nAChR antagonist) and PNU282987 (selective α7nAChR agonist), or in α7nAChR knockout mice. Effect of Ani was also appraised in C2C12 myotubes. Ani reduced mortality and serum potassium and enhanced insulin sensitivity shortly after decompression in animals with crush syndrome, and PNU282987 exerted similar effects. Such effects were counteracted by methyllycaconitine or in α7nAChR knockout mice. Mortality and serum potassium in rats with hyperkalemia were also reduced by Ani. Phosphorylation of Na/K-ATPase was enhanced by Ani in C2C12 myotubes. Inhibition of tyrosine kinase on insulin receptor, phosphoinositide 3-kinase, mammalian target of rapamycin, signal transducer and activator of transcription 3, and Na/K-ATPase counteracted the effect of Ani on extracellular potassium. These findings demonstrated that activation of α7nAChR could decrease on-site mortality in crush syndrome, at least in part based on the decline of serum potassium through insulin signaling-Na/K-ATPase pathway. PMID:27065867

  3. Insights into the structural determinants required for high-affinity binding of chiral cyclopropane-containing ligands to α4β2-nicotinic acetylcholine receptors: an integrated approach to behaviorally active nicotinic ligands.

    PubMed

    Zhang, Han-Kun; Eaton, J Brek; Yu, Li-Fang; Nys, Mieke; Mazzolari, Angelica; van Elk, René; Smit, August B; Alexandrov, Vadim; Hanania, Taleen; Sabath, Emily; Fedolak, Allison; Brunner, Daniela; Lukas, Ronald J; Vistoli, Giulio; Ulens, Chris; Kozikowski, Alan P

    2012-09-27

    Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a cocrystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine a previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. To validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogues of compound 5. The most promising compound, 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral end points in the rodent studies.

  4. Insights into the Structural Determinants Required for High Affinity Binding of Chiral Cyclopropane-Containing Ligands to α4β2-Nicotinic Acetylcholine Receptors; An Integrated Approach to Behaviorally Active Nicotinic Ligands

    PubMed Central

    Zhang, Han-Kun; Eaton, J. Brek; Yu, Li-Fang; Nys, Mieke; Mazzolari, Angelica; van Elk, René; Smit, August B.; Alexandrov, Vadim; Hanania, Taleen; Sabath, Emily; Fedolak, Allison; Brunner, Daniela; Lukas, Ronald J.; Vistoli, Giulio; Ulens, Chris; Kozikowski, Alan P.

    2012-01-01

    Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a co-crystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing, selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine our previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. In order to validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogs of compound 5. The most promising compound 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral endpoints in the rodent studies. PMID:22928944

  5. Development of a photoactivatable allosteric ligand for the m1 muscarinic acetylcholine receptor.

    PubMed

    Davie, Briana J; Sexton, Patrick M; Capuano, Ben; Christopoulos, Arthur; Scammells, Peter J

    2014-10-15

    The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (4), designed to bind irreversibly to an allosteric site of the M1 muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor.

  6. Sodium-dependent high-affinity binding of (/sup 3/H)hemicholinium-3 in the rat brain: a potentially selective marker for presynaptic cholinergic sites

    SciTech Connect

    Vickroy, T.W.; Roeske, W.R.; Yamamura, H.I.

    1984-12-03

    An attempt has been made to describes the membrane binding properties of (/sup 3/H)hemicholinium-3 ((/sup 3/H)HC-3), a selective inhibitor of sodium-dependent high-affinity choline uptake (SDHACU) in cholinergic nerve terminals. Under the described assay conditions. (/sup 3/H)HC-3 binds with a saturable population of high-affinity (apparent K/sub d/ = 1.9 nM CNS membrane sites having the regional distribution: striatum >> hippocampus > cerebral cortex > cerebellum. High-affinity (/sup 3/H)HC-3 binding is entirely dependent upon the presence of sodium chloride (EC/sub 50/ = 35-50 mM) and is markedly reduced when other salts of sodium or monovalent ions are substituted. (/sup 3/H)HC-3 binding is inhibited by choline (K/sub i/ = 6..mu..M) and acetylcholine (K/sub i/ = 35..mu..M) but markedly less sensitive to other cholinergic agents and metabolic inhibitors. In light of the similar ionic dependencies, regional distributions and pharmacological specificities of (/sup 3/H)HC-3 binding and SDHACU, closely associated sites may be involved in both processes. 2 references, 3 figures, 3 tables.

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

    NASA Astrophysics Data System (ADS)

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

    2006-07-01

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

  8. Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

    PubMed Central

    Gready, J. E.; Ranganathan, S.; Schofield, P. R.; Matsuo, Y.; Nishikawa, K.

    1997-01-01

    Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested. PMID:9144769

  9. In vivo effects of 3-iodocytisine: pharmacological and genetic analysis of hypothermia and evaluation of chronic treatment on nicotinic binding sites.

    PubMed

    Zambrano, C A; Marks, M J; Cassels, B K; Maccioni, R B

    2009-09-01

    Several cytisine derivatives have been developed in the search for more selective drugs at nicotinic acetylcholine receptors (nAChR). Binding experiments in transfected cell lines showed that the iodination of cytisine in the position 3 of the pyridone ring increased potency at alpha7-nAChR and to a lesser extent at the alpha4beta2 subtypes, both of which are widely expressed in the brain. However, no in vivo studies have been published on this compound. Inhibition curves presented here using wild type, beta2, and beta4-null mutant mice confirm that 3-IC binds to alpha4beta2 *, alpha7 * and alpha3beta4 * receptors with higher affinity than cytisine (asterisk indicates the receptor may contain additional subunits, Lukas et al., 1999). Intraperitoneal injection of 3-iodocytisine (3-IC) induced considerable dose-dependent hypothermia in DBA/2J and C57BL/6J mice. This response was blocked by mecamylamine and partially inhibited by hexamethonium. beta4-null mice displayed significantly less 3-IC-induced hypothermia than wild-type mice, beta2-null mice were somewhat less affected than wild types, while responses of alpha7 *-null mice were similar to wild types. Mice treated chronically with 3-IC display a marked increase in alpha7 * and alpha4beta2 * binding sites determined by radioligand binding in membrane preparations from cerebral cortex and hippocampus. Quantitative autoradiographic analysis of 28 brain regions of mice treated with 3-IC was consistent with the membrane binding, detecting an increase of cytisine-sensitive [(125)I]epibatidine binding sites, while cytisine-resistant [(125)I]epibatidine sites were unchanged. [(125)I]alpha-Bungarotoxin binding sites also exhibited up-regulation. These results give a first evaluation of in vivo consequences of 3-IC as a potent agonist with marked effects on mice.

  10. Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Zhao, Guangyu; Chen, Shoucong; Liu, Feng; Sun, Ying; Zhang, Li

    2009-07-01

    The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug-HSA complex, and the effective quenching constants ( Ka) were 2.852 × 10 4, 2.765 × 10 4, 2.638 × 10 4 and 2.542 × 10 4 M -1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be -3.634 kJ mol -1 and 72.66 J mol -1 K -1 according to van't Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Förster's theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug-HSA complex was also discussed.

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

    NASA Astrophysics Data System (ADS)

    Clifford, Jacob; Adami, Christoph

    2015-10-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

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

    PubMed

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

    2014-01-01

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

  13. Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites.

    PubMed

    Kaufmann, S H; Shaper, J H

    1984-03-01

    The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.

  14. Characterization of ruthenium red-binding sites of the Ca(2+)-ATPase from sarcoplasmic reticulum and their interaction with Ca(2+)-binding sites.

    PubMed Central

    Corbalan-Garcia, S; Teruel, J A; Gomez-Fernandez, J C

    1992-01-01

    Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5'-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites. PMID:1280106

  15. Fluorescence energy transfer between points in G-actin: the nucleotide-binding site, the metal-binding site and Cys-373 residue.

    PubMed

    Miki, M; Wahl, P

    1985-04-05

    Fluorescence energy transfers were studied in order to investigate the spatial relationships between the nucleotide-binding site, the metal-binding site and the Cys-373 residue in the G-actin molecule. When 1-N6-ethenoadenosine-5'-triphosphate (epsilon-ATP) in the nucleotide-binding site and Co2+ or Ni2+ in the metal-binding site were used as fluorescence donor and acceptor, respectively, the fluorescence intensity of epsilon-ATP was perfectly quenched by Ni2+ or Co2+. This indicated that the nucleotide-binding site is very close to the metal-binding site; the distance should be less than 10 A. When N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to Cys-373 residue and Co2+ in the metal-binding site were used as a fluorescence donor and an acceptor, respectively, the transfer efficiency was equal to 5 +/- 1%. The corresponding distance was calculated to be 23-32 A, assuming a random orientation factor K2 = 2/3.

  16. Using an α-bungarotoxin binding site tag to study GABA A receptor membrane localization and trafficking.

    PubMed

    Brady, Megan L; Moon, Charles E; Jacob, Tija C

    2014-03-28

    It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

  17. Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites.

    PubMed

    Schober, Douglas A; Croy, Carrie H; Xiao, Hongling; Christopoulos, Arthur; Felder, Christian C

    2014-07-01

    In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dim​ethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-β-hydroxy-17-α-ethynyl-δ-4-androstano[3,2-b]pyrimido[1,2-a]benzimidazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic

  18. 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

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

    PubMed

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

    2001-12-01

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

  20. 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

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

    PubMed Central

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

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  2. Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos.

    PubMed Central

    Zeng, C; Pinsonneault, J; Gellon, G; McGinnis, N; McGinnis, W

    1994-01-01

    How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifically regulated by the Deformed homeotic protein. This 120 bp element (module E) is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays show that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affinity for Dfd protein generate parallel changes in the regulatory activity of module E in transgenic embryos, strong evidence that the in vitro-defined binding site is a direct target of Dfd protein in embryos. However, a monomer or multimer of the Dfd binding region alone is not sufficient to supply Dfd-dependent, segment-specific reporter gene expression. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element. The Dfd binding site and the putative cofactor binding site(s) in the region of the inverted repeat are both necessary and in combination sufficient for the function of module E. Images PMID:7910795

  3. 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

  4. Gibbs Recursive Sampler: finding transcription factor binding sites.

    PubMed

    Thompson, William; Rouchka, Eric C; Lawrence, Charles E

    2003-07-01

    The Gibbs Motif Sampler is a software package for locating common elements in collections of biopolymer sequences. In this paper we describe a new variation of the Gibbs Motif Sampler, the Gibbs Recursive Sampler, which has been developed specifically for locating multiple transcription factor binding sites for multiple transcription factors simultaneously in unaligned DNA sequences that may be heterogeneous in DNA composition. Here we describe the basic operation of the web-based version of this sampler. The sampler may be acces-sed at http://bayesweb.wadsworth.org/gibbs/gibbs.html and at http://www.bioinfo.rpi.edu/applications/bayesian/gibbs/gibbs.html. An online user guide is available at http://bayesweb.wadsworth.org/gibbs/bernoulli.html and at http://www.bioinfo.rpi.edu/applications/bayesian/gibbs/manual/bernoulli.html. Solaris, Solaris.x86 and Linux versions of the sampler are available as stand-alone programs for academic and not-for-profit users. Commercial licenses are also available. The Gibbs Recursive Sampler is distributed in accordance with the ISCB level 0 guidelines and a requirement for citation of use in scientific publications.

  5. Mutations and Binding Sites of Human Transcription Factors

    PubMed Central

    Kamanu, Frederick Kinyua; Medvedeva, Yulia A.; Schaefer, Ulf; Jankovic, Boris R.; Archer, John A. C.; Bajic, Vladimir B.

    2012-01-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, “insertions” are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. PMID:22670148

  6. Energetic localization of saxitoxin in its channel binding site.

    PubMed Central

    Choudhary, Gaurav; Shang, Lisa; Li, Xiufeng; Dudley, Samuel C

    2002-01-01

    Saxitoxin (STX) selectively blocks the voltage-gated sodium channel at the outer vestibule lined by P-loops of the four domains. Neosaxitoxin has an additional -OH group at the N1 position of the 1,2,3 guanidinium (N1-OH) that interacts with domains I and IV of the Na(+) channel. Determination of a second toxin interaction with the channel would fix the location of STX. Gonyautoxin 2,3 and Gonyautoxin 1,4 are C-11 sulfated derivatives of saxitoxin and neosaxitoxin, respectively. We used these variants to constrain the STX docking orientation by energetically localizing the C-11 sulfate in the outer vestibule. Interactions between the C-11 sulfate and each of the four domains of the channel were determined by a systematic approach to mutant cycle analysis in which all known carboxyl groups important for site 1 toxin binding were neutralized, allowing energetic triangulation of the toxin sulfate and overcoming some limitations of mutant cycles. Toxin IC(50)s were measured by two-electrode voltage clamp from Xenopus oocytes injected with the channel mRNA. Three unique types of analysis based on the coupling results localized the C-11 sulfate between domains III and IV. Combined with our previous report, the data establish the orientation of STX in the outer vestibule and confirm the clockwise arrangement of the channel domains. PMID:12124273

  7. Computational Characterization and Prediction of Estrogen Receptor Coactivator Binding Site Inhibitors

    DTIC Science & Technology

    2005-09-01

    Gutendorf, andJ. Westendorf. 2000. Endocrine disruptors in fried meat: PhIP is an estrogen. Proceedings of the American Association for Cancer...binding site of the ERa LBD [3-5]. Because these studies have focused on the estradiol binding site, new potential ER disruptors that bind in the co...activator site have been missed. Our proposal focuses on developing a new computational approach to predict therapeutically useful ERa disruptors by

  8. 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.

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

    PubMed Central

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

    2013-01-01

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

  10. Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin.

    PubMed

    Steiner, J P; Bennett, V

    1988-10-05

    Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.

  11. Acetylcholinesterease and Acetylcholine Receptor

    DTIC Science & Technology

    1987-08-28

    trimethyl site, contiguous with the aryl site. The hydroxyl group decreases binding in phenol and the nitrophenols , in accord with its negative c value...prevents reaction of substrates, by preventing utilization of the enzymic mechanism. However, pretreatment with DFP, phosphorylating the serine- hydroxyl...absence of treatment with DFP (Figures 10 and 11). Phosphorylation of the serine-hydroxyl had no effect on action of [14C]-BrPin at the trimethyl site

  12. Nicotine-morphine interactions at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors.

    PubMed

    Talka, Reeta; Salminen, Outi; Whiteaker, Paul; Lukas, Ronald J; Tuominen, Raimo K

    2013-02-15

    Nicotine and opioids share several behavioral and rewarding properties. Although both opioids and nicotine have their own specific mechanism of action, there is empirical and experimental evidence of interactions between these drugs. We studied receptor-level interactions of nicotine and morphine at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors. [(3)H]epibatidine displacement was used to determine if morphine binds competitively to nicotinic acetylcholine receptors. Functional interactions of morphine and nicotine were studied with calcium fluorometry and (86)Rb(+) efflux assays. Morphine displaced [(3)H]epibatidine from nicotinic agonist binding sites in all cell lines studied. The Ki values for morphine were 13.2μM in SH-EP1-hα4β2 cells, 0.16μM and 126μM in SH-SY5Y cells and 43.7μM in SH-EP1-hα7 cells. In SH-EP1-hα4β2 cells expressing α4β2 nicotinic acetylcholine receptors, morphine acted as a partial agonist of (86)Rb(+) efflux comparable to cytisine (with EC50 values of 53.3μM for morphine and 5.38μM for cytisine). The effect of morphine was attenuated concentration-dependently by the nicotinic antagonist mecamylamine. In the SH-SY5Y cell line expressing several subtypes of nicotinic acetylcholine receptors morphine had an inhibitory effect on nicotine induced (86)Rb(+) ion efflux mediated by α3(⁎) nicotinic acetylcholine receptors. These results suggest that morphine acts as a partial agonist at α4β2 nicotinic acetylcholine receptors and as a weak antagonist at α3(⁎) nicotinic acetylcholine receptors.

  13. Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

    PubMed

    Bauer, Amy L; Hlavacek, William S; Unkefer, Pat J; Mu, Fangping

    2010-11-18

    An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF). Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

  14. Structural identification of DnaK binding sites within bovine and sheep bactenecin Bac7.

    PubMed

    Zahn, Michael; Kieslich, Bjorn; Berthold, Nicole; Knappe, Daniel; Hoffmann, Ralf; Strater, Norbert

    2014-04-01

    Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified.

  15. Duplicate gene divergence by changes in microRNA binding sites in Arabidopsis and Brassica.

    PubMed

    Wang, Sishuo; Adams, Keith L

    2015-02-02

    Gene duplication provides large numbers of new genes that can lead to the evolution of new functions. Duplicated genes can diverge by changes in sequences, expression patterns, and functions. MicroRNAs play an important role in the regulation of gene expression in many eukaryotes. After duplication, two paralogs may diverge in their microRNA binding sites, which might impact their expression and function. Little is known about conservation and divergence of microRNA binding sites in duplicated genes in plants. We analyzed microRNA binding sites in duplicated genes in Arabidopsis thaliana and Brassica rapa. We found that duplicates are more often targeted by microRNAs than singletons. The vast majority of duplicated genes in A. thaliana with microRNA binding sites show divergence in those sites between paralogs. Analysis of microRNA binding sites in genes derived from the ancient whole-genome triplication in B. rapa also revealed extensive divergence. Paralog pairs with divergent microRNA binding sites show more divergence in expression patterns compared with paralog pairs with the same microRNA binding sites in Arabidopsis. Close to half of the cases of binding site divergence are caused by microRNAs that are specific to the Arabidopsis genus, indicating evolutionarily recent gain of binding sites after target gene duplication. We also show rapid evolution of microRNA binding sites in a jacalin gene family. Our analyses reveal a dynamic process of changes in microRNA binding sites after gene duplication in Arabidopsis and highlight the role of microRNA regulation in the divergence and contrasting evolutionary fates of duplicated genes.

  16. Duplicate Gene Divergence by Changes in MicroRNA Binding Sites in Arabidopsis and Brassica

    PubMed Central

    Wang, Sishuo; Adams, Keith L.

    2015-01-01

    Gene duplication provides large numbers of new genes that can lead to the evolution of new functions. Duplicated genes can diverge by changes in sequences, expression patterns, and functions. MicroRNAs play an important role in the regulation of gene expression in many eukaryotes. After duplication, two paralogs may diverge in their microRNA binding sites, which might impact their expression and function. Little is known about conservation and divergence of microRNA binding sites in duplicated genes in plants. We analyzed microRNA binding sites in duplicated genes in Arabidopsis thaliana and Brassica rapa. We found that duplicates are more often targeted by microRNAs than singletons. The vast majority of duplicated genes in A. thaliana with microRNA binding sites show divergence in those sites between paralogs. Analysis of microRNA binding sites in genes derived from the ancient whole-genome triplication in B. rapa also revealed extensive divergence. Paralog pairs with divergent microRNA binding sites show more divergence in expression patterns compared with paralog pairs with the same microRNA binding sites in Arabidopsis. Close to half of the cases of binding site divergence are caused by microRNAs that are specific to the Arabidopsis genus, indicating evolutionarily recent gain of binding sites after target gene duplication. We also show rapid evolution of microRNA binding sites in a jacalin gene family. Our analyses reveal a dynamic process of changes in microRNA binding sites after gene duplication in Arabidopsis and highlight the role of microRNA regulation in the divergence and contrasting evolutionary fates of duplicated genes. PMID:25644246

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

    PubMed

    Weill, Nathanaël; Rognan, Didier

    2010-01-01

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

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

    PubMed

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

    2015-01-02

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

  19. New ligands with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors. Synthesis, receptor binding, and 3D-QSAR modeling.

    PubMed

    Audouze, Karine; Nielsen, Elsebet Østergaard; Olsen, Gunnar M; Ahring, Philip; Jørgensen, Tino Dyhring; Peters, Dan; Liljefors, Tommy; Balle, Thomas

    2006-06-01

    A new series of piperazines, diazepanes, diazocanes, diazabicyclononanes, and diazabicyclodecanes with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors were synthesized on the basis of results from a previous computational study. A predictive 3D-QSAR model was developed using the GRID/GOLPE approach (R2 = 0.94, Q2 = 0.83, SDEP = 0.34). The SAR was interpreted in terms of contour maps of the PLS coefficients and in terms of a homology model of the alpha4beta2 subtype of the nicotinic acetylcholine receptors. The results reveal that hydrogen bonding from both hydrogens on the protonated amine and from the pyridine nitrogen to a water molecule as well as van der Waals interactions between the substituent bearing the protonated amine and the receptor is of importance for ligand affinity. The combination of 3D-QSAR and homology modeling proved successful for the interpretation of structure-affinity relationships as well as the validation of the individual modeling approaches.

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

    SciTech Connect

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

    2006-04-19

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

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

    SciTech Connect

    James, I.F.; Goldstein, A.

    1984-05-01

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

  2. Regulation of the distribution and function of [(125)I]epibatidine binding sites by chronic nicotine in mouse embryonic neuronal cultures.

    PubMed

    Zambrano, Cristian A; Salamander, Rakel M; Collins, Allan C; Grady, Sharon R; Marks, Michael J

    2012-08-01

    Chronic nicotine produces up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) (* denotes that an additional subunit may be part of the receptor). However, the extent of up-regulation to persistent ligand exposure varies across brain regions. The aim of this work was to study the cellular distribution and function of nAChRs after chronic nicotine treatment in primary cultures of mouse brain neurons. Initially, high-affinity [(125)I]epibatidine binding to cell membrane homogenates from primary neuronal cultures obtained from diencephalon and hippocampus of C57BL/6J mouse embryos (embryonic days 16-18) was measured. An increase in α4β2*-nAChR binding sites was observed in hippocampus, but not in diencephalon, after 24 h of treatment with 1 μM nicotine. However, a nicotine dose-dependent up-regulation of approximately 3.5- and 0.4-fold in hippocampus and diencephalon, respectively, was found after 96 h of nicotine treatment. A significant fraction of total [(125)I]epibatidine binding sites in both hippocampus (45%) and diencephalon (65%) was located on the cell surface. Chronic nicotine (96 h) up-regulated both intracellular and surface binding in both brain regions without changing the proportion of those binding sites compared with control neurons. The increase in surface binding was not accompanied by an increase in nicotine-stimulated Ca(2+) influx, suggesting persistent desensitization or inactivation of receptors at the plasma membrane occurred. Given the differences observed between hippocampus and diencephalon neurons exposed to nicotine, multiple mechanisms may play a role in the regulation of nAChR expression and function.

  3. TEMPLE: analysing population genetic variation at transcription factor binding sites.

    PubMed

    Litovchenko, Maria; Laurent, Stefan

    2016-11-01

    Genetic variation occurring at the level of regulatory sequences can affect phenotypes and fitness in natural populations. This variation can be analysed in a population genetic framework to study how genetic drift and selection affect the evolution of these functional elements. However, doing this requires a good understanding of the location and nature of regulatory regions and has long been a major hurdle. The current proliferation of genomewide profiling experiments of transcription factor occupancies greatly improves our ability to identify genomic regions involved in specific DNA-protein interactions. Although software exists for predicting transcription factor binding sites (TFBS), and the effects of genetic variants on TFBS specificity, there are no tools currently available for inferring this information jointly with the genetic variation at TFBS in natural populations. We developed the software Transcription Elements Mapping at the Population LEvel (TEMPLE), which predicts TFBS, evaluates the effects of genetic variants on TFBS specificity and summarizes the genetic variation occurring at TFBS in intraspecific sequence alignments. We demonstrate that TEMPLE's TFBS prediction algorithms gives identical results to PATSER, a software distribution commonly used in the field. We also illustrate the unique features of TEMPLE by analysing TFBS diversity for the TF Senseless (SENS) in one ancestral and one cosmopolitan population of the fruit fly Drosophila melanogaster. TEMPLE can be used to localize TFBS that are characterized by strong genetic differentiation across natural populations. This will be particularly useful for studies aiming to identify adaptive mutations. TEMPLE is a java-based cross-platform software that easily maps the genetic diversity at predicted TFBSs using a graphical interface, or from the Unix command line.

  4. 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

  5. Late evolutionary appearance of 'peripheral-type' binding sites for benzodiazepines.

    PubMed

    Bolger, G T; Weissman, B A; Lueddens, H; Basile, A S; Mantione, C R; Barrett, J E; Witkin, J M; Paul, S M; Skolnick, P

    1985-07-15

    Four classes of non-mammalian vertebrates were examined for the presence of both 'brain-specific' and 'peripheral-type' binding sites for benzodiazepines in the central nervous system. 'Brain-specific' binding sites for benzodiazepines were found in the central nervous systems of all non-mammalian vertebrates studied. However, in contrast to mammals, either very low or undetectable levels of 'peripheral-type' binding sites for benzodiazepines were observed in the central nervous systems of these non-mammalian vertebrates. Furthermore, the density of 'peripheral-type' binding sites for benzodiazepines in non-mammalian vertebrate heart was less than or equal to 2% of that found in mammalian cardiac tissue. These findings suggest a very late evolutionary appearance of 'peripheral-type' binding sites for benzodiazepines, implying that these sites may have (a) highly specialized function(s) in both peripheral tissues and the central nervous system.

  6. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

    The muscarinic acetylcholine receptor, identified by tritiated L-quinuclidinyl benzilate (L-(/sup 3/H)QNB) binding, was solubilized from porcine atrial membranes using a 5:1 (w/w) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol L-(/sup 3/H)QNB sites per gram of protein, representing 75-98% total site recovery and a two- to three-fold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for L-(/sup 3/H)QNB as determined by the three methods varied from 230 to 450 pM depending on the method and temperature. The interaction of alkyl quanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkyl guanidines with alkyl chain lengths from one to ten carbons displaced (/sup 3/H)L-quinuclidinyl bensilate ((/sup 3/H)L-QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of -1n K/sub i/ versus alkyl carbon chain number, a value of -(473 +/- 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The synthesis and properties of a radiolabeled muscarinic antagonist photoaffinity probe, (/sup 3/H) p-azidoatropine methyl iodide were reported.

  7. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    PubMed

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  8. Selectivity of ORC binding sites and the relation to replication timing, fragile sites, and deletions in cancers

    PubMed Central

    Miotto, Benoit; Ji, Zhe; Struhl, Kevin

    2016-01-01

    The origin recognition complex (ORC) binds sites from which DNA replication is initiated. We address ORC binding selectivity in vivo by mapping ∼52,000 ORC2 binding sites throughout the human genome. The ORC binding profile is broader than those of sequence-specific transcription factors, suggesting that ORC is not bound or recruited to specific DNA sequences. Instead, ORC binds nonspecifically to open (DNase I-hypersensitive) regions containing active chromatin marks such as H3 acetylation and H3K4 methylation. ORC sites in early and late replicating regions have similar properties, but there are far more ORC sites in early replicating regions. This suggests that replication timing is due primarily to ORC density and stochastic firing of origins. Computational simulation of stochastic firing from identified ORC sites is in accord with replication timing data. Large genomic regions with a paucity of ORC sites are strongly associated with common fragile sites and recurrent deletions in cancers. We suggest that replication origins, replication timing, and replication-dependent chromosome breaks are determined primarily by the genomic distribution of activator proteins at enhancers and promoters. These activators recruit nucleosome-modifying complexes to create the appropriate chromatin structure that allows ORC binding and subsequent origin firing. PMID:27436900

  9. Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

    PubMed Central

    Ni, Xiaochun; Zhang, Yong E.; Nègre, Nicolas; Chen, Sidi; Long, Manyuan; White, Kevin P.

    2012-01-01

    Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes. PMID:23139640

  10. 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.

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

    SciTech Connect

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

    1991-01-01

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

  12. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    PubMed

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

  13. 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.

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

    SciTech Connect

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

    2013-10-15

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

  15. Modulation of nicotinic acetylcholine receptors by strychnine

    PubMed Central

    García-Colunga, Jesús; Miledi, Ricardo

    1999-01-01

    Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex. PMID:10097172

  16. Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

    SciTech Connect

    Muccioli, G.; Guardabassi, A.; Pattono, P. )

    1990-03-01

    The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrence of prolactin specific binding sites in Xenopus laevis choroid plexus cells.

  17. 2-([sup 125]I) iodomelatonin binding sites in rat adrenals: Pharmacological characteristics and subcellular distribution

    SciTech Connect

    Persengiev, S.P. )

    1992-01-01

    Specific binding sites for 2-[[sup 125]I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[[sup 125]I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin > 2-iodomelatonin > melatonin > 5-methoxytryptamine > 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear and mitochondrial subcellular fractions.

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

    SciTech Connect

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

    1988-01-01

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

  19. In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

    PubMed Central

    Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme; Leblanc, Benjamin; Gut, Ivo; Gut, Marta; Duret, Laurent

    2017-01-01

    In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis. PMID:28336543

  20. In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites.

    PubMed

    Grey, Corinne; Clément, Julie A J; Buard, Jérôme; Leblanc, Benjamin; Gut, Ivo; Gut, Marta; Duret, Laurent; de Massy, Bernard

    2017-04-01

    In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis.

  1. Autoradiographic localization of endothelin-1 binding sites in the cardiovascular and respiratory systems

    SciTech Connect

    Power, R.F.; Wharton, J.; Zhao, Y.; Bloom, S.R.; Polak, J.M.

    1989-01-01

    Specific high-affinity binding sites for endothelin-1 (ET-1) have been demonstrated in peripheral tissues using the technique of in vitro receptor autoradiography. Binding was time dependent and saturable and inhibited by coincubation with an excess of unlabeled ET-1 but resistant to dissociation. Binding sites were localized to blood vessels of all sizes including coronary arteries, intrapulmonary vessels, and intrarenal and intrasplenic arteries. In addition, high-affinity binding sites were identified on airway smooth muscle, over alveolar septa, and on nerve trunks. Scatchard analysis of the data revealed a Bmax of 250 amol/mm2 and a Kd of 0.1 nM for the binding of rat tracheal smooth muscle, with similar values for porcine coronary artery. The localization of binding sites is consistent with the known effects of ET-1 and suggests a direct action on specific receptors.

  2. 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.

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

    PubMed

    Bernard, Pascal; Vanoosthuyse, Vincent

    2015-01-01

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

  4. Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

    PubMed

    Rayes, Diego; De Rosa, María José; Sine, Steven M; Bouzat, Cecilia

    2009-05-06

    Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

  5. Interaction of the muscarinic acetylcholine receptor M₂ subtype with G protein Gα(i/o) isotypes and Gβγ subunits as studied with the maltose-binding protein-M₂-Gα(i/o) fusion proteins expressed in Escherichia coli.

    PubMed

    Ichiyama, Susumu; Nemoto, Reiko; Tanabe, Hiroaki; Haga, Tatsuya

    2014-11-01

    We expressed the fusion proteins of the muscarinic acetylcholine receptor M2 subtype (M2 receptor) with a maltose-binding protein (MBP) and various G protein α subunits (Gα(i1-i3/o)) at its N- and C-terminals, respectively (MBP-M2-Gα(i/o)), in Escherichia coli, and examined the effect of G protein βγ subunits (Gβγ) on the receptor-Gα interaction as assessed by agonist- and GDP-dependent [(35)S]GTPγS binding of the fusion proteins. We found that (i) Gβγ promoted both the agonist-dependent and -independent [(35)S]GTPγS binding with little effect on the guanine nucleotide-sensitive high-affinity agonist binding, (ii) the specific [(35)S]GTPγS binding activity was much greater for MBP-M2-Gα(oA) than for MBP-M2-Gα(i1-i3) in the absence of Gβγ, whereas Gβγ preferentially promoted the agonist-dependent decrease in the affinity for GDP of MBP-M2-Gα(i1-i3) rather than of MBP-M2-Gα(oA), and (iii) the proportion of agonist-dependent [(35)S]GTPγS binding was roughly 50% irrespective of species of Gα and the presence or absence of Gβγ. These results demonstrate that receptor-Gα fusion proteins expressed in E. coli could be useful for studies of receptor-G interaction.

  6. Are high-affinity progesterone binding site(s) from porcine liver microsomes members of the sigma receptor family?

    PubMed

    Meyer, C; Schmieding, K; Falkenstein, E; Wehling, M

    1998-04-24

    Membrane progesterone binding sites have been purified recently from pig liver. Since progesterone is considered as an endogenous sigma (sigma) receptor ligand, these sites were characterized pharmacologically by ligands selective for sigma receptor and dopamine receptor binding sites, and by other drugs from distinct pharmacological classes. Binding studies using the radioligand [3H]progesterone were done in crude membrane preparations and solubilized fractions to determine half-maximal inhibitory concentration (IC50) values, from which inhibitory constants (Ki values) were calculated. Radioligand binding was inhibited by the sigma receptor ligands haloperidol, carbetapentane citrate, 1,3-Di(2-tolyl)guanidine (DTG), R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2 aminopropane HCl (R(-)-PPAAP HCl), or sigma receptor antagonists like (+)-3-(3-hydroxyphenyl)-N-propylpiperidine HCl (R(+)-PPP HCl) and cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]-9H-carbazole dihydrochloride (rimcazole 2HCl). The hierarchy of inhibitory action was not fully compatible with either sigma receptor class I (moderate affinity of pentazocine, diphenylhydantoin (phenytoin) insensitivity) or II sites (high affinity of carbetapentane). The data thus suggest that progesterone binding sites in porcine liver membranes are related to the sigma receptor binding site superfamily, but may represent a particular species with progesterone specificity.

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

    SciTech Connect

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

    2002-03-08

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

  8. Analysis of transcription-factor binding-site evolution by using the Hamilton-Jacobi equations

    NASA Astrophysics Data System (ADS)

    Ancliff, Mark; Park, Jeong-Man

    2016-12-01

    We investigate a quasi-species mutation-selection model of transcription-factor binding-site evolution. By considering the mesa and the crater fitness landscapes designed to describe these binding sites and point mutations, we derive an evolution equation for the population distribution of binding sequences. In the long-length limit, the evolution equation is replaced by a Hamilton-Jacobi equation which we solve for the stationary state solution. From the stationary solution, we derive the population distributions and find that an error threshold, separating populations in which the binding site does or does not evolve, only exists for certain values of the fitness parameters. A phase diagram in this parameter space is derived and shows a critical line below which no error threshold exists. We also investigate the evolution of multiple binding sites for the same transcription factor. For two binding sites, we perform an analysis similar to that for a single site and determine a phase diagram showing different phases with both, one, or no binding sites selected. In the phase diagram, the phase boundary between the one-or-two selected site phases is qualitatively different for the mesa and the crater fitness landscapes. As fitness benefits for a second bound transcription factor tend to zero, the minimum mutation rate at which the two-site phase occurs diverges in the mesa landscape whereas the mutation rate at the phase boundary tends to a finite value for the crater landscape.

  9. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  10. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

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

    PubMed

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

    2012-10-15

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

  12. 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

  13. Incorporating replacement free energy of binding-site waters in molecular docking.

    PubMed

    Sun, Hanzi; Zhao, Lifeng; Peng, Shiming; Huang, Niu

    2014-09-01

    Binding-site water molecules play a crucial role in protein-ligand recognition, either being displaced upon ligand binding or forming water bridges to stabilize the complex. However, rigorously treating explicit binding-site waters is challenging in molecular docking, which requires to fully sample ensembles of waters and to consider the free energy cost of replacing waters. Here, we describe a method to incorporate structural and energetic properties of binding-site waters into molecular docking. We first developed a solvent property analysis (SPA) program to compute the replacement free energies of binding-site water molecules by post-processing molecular dynamics trajectories obtained from ligand-free protein structure simulation in explicit water. Next, we implemented a distance-dependent scoring term into DOCK scoring function to take account of the water replacement free energy cost upon ligand binding. We assessed this approach in protein targets containing important binding-site waters, and we demonstrated that our approach is reliable in reproducing the crystal binding geometries of protein-ligand-water complexes, as well as moderately improving the ligand docking enrichment performance. In addition, SPA program (free available to academic users upon request) may be applied in identifying hot-spot binding-site residues and structure-based lead optimization.

  14. A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

    PubMed

    Barchad-Avitzur, Ofra; Priest, Michael F; Dekel, Noa; Bezanilla, Francisco; Parnas, Hanna; Ben-Chaim, Yair

    2016-10-04

    G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

  15. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  16. Detecting O2 binding sites in protein cavities

    PubMed Central

    Kitahara, Ryo; Yoshimura, Yuichi; Xue, Mengjun; Kameda, Tomoshi; Mulder, Frans A. A.

    2016-01-01

    Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins. PMID:26830762

  17. Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum.

    PubMed

    Santos, R G; Diniz, C R; Cordeiro, M N; De Lima, M E

    1999-12-01

    Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 +/- 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10(-4) g/l and 1.85 x 10(-5) g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (omega-Aga IA and IB) and its competition with [125I]-omega-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

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

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  19. A permanent ion binding site located between two gates of the Shaker K+ channel.

    PubMed

    Harris, R E; Larsson, H P; Isacoff, E Y

    1998-04-01

    K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the selectivity filter.

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

    PubMed

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

    2016-11-01

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

  1. A permanent ion binding site located between two gates of the Shaker K+ channel.

    PubMed Central

    Harris, R E; Larsson, H P; Isacoff, E Y

    1998-01-01

    K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the selectivity filter. PMID:9545043

  2. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  3. 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

  4. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

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

    PubMed

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

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

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

    PubMed

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

    2013-11-19

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

  7. Kinetic and Equilibrium Analysis of Estradiol in Uterus: A Model of Binding-Site Distribution in Uterine Cells

    PubMed Central

    Williams, David; Gorski, Jack

    1972-01-01

    Kinetic and equilibrium binding studies indicate that the process by which the complex of estradiol-binding protein is transferred to the cell nuclei is very rapid and is readily reversible in intact cells; that is, the cytosol and nuclear binding sites are in a rapidly reversible equilibrium. Binding of the hormone appears to shift this equilibrium such that a large percent of the filled binding sites become associated with the nuclear fraction. A model is presented to show that the quantity of filled nuclear binding sites present at any estradiol concentration can be determined strictly by the initial binding between the hormone and the cytosol binding sites. PMID:4508334

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

    SciTech Connect

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

    2003-08-28

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

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

    PubMed Central

    Joseph, Thomas T.

    2016-01-01

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

  10. Genome wide features, distribution and correlations of NF-Y binding sites.

    PubMed

    Zambelli, Federico; Pavesi, Giulio

    2016-10-18

    NF-Y is a trimeric transcription factor that binds on DNA the CCAAT-box motif. In this article we reviewed and complemented with additional bioinformatic analysis existing data on genome-wide NF-Y binding characterization in human, reaching the following main conclusions: (1) about half of NF-Y binding sites are located at promoters, about 60-80 base pairs from transcription start sites; NF-Y binding to distal genomic regions takes place at inactive chromatin loci and/or DNA repetitive elements more often than active enhancers; (2) on almost half of its binding sites, regardless of their genomic localization (promoters or distal regions), NF-Y finds on DNA more than one CCAAT-box, and most of those multiple CCAAT binding loci present precise spacing and organization of the elements composing them; (3) there exists a well defined class of transcription factors that show genome-wide co-localization with NF-Y. Some of them lack their canonical binding site in binding regions overlapping with NF-Y, hence hinting at NF-Y mediated recruitment, while others show a precise positioning on DNA of their binding sites with respect to the CCAAT box bound by NF-Y. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

  11. Binding of [3H]SCH23390 to a non-dopaminergic site in bovine kidney.

    PubMed

    Hollis, C M; Turner, J D; Strange, P G

    1992-05-08

    Binding of the D1 dopamine receptor antagonist [3H]SCH23390 to bovine renal cortical membranes has been studied. Specific binding of [3H]SCH23390 was saturable and reversible and stereoisomers of SCH23390 competed stereoselectively. In contrast, competition with the isomers of butaclamol was not stereoselective and dopamine failed to compete for the [3H]SCH23390 binding site. The site is therefore not a D1 dopamine receptor. Competition studies with a very wide range of compounds failed to define the nature of the [3H]SCH23390 binding sites in renal cortex whereas in parallel studies the characteristics of [3H]SCH23390 binding in caudate nucleus were entirely consistent with those of D1 dopamine receptors. The nature of [3H]SCH23390 binding in preparations of tubular and glomerular membranes was found to be virtually identical to those of crude renal cortical membranes indicating lack of compartmentation of these sites. Autoradiographic studies of [3H]SCH23390 binding in bovine kidney showed significantly higher levels of binding sites in renal cortex compared with renal medulla and this was confirmed by direct ligand binding studies.

  12. Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites

    SciTech Connect

    Itzhak, Y.

    1988-01-01

    The pharmacological specificity of representative psychotomimetic agents such a phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with (/sup 3/H)-1-(1-(3-hydroxyphenyl) cyclohexyl) piperidine ((/sup 3/H)PCP-3-OH), (+)(/sup 3/H)-N-allylnormetazocine ((+)(/sup 3/H)SKF 10047) and (+) (/sup 3/H)-3-(3-hydroxy-phenyl)-N-(1-propyl) piperidine and ((+)(/sup 3/H)-3-PPP). PCP analogs inhibit potently high affinity (/sup 3/H)PCP-3-OH binding and (+)(/sup 3/H)SKF 10047 binding, moderately the low affinity binding component of (/sup 3/H)PCP-3-OH and very weakly (+) (/sup 3/H)-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+)(/sup 3/H)SKF 10047, high affinity (/sup 3/H)PCP-3-OH and (+)(/sup 3/H)-3-PCP-3-OH binding. The antipsychotic agents display high affinity for (+)(/sup 3/H)-3-PPP binding sites, moderate affinity for (+)(/sup 3/H)SKF 10047 sites and have no effect on either the high or low affinity (/sup 3/H)PCP-3-OH binding. 20 references, 3 figures, 2 tables.

  13. Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site

    PubMed Central

    Águila, Sonia; Teruel-Montoya, Raúl; Vicente, Vicente; Corral, Javier; Martínez-Martínez, Irene

    2016-01-01

    Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin. PMID:27322195

  14. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

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

    SciTech Connect

    Crankshaw, D.; Gaspar, V.; Pliska, V. )

    1990-01-01

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

  16. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

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

  17. Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells.

    PubMed

    Calvo, J R; Rafii-el-Idrissi, M; Pozo, D; Guerrero, J M

    1995-04-01

    This paper reviews the evidence that supports the hypothesis of the existence of specific binding sites for melatonin on immune cells. These binding sites have been described in human blood lymphocytes and granulocytes, and thymus, spleen, and bursa of Fabricius from different rodents and birds. The dissociation constant values of these binding sites are in the 0.1-1 nM range, suggesting that melatonin may play a physiological role in lymphocyte regulation. Moreover, melatonin binding sites appear to be modulated by guanine nucleotides. Therefore, in addition to other mechanisms described for the regulation of immune function by melatonin, a direct mechanism of regulation can be involved via binding of melatonin by immunocompetent cells.

  18. Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

    SciTech Connect

    Leslie, R.A.; McDonald, T.J.; Robertson, H.A.

    1988-09-01

    Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared (/sup 125/I)PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of (/sup 125/I)PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of (/sup 125/I)PYY binding sites throughout the rat brain was seen to be similar to the distribution of (/sup 125/I)NPY binding sites.

  19. Cluster Analysis of p53 Binding Site Sequences Reveals Subsets with Different Functions

    PubMed Central

    Lim, Ji-Hyun; Latysheva, Natasha S.; Iggo, Richard D.; Barker, Daniel

    2016-01-01

    p53 is an important regulator of cell cycle arrest, senescence, apoptosis and metabolism, and is frequently mutated in tumors. It functions as a tetramer, where each component dimer binds to a decameric DNA region known as a response element. We identify p53 binding site subtypes and examine the functional and evolutionary properties of these subtypes. We start with over 1700 known binding sites and, with no prior labeling, identify two sets of response elements by unsupervised clustering. When combined, they give rise to three types of p53 binding sites. We find that probabilistic and alignment-based assessments of cross-species conservation show no strong evidence of differential conservation between types of binding sites. In contrast, functional analysis of the genes most proximal to the binding sites provides strong bioinformatic evidence of functional differentiation between the three types of binding sites. Our results are consistent with recent structural data identifying two conformations of the L1 loop in the DNA binding domain, suggesting that they reflect biologically meaningful groups imposed by the p53 protein structure. PMID:27812278

  20. Molecular forms and subunit structure of the acetylcholine receptor in the central nervous system of insects.

    PubMed

    Breer, H; Kleene, R; Hinz, G

    1985-12-01

    The nicotinic acetylcholine receptor as probed by alpha-bungarotoxin binding has been isolated from detergent-solubilized ganglionic membrane preparations from the insect, Locusta migratoria. The isolation and characterization of the receptor protein was achieved by preparation of membrane fragments, extraction by sodium deoxycholate, centrifugation on sucrose density gradient, affinity chromatography, gel electrophoresis, and immunoblotting. The purified receptor protein migrated as a single band on polyacrylamide when native (Mr = 250,000 to 300,000) but also under denaturing conditions (Mr = 65,000) and cross-reacted with some monoclonal antibodies against the Torpedo receptor. In immunohistochemical approaches using polyclonal antibodies the acetylcholine receptor antigenic sites could topochemically be identified at very distinct zones in the neuropil of locust ganglia. The results suggest that the acetylcholine receptor in the central nervous system of insects represents an oligomeric complex composed of four identical or very similar subunits and thus may represent a prototype of the recently proposed homo-oligomeric ancestral acetylcholine receptor.

  1. Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites.

    PubMed Central

    Beachy, P A; Varkey, J; Young, K E; von Kessler, D P; Sun, B I; Ekker, S C

    1993-01-01

    Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity. Images PMID:8105373

  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. Host-Guest Binding-Site-Tunable Self-Assembly of Stimuli-Responsive Supramolecular Polymers.

    PubMed

    Yao, Hao; Qi, Miao; Liu, Yuyang; Tian, Wei

    2016-06-13

    Despite the remarkable progress made in controllable self-assembly of stimuli-responsive supramolecular polymers (SSPs), a basic issue that has not been consideration to date is the essential binding site. The noncovalent binding sites, which connect the building blocks and endow supramolecular polymers with their ability to respond to stimuli, are expected to strongly affect the self-assembly of SSPs. Herein, the design and synthesis of a dual-stimuli thermo- and photoresponsive Y-shaped supramolecular polymer (SSP2) with two adjacent β-cyclodextrin/azobenzene (β-CD/Azo) binding sites, and another SSP (SSP1) with similar building blocks, but only one β-CD/Azo binding site as a control, are described. Upon gradually increasing the polymer solution temperature or irradiating with UV light, SSP2 self-assemblies with a higher binding-site distribution density; exhibits a flower-like morphology, smaller size, and more stable dynamic aggregation process; and greater controllability for drug-release behavior than those observed with SSP1 self-assemblies. The host-guest binding-site-tunable self-assembly was attributed to the positive cooperativity generated among adjacent binding sites on the surfaces of SSP2 self-assemblies. This work is beneficial for precisely controlling the structural parameters and controlled release function of SSP self-assemblies.

  4. A functional study of concanavalin A-histamine binding site overlap in Tetrahymena phagocytosis test.

    PubMed

    Csaba, G; Darvas, Z; László, V

    1983-01-01

    Treatment with histamine stimulated the phagocytotic activity of the Tetrahymena to a measurable degree, which was still demonstrable after a week (about 40 generations). Concanavalin A, which binds to the same membrane binding site as histamine, inhibited the stimulatory action of subsequently added histamine, but did not in itself influence phagocytotic activity in any way. The inhibitory effect of Con A on the histamine binding site proved to be dose-dependent. These observations stress the importance of investigating the functional context--as sole realistic measure--of receptor--ligand bindings.

  5. Defining the Plasticity of Transcription Factor Binding Sites by Deconstructing DNA Consensus Sequences: The PhoP-Binding Sites among Gamma/Enterobacteria

    PubMed Central

    Harari, Oscar; Park, Sun-Yang; Huang, Henry; Groisman, Eduardo A.; Zwir, Igor

    2010-01-01

    Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs) using a machine learning method inspired by the “Divide & Conquer” strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target genes and/or the

  6. Multi-site substrate binding and interplay in barley alpha-amylase 1.

    PubMed

    Nielsen, Morten Munch; Seo, Eun-Seong; Bozonnet, Sophie; Aghajari, Nushin; Robert, Xavier; Haser, Richard; Svensson, Birte

    2008-07-23

    Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multi-site substrate interactions are functionally significant. In barley alpha-amylase both Tyr380, situated on a remote non-catalytic domain, and Tyr105 in subsite -6 of the active site cleft are principal carbohydrate binding residues. The dual active site/secondary site mutants Y105A/Y380A and Y105A/Y380M show that each of Tyr380 and Tyr105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr105 predominates in oligosaccharide hydrolysis. Additional delicate structure/function relationships of the secondary site are uncovered using Y380A/H395A, Y380A, and H395A AMY1 mutants.

  7. A Predicted Binding Site for Cholesterol on the GABAA Receptor

    PubMed Central

    Hénin, Jérôme; Salari, Reza; Murlidaran, Sruthi; Brannigan, Grace

    2014-01-01

    Modulation of the GABA type A receptor (GABAAR) function by cholesterol and other steroids is documented at the functional level, yet its structural basis is largely unknown. Current data on structurally related modulators suggest that cholesterol binds to subunit interfaces between transmembrane domains of the GABAAR. We construct homology models of a human GABAAR based on the structure of the glutamate-gated chloride channel GluCl of Caenorhabditis elegans. The models show the possibility of previously unreported disulfide bridges linking the M1 and M3 transmembrane helices in the α and γ subunits. We discuss the biological relevance of such disulfide bridges. Using our models, we investigate cholesterol binding to intersubunit cavities of the GABAAR transmembrane domain. We find that very similar binding modes are predicted independently by three approaches: analogy with ivermectin in the GluCl crystal structure, automated docking by AutoDock, and spontaneous rebinding events in unbiased molecular dynamics simulations. Taken together, the models and atomistic simulations suggest a somewhat flexible binding mode, with several possible orientations. Finally, we explore the possibility that cholesterol promotes pore opening through a wedge mechanism. PMID:24806926

  8. Identification of 5-hydroxytryptamine1D binding sites in sheep caudate nucleus membranes.

    PubMed

    Pauwels, P J; Palmier, C; Briley, M

    1993-08-03

    Radioligand binding measurements were performed in membranes of sheep caudate nucleus using [3H]5-hydroxytryptamine (5-HT). [3H]5-HT labeled a population of high affinity binding sites with a Kd of 1.9 +/- 0.1 nM and a Bmax of 19.8 +/- 2.2 fmol/mg tissue. Combined 5-HTID/E binding sites were the predominant 5-HT1 subtype, accounting for 78% of the total population of 5-HT1 binding sites. 5-Carboxamidotryptamine (5-CT) and sumatriptan yielded inhibition curves which best fitted a two-site model with high affinity values of 0.8 and 10.1 nM, and 1000 and 206 nM for their low affinity components. The proportion of the high affinity 5-CT and sumatriptan binding sites was 79 and 72%. The binding affinity profile of 5-HT1D binding sites [5-CT > 5-HT > d-LSD > 5-MeOT > sumatriptan > RU 24,969 > metergoline > tryptamine = rauwolscine = methylsergide > yohimbine = methiothepin > TFMPP = 8-OH-DPAT > 2-methyl-5-HT > mCPP = quipazine = CP 93,129 > ketanserin > (-)-propranolol = haloperidol = ipsapirone] compares well to that reported for 5-HT1D receptor sites in human caudate and cortex (correlation coefficient: 0.99 and 0.98). The present results indicate that sheep caudate nucleus is a valid tissue for studying interaction of compounds with 5-HT1D binding sites in the relative absence of 5-HT1E binding sites.

  9. Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae

    PubMed Central

    Lang, Michael; Juan, Elvira

    2010-01-01

    There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to sites consisting of the multiple trinucleotide repeat . The ADF-1 recognition sites with high binding affinity differ from this trinucleotide repeat consensus sequence and a new consensus sequence is proposed for the high-affinity ADF-1 binding sites. In vitro transcription experiments with the D. funebris and D. virilis ADF-1 binding regions revealed that stronger ADF-1 binding to the expanded D. funebris ADF-1 binding region only moderately lead to increased transcriptional activity of the Adh gene. The potential of this regional expansion is discussed in the context of different ADF-1 cellular concentrations and maintenance of the ADF-1 stimulus. Altogether, evolutionary change of ADF-1 binding regions involves both, rearrangements of complex binding site cluster and also nucleotide substitutions within sites that lead to different binding affinities. PMID:20542916

  10. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase

    SciTech Connect

    Ravilious, Geoffrey E.; Jez, Joseph M.

    2012-08-31

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

  11. Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

    PubMed Central

    Wang, S W; Speck, N A

    1992-01-01

    The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes. Images PMID:1309596

  12. Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

    PubMed Central

    2013-01-01

    Background Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. Results We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). Conclusions Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3. PMID:24001316

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

    PubMed Central

    Weyer, Sven; Pääbo, Svante

    2016-01-01

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

  14. Carbonic anhydrase binding site parameterization in OPLS-AA force field.

    PubMed

    Bernadat, Guillaume; Supuran, Claudiu T; Iorga, Bogdan I

    2013-03-15

    The parameterization of carbonic anhydrase binding site in OPLS-AA force field was performed using quantum chemistry calculations. Both OH2 and OH(-) forms of the binding site were considered, showing important differences in terms of atomic partial charges. Three different parameterization protocols were used, and the results obtained highlighted the importance of including an extended binding site in the charge calculation. The force field parameters were subsequently validated using standard molecular dynamics simulations. The results presented in this work should greatly facilitate the use of molecular dynamics simulations for studying the carbonic anhydrase, and more generally, the metallo-enzymes.

  15. Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions.

    PubMed

    Chatagnon, Amandine; Perriaud, Laury; Nazaret, Nicolas; Croze, Séverine; Benhattar, Jean; Lachuer, Joël; Dante, Robert

    2011-11-01

    Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

  16. Nicotinic acetylcholine receptor from chick optic lobe.

    PubMed Central

    Norman, R I; Mehraban, F; Barnard, E A; Dolly, J O

    1982-01-01

    An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors. Images PMID:6175967

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

    PubMed Central

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

    2016-01-01

    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. PMID:26721389

  18. 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.

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  1. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  2. Distribution of cholecystokinin receptor binding sites in the human brain: an autoradiographic study

    SciTech Connect

    Dietl, M.M.; Probst, A.; Palacios, J.M.

    1987-01-01

    Cholecystokinin (CCK) binding sites were localized by in vitro autoradiography in human postmortem brain materials from 12 patients without reported neurological diseases using (125I)Bolton-Hunter CCK octapeptide (BHCCK-8) as a ligand. The pharmacological characteristics of BHCCK-8 binding to mounted tissue sections were comparable to those previously reported in the rat. CCK-8 being the most potent displacer, followed by caerulein, CCK-4, and gastrin I. The distribution of BHCCK-8 binding sites was heterogeneous. These sites were highly concentrated in a limited number of gray matter areas and nuclei. The highest binding densities were seen in the glomerular and external plexiform layers of the olfactory bulb. BHCCK-8 binding sites were also enriched in the neocortex, where they presented a laminar distribution with low levels in lamina I, moderate concentration in laminae II to IV, high density in lamina V, and low levels in lamina VI. A different laminar distribution was seen in the visual cortex, where a low receptor density was observed in lamina IV but higher density in laminae II and VI. In the basal ganglia the nucleus accumbens, caudatus, and the putamen presented moderate to high densities of binding sites, while the globus pallidus lacked sites of BHCCK-8 binding. In the limbic system the only area presenting moderate to high density was the amygdaloid complex, particularly in the granular nucleus, while most of the thalamic nuclei were extremely poor or lacked BHCCK-8 binding. The hippocampal formation showed low (CA1-3) to moderate (subiculum) densities. Midbrain areas generally disclosed very low levels of BHCCK-8 binding sites. The pontine gray and the nucleus reticularis tegmenti pontis showed a relatively high density of CCK-8 receptor specific binding.

  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. 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

  5. Specific binding of a dichloroacetamide herbicide safener in maize at a site that also binds thiocarbamate and chloroacetanilide herbicides.

    PubMed

    Walton, J D; Casida, J E

    1995-09-01

    Dichloroacetamide safeners such as N,N-diallyl-2,2-dichloroacetamide and (R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidine protect maize (Zea mays) against injury from thiocarbamate and chloroacetanilide herbicides. Binding activity of tritium-labeled (R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidine (15 Ci/mmol; referred to as [3H]Saf) was characterized in extracts of etiolated maize seedlings. The binding is saturable, involves a single class of binding sites (Kd 0.12 microM; maximal binding in coleoptiles 0.53 nmol/g fresh weight, equivalent to 55 pmol/mg protein), and is sensitive to boiling and protease treatment. Binding in etiolated maize seedlings is highest in the coleoptile and lowest in the leaves. Binding of [3H]Saf also occurs in etiolated sorghum (Sorghum bicolor) shoots but not several other cereals. There is a good correlation between known safener effectiveness and the concentration that inhibits [3H]Saf binding half-maximally among 21 dichloroacetamides and related compounds. N,N-Diallyl-2,2-dichloroacetamide had the lowest inhibitor concentration that reduces specific binding by 50% (IC50), 0.01 microM. [3H]Saf binding is inhibited by 4 chloroacetanilide herbicides with IC50 values of 0.07 to 0.48 microM and by 12 thiocarbamate herbicides and analogs with IC50 values of 0.06 to 2.3 microM. The inhibition of [3H]Saf binding by alachlor and S-ethyl dipropylthiocarbamate is competitive.

  6. Theory and simulation of diffusion-influenced, stochastically gated ligand binding to buried sites

    PubMed Central

    Barreda, Jorge L.; Zhou, Huan-Xiang

    2011-01-01

    We consider the diffusion-influenced rate coefficient of ligand binding to a site located in a deep pocket on a protein; the binding pocket is flexible and can reorganize in response to ligand entrance. We extend to this flexible protein-ligand system a formalism developed previously [A. M. Berezhkovskii, A, Szabo, and H.-X. Zhou, J. Chem. Phys. 135, 075103 (2011)10.1063/1.3609973] for breaking the ligand-binding problem into an exterior problem and an interior problem. Conformational fluctuations of a bottleneck or a lid and the binding site are modeled as stochastic gating. We present analytical and Brownian dynamics simulation results for the case of a cylindrical pocket containing a binding site at the bottom. Induced switch, whereby the conformation of the protein adapts to the incoming ligand, leads to considerable rate enhancement. PMID:22010732

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

    SciTech Connect

    Orlow, S.J.; Hotchkiss, S.; Pawelek, J.M. )

    1990-01-01

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

  8. Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

    PubMed

    Payvar, F; Firestone, G L; Ross, S R; Chandler, V L; Wrange, O; Carlstedt-Duke, J; Gustafsson, J A; Yamamoto, K R

    1982-01-01

    Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

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

    SciTech Connect

    Buck, S.H.; Burcher, E.

    1985-11-01

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

  10. 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.

  11. 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.

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

    PubMed Central

    Hong, Lian; Simon, John D.

    2008-01-01

    Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of understanding of this field. PMID:17580858

  13. Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors.

    PubMed

    Osipov, Alexey V; Rucktooa, Prakash; Kasheverov, Igor E; Filkin, Sergey Yu; Starkov, Vladislav G; Andreeva, Tatyana V; Sixma, Titia K; Bertrand, Daniel; Utkin, Yuri N; Tsetlin, Victor I

    2012-02-24

    In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.

  14. 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.

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

    PubMed Central

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

    2013-01-01

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

  16. Autoradiographic localization of [3H]thiocolchicoside binding sites in the rat brain and spinal cord.

    PubMed

    Balduini, W; De Angelis, V; Mazzoni, E; Depoortere, H; Cattabeni, F; Cimino, M

    2001-06-01

    Thiocolchicoside is used in humans as a myorelaxant drug with anti-inflammatory and analgesic activity. Recently we established the experimental conditions that allowed the identification of [3H]thiocolchicoside binding sites in synaptic membranes of rat spinal cord and cerebral cortex. The pharmacological characterization of these sites indicated that GABA and several of its agonists and antagonists, as well as strychnine, were able to interact with [3H]thiocolchicoside binding in a dose-dependent manner and with different affinities. In order to gain more insight into the nature and the anatomical distribution of the binding sites labeled by [3H]thiocolchicoside, in the present study we examined the localization of these sites on parasagittal and coronal sections of the rat brain and spinal cord, respectively, using receptor autoradiography. In the spinal cord an intense signal was observed in the gray matter, with the highest density occurring in the superficial layers of the dorsal horns. Strychnine completely displaced [3H]thiocolchicoside binding, whereas GABA only partially removed the radioligand from its binding sites. In the brain, specific binding occurred in several areas and was displaced by both GABA and strychnine. The distribution of [3H]thiocolchicoside binding sites in brain sections, however, did not match that found for [3H]muscimol. Furthermore, cold thiocolchicoside was not able to completely displace [3H]muscimol binding, and showed a different efficacy in the various areas labeled by the radioligand. We conclude that thiocolchicoside may interact with a subpopulation of GABA(A) receptors having low-affinity binding sites for GABA. Furthermore, the observed sensitivity to strychnine in the spinal cord indicates an interaction also with strychnine-sensitive glycine receptors, suggesting that the pharmacological effects of thiocolchicoside may be the result of its interaction with different receptor populations.

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

    PubMed

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

    2015-09-01

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

  18. 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

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

    PubMed

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

    2016-11-28

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

  20. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    PubMed Central

    Liu, Jiajian; Stormo, Gary D

    2005-01-01

    Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data. PMID:16014175

  3. Mechanism for binding site diversity on ankyrin. Comparison of binding sites on ankyrin for neurofascin and the Cl-/HCO3- anion exchanger.

    PubMed

    Michaely, P; Bennett, V

    1995-12-29

    Ankyrins are a family of spectrin-binding proteins that associate with at least seven distinct membrane proteins, including ion transporters and cell adhesion molecules. The membrane-binding domain of ankyrin is comprised of a tandem array of 24 ANK repeats organized into four 6-repeat folding domains. Tandem arrays of ANK repeats have been proposed to mediate protein interactions in a variety of proteins including factors involved in the regulation of transcription and the cell cycle. This report provides several new insights into the versatility of ANK repeats of ankyrin in protein recognition, using neurofascin and the Cl-/HCO3- anion exchanger as model ligands and ankyrinR as the prototypic ankyrin. Different combinations of ANK repeat domains from this ankyrin form two distinct, high affinity binding sites for neurofascin. One site requires both repeat domains 3 and 4. The other site involves both repeat domains 2 and 3, although domain 2 has significant activity alone. The sites appear to be independent with Kd values of 3 and 14 nM, respectively. Both the Cl-/HCO3- anion exchanger and neurofascin can interact simultaneously with repeat domains 3 and 4, because neurofascin is unable to displace binding of the anion exchanger cytoplasmic domain to domains 3 and 4, despite having a 3-5-fold higher affinity. These results demonstrate two levels of diversity in the binding sites on ankyrin: one resulting from different combinations of ANK repeat domains and another from different determinants within the same combination of repeat domains. One consequence of this diversity is that ankyrin can accommodate two neurofascin molecules as well as the anion exchanger through interactions mediated by ANK repeats. The ability of ankyrin to simultaneously associate with multiple types of membrane proteins is an unanticipated finding with implications for the assembly of integral membrane proteins into specialized regions of the plasma membrane.

  4. Dynamic coupling of regulated binding sites and cycling myosin heads in striated muscle.

    PubMed

    Campbell, Kenneth S

    2014-03-01

    In an activated muscle, binding sites on the thin filament and myosin heads switch frequently between different states. Because the status of the binding sites influences the status of the heads, and vice versa, the binding sites and myosin heads are dynamically coupled. The functional consequences of this coupling were investigated using MyoSim, a new computer model of muscle. MyoSim extends existing models based on Huxley-type distribution techniques by incorporating Ca(2+) activation and cooperative effects. It can also simulate arbitrary cross-bridge schemes set by the researcher. Initial calculations investigated the effects of altering the relative speeds of binding-site and cross-bridge kinetics, and of manipulating cooperative processes. Subsequent tests fitted simulated force records to experimental data recorded using permeabilized myocardial preparations. These calculations suggest that the rate of force development at maximum activation is limited by myosin cycling kinetics, whereas the rate at lower levels of activation is limited by how quickly binding sites become available. Additional tests investigated the behavior of transiently activated cells by driving simulations with experimentally recorded Ca(2+) signals. The unloaded shortening profile of a twitching myocyte could be reproduced using a model with two myosin states, cooperative activation, and strain-dependent kinetics. Collectively, these results demonstrate that dynamic coupling of binding sites and myosin heads is important for contractile function.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  6. Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1

    PubMed Central

    Capper, Michael J.; O’Neill, Paul M.; Fisher, Nicholas; Strange, Richard W.; Moss, Darren; Ward, Stephen A.; Berry, Neil G.; Lawrenson, Alexandre S.; Hasnain, S. Samar; Biagini, Giancarlo A.; Antonyuk, Svetlana V.

    2015-01-01

    Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Qo site (one of two potential binding sites within cytochrome bc1) using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Qo site but bind at the Qi site. The discovery that these compounds bind at the Qi site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Qi also explains the ability of this class to overcome parasite Qo-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles. PMID:25564664

  7. Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

    PubMed Central

    Marver, D; Chiu, W; Wolff, M E; Edelman, I S

    1976-01-01

    A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan. PMID:1069998

  8. Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

    SciTech Connect

    Large, T.H.; Cho, N.J.; De Mello, F.G.; Klein, W.L.

    1985-07-25

    Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.

  9. Human retina contains polyamine sensitive [3H]-ifenprodil binding sites: implications for neuroprotection?

    PubMed Central

    Sharif, N; Xu, S

    1999-01-01

    AIMS—This study characterised the pharmacology of [3H]-ifenprodil binding to the polyamine binding sites (PBS) on the N-methyl-D-aspartate (NMDA) receptor channel complex on human retinas. These data were correlated with the known neuroprotective effects of ifenprodil and eliprodil.
METHODS—Specific binding of [3H]-ifenprodil (under sigma site blockade) was investigated using human retinal homogenates and radioligand binding techniques. Scatchard and competition analyses were utilised to define the pharmacology of the [3H]-ifenprodil binding sites.
RESULTS—Specific binding of [3H]-ifenprodil comprised 73% (SEM 3%) of total and reflected interaction with two affinity sites (Kds = 0.39 and 4.3 µM) of different densities (Bmax = 14.4 and 105 pmol/ mg protein) (n = 5). The rank order of affinity of compounds competing for [3H]-ifenprodil binding to the high affinity PBS was: ifenprodil > eliprodil > arcaine > spermine > diaminodecane > spermidine > putrescine >> MK-801 (n = 3-7). However, [3H]-ifenprodil binding was minimally inhibited by glutamate, NMDA, and kainate.
CONCLUSION—These studies have shown, for the first time, the presence of specific [3H]-ifenprodil binding sites in the human retina with pharmacological characteristics of PBS associated with the NMDA receptor ionophore complex. The neuroprotective effects of eliprodil and ifenprodil may, in part, be mediated via these [3H]-ifenprodil labelled sites.

 Keywords: retina; neuroprotection; eliprodil; NMDA receptors PMID:10396205

  10. RPI-Bind: a structure-based method for accurate identification of RNA-protein binding sites.

    PubMed

    Luo, Jiesi; Liu, Liang; Venkateswaran, Suresh; Song, Qianqian; Zhou, Xiaobo

    2017-04-04

    RNA and protein interactions play crucial roles in multiple biological processes, while these interactions are significantly influenced by the structures and sequences of protein and RNA molecules. In this study, we first performed an analysis of RNA-protein interacting complexes, and identified interface properties of sequences and structures, which reveal the diverse nature of the binding sites. With the observations, we built a three-step prediction model, namely RPI-Bind, for the identification of RNA-protein binding regions using the sequences and structures of both proteins and RNAs. The three steps include 1) the prediction of RNA binding regions on protein, 2) the prediction of protein binding regions on RNA, and 3) the prediction of interacting regions on both RNA and protein simultaneously, with the results from steps 1) and 2). Compared with existing methods, most of which employ only sequences, our model significantly improves the prediction accuracy at each of the three steps. Especially, our model outperforms the catRAPID by >20% at the 3(rd) step. All of these results indicate the importance of structures in RNA-protein interactions, and suggest that the RPI-Bind model is a powerful theoretical framework for studying RNA-protein interactions.

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

    SciTech Connect

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

    1986-05-01

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

  12. Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II

    PubMed Central

    Nettles, Whitnee L.; Song, He; Farquhar, Erik R.; Fitzkee, Nicholas C.; Emerson, Joseph P.

    2015-01-01

    Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo-form of human carbonic anhydrase II (CA) binds two equivalents of copper(II) with high affinity. The Cu2+ ions bind independently forming two non-coupled type-II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we have identified an N-terminal Cu2+ binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four to five coordinate N-terminal Cu2+ binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-ATCUN and copper(II)-beta-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu2+ binding data to the high-affinity CuA site, we have derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu2+ binding to the CuB site of CA to be 2 × 109 and −17.4 kcal/mol, respectively. PMID:26010488

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

    PubMed

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

    1999-02-01

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

  14. Transcriptional start and MetR binding sites on the Escherichia coli metH gene.

    PubMed

    Marconi, R; Wigboldus, J; Weissbach, H; Brot, N

    1991-03-29

    The 5' upstream region of the Escherichia coli metH gene has been sequenced. Primer extension analysis revealed a transcription start site at 324 bases upstream of the initiator codon. An 8 base sequence homologous to the MetR binding region on the E. coli metE gene is present 217 bp downstream of the transcription start site. Gel retardation experiments showed that purified MetR protein could bind to a 30 base oligonucleotide containing the putative MetR binding region. No "met box" was present which explains the relative lack of regulation of the expression of the metH gene by methionine.

  15. Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

    PubMed Central

    Haycocks, James R. J.; Grainger, David C.

    2016-01-01

    A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality. PMID:27258043

  16. 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.

  17. External Imaging of Cerebral Muscarinic Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Eckelman, William C.; Reba, Richard C.; Rzeszotarski, Waclaw J.; Gibson, Raymond E.; Hill, Thomas; Holman, B. Leonard; Budinger, Thomas; Conklin, James J.; Eng, Robert; Grissom, Michael P.

    1984-01-01

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  18. External imaging of cerebral muscarinic acetylcholine receptors

    SciTech Connect

    Eckelman, W.C.; Reba, R.C.; Rzeszotarski, W.J.; Gibson, R.E.; Hill, T.; Holman, B.L.; Budinger, T.; Conklin, J.J.; Eng, R.; Grissom, M.P.

    1984-01-20

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  19. Mapping of anion binding sites on cytochrome c by differential chemical modification of lysine residues.

    PubMed Central

    Osheroff, N; Brautigan, D L; Margoliash, E

    1980-01-01

    The carbonate binding site on horse cytochrome c was mapped by comparing the yields of carboxydinitrophenyl-cytochromes c, each with a single carboxydinitrophenyl-substituted lysine residue per molecule, when the modification reaction was carried out in the presence and absence of carbonate. The site is located on the "left surface" of the protein and consists of lysine residues 72 and/or 73 as well as 86 and/or 87 (Carbonate Site). Although one of the binding sites for phosphate on cytochrome c (Phosphat Site I) is located near the carbonate site, the sites are distinctly different since carbonate does not displace bound phosphate, as monitored by 31P NMR. Furthermore, citrate interacts with Phosphate Site I with high affinity, whereas chloride, acetate, borate, and cacodylate have a much lower affinity for this site, if they bind to it at all. The affinity of phosphate for Phosphate Site I (KD = 2 X 10(-4) M) is at least 1 order of magnitude higher than it is for other sites of interaction. Images PMID:6254024

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

    PubMed

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

    2016-03-27

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

  1. Discovery of Fur binding site clusters in Escherichia coli by information theory models

    PubMed Central

    Chen, Zehua; Lewis, Karen A.; Shultzaberger, Ryan K.; Lyakhov, Ilya G.; Zheng, Ming; Doan, Bernard; Storz, Gisela; Schneider, Thomas D.

    2007-01-01

    Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints (∼83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur–DNA recognition mechanism may be conserved for even distantly related bacteria. PMID:17921503

  2. 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.

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

    SciTech Connect

    Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.; Mantyh, P.W. )

    1988-09-01

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

  4. Photoaffinity crosslinking of etorphine with opioid binding sites in the bovine adrenal medulla

    SciTech Connect

    Cantau, P.; Bourhim, N.; Giraud, P.; Oliver, C.; Castanas, E.

    1987-04-01

    The covalent crosslinking of (/sup 3/H)etorphine with opioid binding sites in the bovine adrenal medulla is reported. Of all the radiolabeled opiates tested (ethylketocyclazocine, etorphine, (D-Ala2, D-Leu5)enkephalin, (D-Ala2, Me-Phe4, Gly5-ol)enkephalin only etorphine could be crosslinked under uv irradiation. In our conditions (black uv lamp, 160 W, peak mean 360 nm, from a distance of 10 cm) maximum covalent binding was observed after a 10-min irradiation. Protein concentration was a crucial factor for the irreversible/total binding ratio. A good ratio (50%) was obtained at protein concentrations of about 1.0 mg/ml. Covalent binding of nonmodified opiates could be of interest for the biochemical characterization of their binding sites.

  5. Dual function of a nuclear factor I binding site in MMTV transcription regulation.

    PubMed Central

    Buetti, E; Kühnel, B; Diggelmann, H

    1989-01-01

    Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element. Images PMID:2542892

  6. High and low affinity binding sites for endothelin on cultured rat glomerular mesangial cells.

    PubMed

    Badr, K F; Munger, K A; Sugiura, M; Snajdar, R M; Schwartzberg, M; Inagami, T

    1989-06-15

    Endothelin contracts glomerular mesangial cells, thereby influencing glomerular size and filtration rate. Here, we demonstrate the presence of two ET-specific binding sites on cultured rat mesangial cells with Kds of 0.76 and 44.70 nM, and maximal binding capacity (Bmax) values of 6.78 x 10(2) and 27.60 x 10(2) binding sites/cell, respectively. Binding of [125I]-ET was maximal at 120 min at 4 degrees C, stable for the subsequent 60 min, and selective. No competition for binding was observed with greater than 1000-fold concentrations of atrial natriuretic peptide, angiotensin II, arginine vasopressin, nicardipine, or nifedipine. The presence of specific receptors for ET on glomerular mesangial cells suggests a major role for this peptide in the regulation of glomerular filtration rate.

  7. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.

    PubMed

    Wang, Liangyan; Lu, Huizhi; Wang, Yunguang; Yang, Su; Xu, Hong; Cheng, Kaiying; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2017-03-01

    Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

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

    SciTech Connect

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

    1987-04-01

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

  9. The effect of saturation of ACE binding sites on the pharmacokinetics of enalaprilat in man.

    PubMed Central

    Wade, J R; Meredith, P A; Hughes, D M; Elliott, H L

    1992-01-01

    1. Eight healthy male volunteers received oral enalapril, 10 mg, in the presence and absence of pretreatment with captopril, 50 mg, twice daily for 5 days. 2. Enalaprilat pharmacokinetics were characterised after both doses of enalapril to investigate the effect of saturating ACE binding sites by pretreatment with captopril. 3. The pharmacokinetics of enalaprilat were best described by a one compartment model with zero order input incorporating saturable binding to plasma and tissue ACE. 4. Values of AUC (0.72 h) for enalaprilat were 419 +/- 97 and 450 +/- 87 ng ml-1 h in the presence and absence of captopril, respectively. The difference was not statistically significant nor were there any other differences in model parameters. 5. Induction of ACE by captopril resulting in an increase in the number of ACE binding sites, may have obscured any effect of captopril on the occupancy of ACE binding sites by enalapril. PMID:1312853

  10. Endometrial oxytocin binding sites in normal women and in subfertile patients.

    PubMed Central

    Baker, P. N.; Peat, M. L.; Symonds, E. M.; Maynard, P. V.

    1990-01-01

    Specific binding of oxytocin to high affinity sites in endometrial membrane preparations has previously been shown in sheep. Endometrial tissue preparations from 27 'normal' women of proven fertility were incubated with tritiated oxytocin and the existence of significant binding sites in human endometrium was shown. Furthermore, the level of binding sites underwent a cyclical variation with the highest concentration of binding at midcycle. A cyclical pattern of binding site concentration not unlike that found in the normal women was shown in 19 subfertile patients taking clomiphene. However, in 20 subfertile patients not taking clomiphene, no cyclical pattern emerged with significantly lower levels of binding sites in the mid-portion of the cycle (P less than 0.02) and significantly higher levels in the mid-late luteal phase (P less than 0.01), as compared to the normal women. In the mid-portion of the cycle levels were significantly lower in the subfertile patients not taking clomiphene (P less than 0.03) as compared to those taking clomiphene. No significant differences were shown between the normal women and those patients taking clomiphene. PMID:2362885

  11. 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.

  12. Agonist and antagonist protect sulfhydrals in the binding site of the D-1 dopamine receptor

    SciTech Connect

    Sidhu, A.; Kebabian, J.W.; Fishman, P.H.

    1986-05-01

    An iodinated compound (/sup 125/I)-SCH 23982 (8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol) has been characterized as a specific, high affinity (Kd = 0.7 nM) ligand for the D-1 dopamine receptor. The ligand binding site of the D-1 receptor in rat striatum was inactivated by N-ethylmaleimide (NEM) in a time and concentration dependent manner. The inactivation was rapi