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Sample records for ligand binding site

  1. 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

  2. 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. PMID:23762824

  3. 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.

  4. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    PubMed

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function. PMID:26064949

  5. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites

    PubMed Central

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where “nonspecific” interactions contribute to biological function. PMID:26064949

  6. Ligand electronic properties modulate tau filament binding site density

    PubMed Central

    Cisek, Katryna; Jensen, Jordan R.; Honson, Nicolette S.; Schafer, Kelsey N.; Cooper, Grace L.; Kuret, Jeff

    2012-01-01

    Small molecules that bind tau-bearing neurofibrillary lesions are being sought for premortem diagnosis, staging, and treatment of Alzheimer’s disease and other tauopathic neurodegenerative diseases. The utility of these agents will depend on both their binding affinity and binding site density (Bmax). Previously we identified polarizability as a descriptor of protein aggregate binding affinity. To examine its contribution to binding site density, we investigated the ability of two closely related benzothiazole derivatives ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) and ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole)) that differed in polarizability to displace probes of high (Thioflavin S) and low (radiolabeled (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene; IMSB) density sites. Consistent with their site densities, Thioflavin S completely displaced radiolabeled IMSB, but IMSB was incapable of displacing Thioflavin S. Although both benzothiazoles displaced the low Bmax IMSB probe, only the highly polarizable analog displaced near saturating concentrations of the Thioflavin S probe. Quantum calculations showed that high polarizability reflected extensive pi-electron delocalization fostered by the presence of electron donating and accepting groups. These data suggest that electron delocalization promotes ligand binding at a subset of sites on tau aggregates that are present at high density, and that optimizing this aspect of ligand structure can yield tau-directed agents with superior diagnostic and therapeutic performance. PMID:23072817

  7. Organic solvents identify specific ligand binding sites on protein surfaces.

    PubMed

    Liepinsh, E; Otting, G

    1997-03-01

    Enzymes frequently recognize substrates and pharmaceutical drugs through specific binding interactions in deep pockets on the protein surface. We show how the specificity-determining substrate binding site of hen egg-white lysozyme (HEWL) can be readily identified in aqueous solution by nuclear magnetic resonance spectroscopy using small organic solvent molecules as detection probes. Exchange of magnetization between the 1H nuclei of the protein and the ligands through dipole-dipole interactions is observed which allows the modeling of their position and orientation at the binding site. Combined with site-specific binding constants measured by titration experiments with different organic solvents, the method can provide important information for rational drug design. In addition, the lifetime of nonspecific interactions of HEWL with organic solvents is shown to be in the sub-nanosecond time range. PMID:9062927

  8. Rosetta and the Design of Ligand Binding Sites.

    PubMed

    Moretti, Rocco; Bender, Brian J; Allison, Brittany; Meiler, Jens

    2016-01-01

    Proteins that bind small molecules (ligands) can be used as biosensors, signal modulators, and sequestering agents. When naturally occurring proteins for a particular target ligand are not available, artificial proteins can be computationally designed. We present a protocol based on RosettaLigand to redesign an existing protein pocket to bind a target ligand. Starting with a protein structure and the structure of the ligand, Rosetta can optimize both the placement of the ligand in the pocket and the identity and conformation of the surrounding sidechains, yielding proteins that bind the target compound.

  9. Rosetta and the Design of Ligand Binding Sites.

    PubMed

    Moretti, Rocco; Bender, Brian J; Allison, Brittany; Meiler, Jens

    2016-01-01

    Proteins that bind small molecules (ligands) can be used as biosensors, signal modulators, and sequestering agents. When naturally occurring proteins for a particular target ligand are not available, artificial proteins can be computationally designed. We present a protocol based on RosettaLigand to redesign an existing protein pocket to bind a target ligand. Starting with a protein structure and the structure of the ligand, Rosetta can optimize both the placement of the ligand in the pocket and the identity and conformation of the surrounding sidechains, yielding proteins that bind the target compound. PMID:27094285

  10. 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

  11. Prediction of ligand-binding sites of proteins by molecular docking calculation for a random ligand library.

    PubMed

    Fukunishi, Yoshifumi; Nakamura, Haruki

    2011-01-01

    A new approach to predicting the ligand-binding sites of proteins was developed, using protein-ligand docking computation. In this method, many compounds in a random library are docked onto the whole protein surface. We assumed that the true ligand-binding site would exhibit stronger affinity to the compounds in the random library than the other sites, even if the random library did not include the ligand corresponding to the true binding site. We also assumed that the affinity of the true ligand-binding site would be correlated to the docking scores of the compounds in the random library, if the ligand-binding site was correctly predicted. We call this method the molecular-docking binding-site finding (MolSite) method. The MolSite method was applied to 89 known protein-ligand complex structures extracted from the Protein Data Bank, and it predicted the correct binding sites with about 80-99% accuracy, when only the single top-ranked site was adopted. In addition, the average docking score was weakly correlated to the experimental protein-ligand binding free energy, with a correlation coefficient of 0.44.

  12. ProBiS-ligands: a web server for prediction of ligands by examination of protein binding sites

    PubMed Central

    Konc, Janez; Janežič, Dušanka

    2014-01-01

    The ProBiS-ligands web server predicts binding of ligands to a protein structure. Starting with a protein structure or binding site, ProBiS-ligands first identifies template proteins in the Protein Data Bank that share similar binding sites. Based on the superimpositions of the query protein and the similar binding sites found, the server then transposes the ligand structures from those sites to the query protein. Such ligand prediction supports many activities, e.g. drug repurposing. The ProBiS-ligands web server, an extension of the ProBiS web server, is open and free to all users at http://probis.cmm.ki.si/ligands. PMID:24861616

  13. Blind prediction of charged ligand binding affinities in a model binding site

    PubMed Central

    Rocklin, Gabriel J.; Boyce, Sarah E.; Fischer, Marcus; Fish, Inbar; Mobley, David L.; Shoichet, Brian K.; Dill, Ken A.

    2013-01-01

    Predicting absolute protein-ligand binding affinities remains a frontier challenge in ligand discovery and design. This becomes more difficult when ionic interactions are involved, because of the large opposing solvation and electrostatic attraction energies. In a blind test, we examined whether alchemical free energy calculations could predict binding affinities of 14 charged and 5 neutral compounds previously untested as ligands for a cavity binding site in Cytochrome C Peroxidase. In this simplified site, polar and cationic ligands compete with solvent to interact with a buried aspartate. Predictions were tested by calorimetry, spectroscopy, and crystallography. Of the 15 compounds predicted to bind, 13 were experimentally confirmed, while four compounds were false negative predictions. Predictions had an RMSE of 1.95 kcal/mol to the experimental affinities, and predicted poses had an average RMSD of 1.7 Å to the crystallographic poses. This test serves as a benchmark for these thermodynamically rigorous calculations at predicting binding affinities for charged compounds, and gives insights into the existing sources of error, which are primarily electrostatic interactions inside proteins. Our experiments also provide a useful set of ionic binding affinities in a simplified system for testing new affinity prediction methods. PMID:23896298

  14. Proteus and the Design of Ligand Binding Sites.

    PubMed

    Polydorides, Savvas; Michael, Eleni; Mignon, David; Druart, Karen; Archontis, Georgios; Simonson, Thomas

    2016-01-01

    This chapter describes the organization and use of Proteus, a multitool computational suite for the optimization of protein and ligand conformations and sequences, and the calculation of pK α shifts and relative binding affinities. The software offers the use of several molecular mechanics force fields and solvent models, including two generalized Born variants, and a large range of scoring functions, which can combine protein stability, ligand affinity, and ligand specificity terms, for positive and negative design. We present in detail the steps for structure preparation, system setup, construction of the interaction energy matrix, protein sequence and structure optimizations, pK α calculations, and ligand titration calculations. We discuss illustrative examples, including the chemical/structural optimization of a complex between the MHC class II protein HLA-DQ8 and the vinculin epitope, and the chemical optimization of the compstatin analog Ac-Val4Trp/His9Ala, which regulates the function of protein C3 of the complement system. PMID:27094287

  15. 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. PMID:19754879

  16. 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.

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

    PubMed Central

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

    2014-01-01

    We present a method to identify small molecule ligand binding sites and orientations to a given protein crystal structure using GPU-accelerated Hamiltonian replica exchange molecular dynamics simulations. The Hamiltonians used vary from the physical end state of protein interacting with the ligand to a unphysical end state where the ligand does not interact with the protein. As replicas explore the space of Hamiltonians interpolating between these states the ligand can rapidly escape local minima and explore potential binding sites. Geometric restraints keep the ligands within the protein volume, and a potential energy pathway designed to increase phase space overlap between intermediates ensures good mixing. Because of the rigorous statistical mechanical nature of the Hamiltonian exchange framework, we can also extract binding free energy estimates at all putative binding sites, which agree well with free energies computed from occupation probabilities. We present results of this methodology on the T4 lysozyme L99A model system with four ligands, including one non-binder as a control. We find that our methodology identifies the crystallographic binding sites consistently and accurately for the small number of ligands considered here and gives free energies consistent with experiment. We are also able to analyze the contribution of individual binding sites on the overall binding affinity. Our methodology points to near term potential applications in early-stage drug discovery. PMID:24297454

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

    PubMed

    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.

  19. 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

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

    PubMed

    Joseph, Thomas T; Mincer, Joshua S

    2016-01-01

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

  1. The ligand binding site of the synaptosomal choline transporter: a provisional model based on inhibition studies.

    PubMed

    Roberts, E; Tamaru, M

    1992-05-01

    A topographic model of the ligand binding site of the choline transporter was deduced from inhibition studies with the help of CPK molecular models. It is posited that there are two identical or closely similar hydrophilic anionic sites separated from each other by an hinged, essentially planar but conformationally flexible cationic hydrophobic domain. Subsequently to attachment of external choline to either one of the anionic sites, both sites cooperate in enveloping the ligand by a Venus fly-trap mechanism. This leads to rapid configurational changes by which the closed-liganded form of the transporter opens up to the interior to release the bound choline. Intracellular K+, a ligand for the choline-binding site, is proposed to be counter-transported by a reversal of the above mechanism.

  2. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

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

    PubMed Central

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

    2008-01-01

    The T-cell immunoglobulin and mucin domain (TIM) proteins are important regulators of T cell responses. They have been linked to autoimmunity and cancer. Structures of the murine TIM-4 identified a Metal Ion-dependent Ligand Binding Site (MILIBS) in the immunoglobulin (Ig) domain of the TIM family. The characteristic CC’ loop of the TIM domain and the hydrophobic FG loop shaped a narrow cavity where acidic compounds penetrate and coordinate to a metal ion bound to conserved residues in the TIM proteins. The structure of phosphatidylserine bound to the Ig domain showed that the hydrophilic head penetrates into the MILIBS and coordinates with the metal ion, while the aromatic residues on the tip of the FG loop interacted with the fatty acid chains and could insert into the lipid bilayer. Our results also revealed a significant role of the MILIBS in trafficking of TIM-1 to the cell surface. PMID:18083575

  4. The good, the bad and the dubious: VHELIBS, a validation helper for ligands and binding sites

    PubMed Central

    2013-01-01

    Background Many Protein Data Bank (PDB) users assume that the deposited structural models are of high quality but forget that these models are derived from the interpretation of experimental data. The accuracy of atom coordinates is not homogeneous between models or throughout the same model. To avoid basing a research project on a flawed model, we present a tool for assessing the quality of ligands and binding sites in crystallographic models from the PDB. Results The Validation HElper for LIgands and Binding Sites (VHELIBS) is software that aims to ease the validation of binding site and ligand coordinates for non-crystallographers (i.e., users with little or no crystallography knowledge). Using a convenient graphical user interface, it allows one to check how ligand and binding site coordinates fit to the electron density map. VHELIBS can use models from either the PDB or the PDB_REDO databank of re-refined and re-built crystallographic models. The user can specify threshold values for a series of properties related to the fit of coordinates to electron density (Real Space R, Real Space Correlation Coefficient and average occupancy are used by default). VHELIBS will automatically classify residues and ligands as Good, Dubious or Bad based on the specified limits. The user is also able to visually check the quality of the fit of residues and ligands to the electron density map and reclassify them if needed. Conclusions VHELIBS allows inexperienced users to examine the binding site and the ligand coordinates in relation to the experimental data. This is an important step to evaluate models for their fitness for drug discovery purposes such as structure-based pharmacophore development and protein-ligand docking experiments. PMID:23895374

  5. Exploration of Gated Ligand Binding Recognizes an Allosteric Site for Blocking FABP4-Protein Interaction

    PubMed Central

    Li, Yan; Li, Xiang; Dong, Zigang

    2015-01-01

    Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level. PMID:26580122

  6. Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

    PubMed Central

    Liang, J.; Edelsbrunner, H.; Woodward, C.

    1998-01-01

    Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et

  7. Ligand-apomyoglobin interactions. Configurational adaptability of the haem-binding site.

    PubMed Central

    Lind, K E; Moller, J V

    1976-01-01

    1. The interaction of the haem-binding region of apomyoglobin with different ligands was examined by ultrafiltration, equilibrium dialysis and spectrophotometry, to study unspecific features of protein-ligand interactions such as they occur in, for example, serum albumin binding. 2. Apomyoglobin, in contrast with metmyoglobin, binds at pH 7, with a high affinity, one molecule of Bromophenol Blue, bilirubin and protoporphyrin IX, two molecules of n-dodecanoate and n-decyl sulphate and four molecules of n-dodecyl sulphate and n-tetradecyl sulphate. 3. The number of high-affinity sites and/or association constants for the alkyl sulphates are enhanced by an increase of hydrocarbon length, indicating hydrophobic interactions with the protein. 4. Measurements of the temperature-dependence of the association constants of the high-affinity sites imply that the binding processes are largely entropy-driven. 5. Binding studies in the presence of two ligands show that bilirubin plus Bromophenol Blue and dodecanoate plus Bromophenol Blue can be simultaneously bound by apomyoglobin, but with decreased affinities. By contrast, the apomyoglobin-protoporphyrin IX complex does not react with Bromophenol Blue. 6. Optical-rotatory-dispersion measurements show that the laevorotation of apomyoglobin is increased towards that of metmyglobin in the presence of haemin and protoporphyrin IX. Small changes in the optical-rotatory-dispersion spectrum of apomyoglobin are observed in the presence of the other ligands. 7. It is concluded that the binding sites on apomyoglobin probably do not pre-exist but appear to be moulded from predominantly non-polar amino acid residues by reaction with hydrophobic ligands. 8. Comparison with data in the literature indicates that apomyoglobin on a weight basis has a larger hydrophobic area avaialble for binding of ligands than has human serum albumin. On the other hand, the association constants of serum for the ligands used in this study are generally

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

    PubMed

    Tsujikawa, Hiroto; Sato, Kenta; Wei, Cao; Saad, Gul; Sumikoshi, Kazuya; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

    2016-09-01

    We present a new method for predicting protein-ligand-binding sites based on protein three-dimensional structure and amino acid conservation. This method involves calculation of the van der Waals interaction energy between a protein and many probes placed on the protein surface and subsequent clustering of the probes with low interaction energies to identify the most energetically favorable locus. In addition, it uses amino acid conservation among homologous proteins. Ligand-binding sites were predicted by combining the interaction energy and the amino acid conservation score. The performance of our prediction method was evaluated using a non-redundant dataset of 348 ligand-bound and ligand-unbound protein structure pairs, constructed by filtering entries in a ligand-binding site structure database, LigASite. Ligand-bound structure prediction (bound prediction) indicated that 74.0 % of predicted ligand-binding sites overlapped with real ligand-binding sites by over 25 % of their volume. Ligand-unbound structure prediction (unbound prediction) indicated that 73.9 % of predicted ligand-binding residues overlapped with real ligand-binding residues. The amino acid conservation score improved the average prediction accuracy by 17.0 and 17.6 points for the bound and unbound predictions, respectively. These results demonstrate the effectiveness of the combined use of the interaction energy and amino acid conservation in the ligand-binding site prediction. PMID:27400687

  9. Identification of Ligand Binding Sites of Proteins Using the Gaussian Network Model

    PubMed Central

    Tuzmen, Ceren; Erman, Burak

    2011-01-01

    The nonlocal nature of the protein-ligand binding problem is investigated via the Gaussian Network Model with which the residues lying along interaction pathways in a protein and the residues at the binding site are predicted. The predictions of the binding site residues are verified by using several benchmark systems where the topology of the unbound protein and the bound protein-ligand complex are known. Predictions are made on the unbound protein. Agreement of results with the bound complexes indicates that the information for binding resides in the unbound protein. Cliques that consist of three or more residues that are far apart along the primary structure but are in contact in the folded structure are shown to be important determinants of the binding problem. Comparison with known structures shows that the predictive capability of the method is significant. PMID:21283550

  10. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

  11. In silico Identification and Characterization of Protein-Ligand Binding Sites.

    PubMed

    Roche, Daniel Barry; McGuffin, Liam James

    2016-01-01

    Protein-ligand binding site prediction methods aim to predict, from amino acid sequence, protein-ligand interactions, putative ligands, and ligand binding site residues using either sequence information, structural information, or a combination of both. In silico characterization of protein-ligand interactions has become extremely important to help determine a protein's functionality, as in vivo-based functional elucidation is unable to keep pace with the current growth of sequence databases. Additionally, in vitro biochemical functional elucidation is time-consuming, costly, and may not be feasible for large-scale analysis, such as drug discovery. Thus, in silico prediction of protein-ligand interactions must be utilized to aid in functional elucidation. Here, we briefly discuss protein function prediction, prediction of protein-ligand interactions, the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated EvaluatiOn (CAMEO) competitions, along with their role in shaping the field. We also discuss, in detail, our cutting-edge web-server method, FunFOLD for the structurally informed prediction of protein-ligand interactions. Furthermore, we provide a step-by-step guide on using the FunFOLD web server and FunFOLD3 downloadable application, along with some real world examples, where the FunFOLD methods have been used to aid functional elucidation. PMID:27094282

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

    PubMed

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

    2009-12-01

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

  13. Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

    PubMed Central

    Sael, Lee; Kihara, Daisuke

    2012-01-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

  14. Electrostatic coupling to pH-titrating sites as a source of cooperativity in protein-ligand binding.

    PubMed Central

    Spassov, V.; Bashford, D.

    1998-01-01

    This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand-binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin D9k, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain. PMID:9761483

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution. PMID:26561004

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  18. Benzene Probes in Molecular Dynamics Simulations Reveal Novel Binding Sites for Ligand Design.

    PubMed

    Tan, Yaw Sing; Reeks, Judith; Brown, Christopher J; Thean, Dawn; Ferrer Gago, Fernando Jose; Yuen, Tsz Ying; Goh, Eunice Tze Leng; Lee, Xue Er Cheryl; Jennings, Claire E; Joseph, Thomas L; Lakshminarayanan, Rajamani; Lane, David P; Noble, Martin E M; Verma, Chandra S

    2016-09-01

    Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design. PMID:27532490

  19. A search for site-filling ligands in the Mcg Bence-Jones dimer: crystal binding studies of fluorescent compounds.

    PubMed

    Edmundson, A B; Ely, K R; Herron, J N

    1984-07-01

    In trigonal crystals grown in 1.9 M ammonium sulfate buffered at pH 6.2, the Mcg light-chain (Bence-Jones) dimer has a highly aromatic binding cavity accessible to a wide range of hydrophobic and aromatic ligands. A search was made for site-filling ligands by diffusing compounds into the crystals and determining their locations, orientations and relative occupancies by difference Fourier analysis at 2.7-A resolution. 1-Anilinonaphthalene-8-sulfonate, a small ligand in comparison with the rest of the series, initially occupied a site in the main binding cavity. With time, however, this ligand changed its position to the deep binding pocket beyond the floor of the main cavity. The original binding site remained vacant, despite the presence of a large excess of ligand in the soaking solution. Ligands increasing in size from fluorescein to bis(N-methyl)acridine (lucigenin) to dimers of carboxytetramethylrhodamine were found to bind with stringent stereospecificity in the main cavity, but the mode of binding was different in each case. The dimer of the 6-isomer of carboxytetramethylrhodamine, in which the two carboxyl groups are in para positions on the phenyl moiety, proved to be an effective site-filling ligand. The differences in the binding properties of dimers of 5- and 6-carboxytetramethylrhodamine led to an explanation for isomeric discrimination in the binding site. There were extensive conformational changes in the binding cavity to accommodate the ligands, particularly 6-carboxytetramethylrhodamine. The second and third hypervariable loops proved very flexible, and moved in ways to expand the binding site. The side chains of key tyrosine and phenylalanine residues in the site were also highly mobile. Their orientations adjusted to optimize complementarity with the ligands. These conformational adjustments are consistent with the tenets of a limited neo-instructive theory of ligand binding.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-01-01

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein–ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein–ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein–ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems. PMID:26694353

  2. Allosteric modulation of ligand binding to [3H](+)pentazocine-defined sigma recognition sites by phenytoin.

    PubMed

    DeHaven-Hudkins, D L; Ford-Rice, F Y; Allen, J T; Hudkins, R L

    1993-01-01

    The allosteric modulation of sigma recognition sites by phenytoin (diphenylhydantoin) has been demonstrated by the ability of phenytoin to stimulate binding of various [3H] sigma ligands, as well as to slow dissociation from sigma sites and to shift sigma sites from a low- to a high-affinity state. Phenytoin stimulated the binding of the sigma 1- selective ligand [3H](+)pentazocine in a dose-dependent manner. Stimulation of binding at a final concentration of 250 microM phenytoin was associated with a decrease in the KD. The affinities of the sigma reference compounds caramiphen, dextromethorphan, dextrophan, (+)3-PPP and (+)SKF-10,047 were three- to eight-fold higher, while the affinities of benzetimide, BMY-14802, carbetapentane, DTG and haloperidol were unchanged in the presence of 250 microM phenytoin. The relative sensitivity of sigma compounds to allosteric modulation by phenytoin is not a property of all sigma ligands, and may provide an in vitro basis for distinguishing actions of sigma compounds and predicting sigma effects in vivo. PMID:8515681

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

    PubMed

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-12-15

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

  4. eFindSite: Enhanced Fingerprint-Based Virtual Screening Against Predicted Ligand Binding Sites in Protein Models.

    PubMed

    Feinstein, Wei P; Brylinski, Michal

    2014-02-01

    A standard practice for lead identification in drug discovery is ligand virtual screening, which utilizes computing technologies to detect small compounds that likely bind to target proteins prior to experimental screens. A high accuracy is often achieved when the target protein has a resolved crystal structure; however, using protein models still renders significant challenges. Towards this goal, we recently developed eFindSite that predicts ligand binding sites using a collection of effective algorithms, including meta-threading, machine learning and reliable confidence estimation systems. Here, we incorporate fingerprint-based virtual screening capabilities in eFindSite in addition to its flagship role as a ligand binding pocket predictor. Virtual screening benchmarks using the enhanced Directory of Useful Decoys demonstrate that eFindSite significantly outperforms AutoDock Vina as assessed by several evaluation metrics. Importantly, this holds true regardless of the quality of target protein structures. As a first genome-wide application of eFindSite, we conduct large-scale virtual screening of the entire proteome of Escherichia coli with encouraging results. In the new approach to fingerprint-based virtual screening using remote protein homology, eFindSite demonstrates its compelling proficiency offering a high ranking accuracy and low susceptibility to target structure deformations. The enhanced version of eFindSite is freely available to the academic community at http://www.brylinski.org/efindsite. PMID:27485570

  5. Characterization of a Ligand Binding Site in the Human Transient Receptor Potential Ankyrin 1 Pore

    PubMed Central

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

    2013-01-01

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule. PMID:23442958

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

    PubMed

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

    2013-02-19

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

  7. Ligand binding and proton exchange dynamics in site-specific mutants of human myoglobin

    SciTech Connect

    Lambright, D.G.

    1992-01-01

    Site specific mutagenesis was used to make substitutions of four residues in the distal heme pocket of human myoglobin: Val68, His64, Lys45, and Asp60. Strongly diffracting crystals of the conservative mutation K45R in the met aquo form were grown in the trigonal space group P3[sub 2]21 and the X-ray crystal structure determined at 1.6 [angstrom] resolution. The overall structure is similar to that of sperm whale met aquo myoglobin. Several of the mutant proteins were characterized by 2-D NMR spectroscopy. The NMR data suggest the structural changes are localized to the region of the mutation. The dynamics of ligand binding to myoglobin mutants were studied by transient absorption spectroscopy following photolysis of the CO complexes. Transient absorption kinetics and spectra on the ns to ms timescale were measured in aqueous solution from 280 K to 310 K and in 75% glycerol: water from 250 K to 310 K. Two significant basis spectra were obtained from singular value decomposition of the matrix of time dependent spectra. The information was used to obtain approximations for the extent of ligand rebinding and the kinetics of conformational relaxation. Except for K45R, substitutions at Lys45 or Asp60 produce changes in the kinetics for ligand rebinding. Replacement of Lys45 with Arg increases the rate of ligand rebinding from the protein matrix by a factor of 2, but does not alter the rates for ligand escape or entry into the protein or the dynamics of the conformational relaxation. Substitutions at His64 and Val68 influence the kinetics of ligand rebinding and the dynamics of conformational relaxation. The results do not support the hypothesis that ligand migration between the heme pocket and solvent is determined solely by fluctuations of Arg45 and His64 between open and closed conformations of the heme pocket but can be rationalized if ligand diffusion through the protein matrix involves multiple competing pathways.

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

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation. PMID:26506102

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

    PubMed Central

    Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Classification and comparison of ligand-binding sites derived from grid-mapped knowledge-based potentials.

    PubMed

    Hoppe, Christian; Steinbeck, Christoph; Wohlfahrt, Gerd

    2006-03-01

    We describe the application of knowledge-based potentials implemented in the MOE program to compare the ligand-binding sites of several proteins. The binding probabilities for a polar and a hydrophobic probe are calculated on a grid to allow easy comparison of binding sites of superimposed related proteins. The method is fast and simple enough to simultaneously use structural information of multiple proteins of a target family. The method can be used to rapidly cluster proteins into subfamilies according to the similarity of hydrophobic and polar fields of their ligand-binding sites. Regions of the binding site which are common within a protein family can be identified and analysed for the design of family-targeted libraries or those which differ for improvement of ligand selectivity. The field-based hierarchical clustering is demonstrated for three protein families: the ligand-binding domains of nuclear receptors, the ATP-binding sites of protein kinases and the substrate binding sites of proteases. More detailed comparisons are presented for serine proteases of the chymotrypsin family, for the peroxisome proliferator-activated receptor subfamily of nuclear receptors and for progesterone and androgen receptor. The results are in good accordance with structure-based analysis and highlight important differences of the binding sites, which have been also described in the literature.

  13. Modulating protein activity using tethered ligands with mutually exclusive binding sites

    PubMed Central

    Schena, Alberto; Griss, Rudolf; Johnsson, Kai

    2015-01-01

    The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors. PMID:26198003

  14. Binding site structure of one LRP-RAP complex: implications for a common ligand-receptor binding motif.

    PubMed

    Jensen, Gitte A; Andersen, Olav M; Bonvin, Alexandre M J J; Bjerrum-Bohr, Ida; Etzerodt, Michael; Thøgersen, Hans C; O'Shea, Charlotte; Poulsen, Flemming M; Kragelund, Birthe B

    2006-09-29

    The low-density lipoprotein receptor-related protein (LRP) interacts with more than 30 ligands of different sizes and structures that can all be replaced by the receptor-associated protein (RAP). The double module of complement type repeats, CR56, of LRP binds many ligands including all three domains of RAP and alpha2-macroglobulin, which promotes the catabolism of the Abeta-peptide implicated in Alzheimer's disease. To understand the receptor-ligand cross-talk, the NMR structure of CR56 has been solved and ligand binding experiments with RAP domain 1 (RAPd1) have been performed. From chemical shift perturbations of both binding partners upon complex formation, a HADDOCK model of the complex between CR56 and RAPd1 has been obtained. The binding residues are similar to a common binding motif suggested from alpha2-macroglobulin binding studies and provide evidence for an understanding of their mutual cross-competition pattern. The present structural results convey a simultaneous description of both binding partners of an LRP-ligand complex and open a route to a broader understanding of the binding specificity of the LRP receptor, which may involve a general four-residue receptor-ligand recognition motif common to all LRP ligands. The present result may be beneficial in the design of antagonists of ligand binding to the LDL receptor family, and especially of drugs for treatment of Alzheimer's disease.

  15. eMatchSite: Sequence Order-Independent Structure Alignments of Ligand Binding Pockets in Protein Models

    PubMed Central

    Brylinski, Michal

    2014-01-01

    Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4–9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite. PMID

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-31

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

  18. Investigation of the Copper Binding Site and the Role of Histidine as a Ligand in Riboflavin Binding Protein

    PubMed Central

    Smith, Sheila R.; Bencze, Krisztina Z.; Russ, Kristen A.; Wasiukanis, Kristen; Benore-Parsons, Marilee; Stemmler, Timothy L.

    2008-01-01

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 Å, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu–O3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit. PMID:18593109

  19. Observation of multiple, identical binding sites in the exchange of carboxylic acid ligands with CdS nanocrystals.

    PubMed

    Li, Xin; Nichols, Valerie M; Zhou, Dapeng; Lim, Cynthia; Pau, George Shu Heng; Bardeen, Christopher J; Tang, Ming L

    2014-06-11

    We study ligand exchange between the carboxylic acid group and 5.0 nm oleic-acid capped CdS nanocrystals (NCs) using fluorescence resonance energy transfer (FRET). This is the first measurement of the initial binding events between cadmium chalcogenide NCs and carboxylic acid groups. The binding behavior can be described as an interaction between a ligand with single binding group and a substrate with multiple, identical binding sites. Assuming Poissonian binding statistics, our model fits both steady-state and time-resolved photoluminescence (SSPL and TRPL, respectively) data well. A modified Langmuir isotherm reveals that a CdS nanoparticle has an average of 3.0 new carboxylic acid ligands and binding constant, Ka, of 3.4 × 10(5) M(-1).

  20. Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site.

    PubMed

    Alves, Nathan J; Champion, Matthew M; Stefanick, Jared F; Handlogten, Michael W; Moustakas, Demetri T; Shi, Yunhua; Shaw, Bryan F; Navari, Rudolph M; Kiziltepe, Tanyel; Bilgicer, Basar

    2013-07-01

    The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 μM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.

  1. Spatial analysis and quantification of the thermodynamic driving forces in protein-ligand binding: binding site variability.

    PubMed

    Raman, E Prabhu; MacKerell, Alexander D

    2015-02-25

    The thermodynamic driving forces behind small molecule-protein binding are still not well-understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provide an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both nonpolar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the "hydrophobic effect". Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. It is notable to have the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  2. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes. PMID:17168764

  3. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

  4. Label-free molecular beacon system based on DNAs containing abasic sites and fluorescent ligands that bind abasic sites.

    PubMed

    Sato, Yusuke; Nishizawa, Seiichi; Teramae, Norio

    2011-10-01

    A new class of label-free molecular beacon (MB) system based on DNA strands that contain abasic (AP) sites (AP-DNA) and adopt stem-loop structures, in combination with fluorescent ligands that bind these AP sites, has been developed. Unlike a conventional MB, which requires covalent labeling of the MB with a fluorophore and a quencher, the developed system (APMB) does not require covalent attachment of signal transduction units. Detailed sensing functions of a series of APMB systems were examined with the aid of the fluorescent ligand named ATMND to provide insight into the design strategy for APMB systems. The effects of the stem length and the position of the AP site in the stem moiety on the fluorescence response of the APMB system were examined. Genotyping of a G/C SNP of PCR amplification products was successfully demonstrated with the APMB system and blue-fluorescent ATMND as a ligand. The APMB system was further extended to a system that utilized green-fluorescent lumiflavin.

  5. ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

    PubMed

    Konc, Janez; Miller, Benjamin T; Štular, Tanja; Lešnik, Samo; Woodcock, H Lee; Brooks, Bernard R; Janežič, Dušanka

    2015-11-23

    Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

  6. Binding site and ligand flexibility revealed by high resolution crystal structures of GluK1 competitive antagonists

    PubMed Central

    Alushin, Gregory M.; Jane, David; Mayer, Mark L.

    2010-01-01

    The availability of crystal structures for the ligand binding domains of ionotropic glutamate receptors, combined with their key role in synaptic function in the normal and diseased brain, offers a unique selection of targets for pharmaceutical research compared to other drug targets for which the atomic structure of the ligand binding sites is not known. Currently only a few antagonist structures have been solved, and these reveal ligand specific conformational changes that hinder rational drug design. Here we report high resolution crystal structures for three kainate receptor GluK1 antagonist complexes which reveal new and unexpected modes of binding, highlighting the continued need for experimentally determined receptor-ligand complexes. PMID:20558186

  7. ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex

    PubMed Central

    Anand, Praveen; Nagarajan, Deepesh; Mukherjee, Sumanta; Chandra, Nagasuma

    2014-01-01

    Most physiological processes in living systems are fundamentally regulated by protein–ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are calculated by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide a ranked list of residues to the user in order to obtain loss-of-function mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/. PMID:25685322

  8. Automatic generation of bioinformatics tools for predicting protein–ligand binding sites

    PubMed Central

    Banno, Masaki; Ueki, Kokoro; Saad, Gul; Shimizu, Kentaro

    2016-01-01

    Motivation: Predictive tools that model protein–ligand binding on demand are needed to promote ligand research in an innovative drug-design environment. However, it takes considerable time and effort to develop predictive tools that can be applied to individual ligands. An automated production pipeline that can rapidly and efficiently develop user-friendly protein–ligand binding predictive tools would be useful. Results: We developed a system for automatically generating protein–ligand binding predictions. Implementation of this system in a pipeline of Semantic Web technique-based web tools will allow users to specify a ligand and receive the tool within 0.5–1 day. We demonstrated high prediction accuracy for three machine learning algorithms and eight ligands. Availability and implementation: The source code and web application are freely available for download at http://utprot.net. They are implemented in Python and supported on Linux. Contact: shimizu@bi.a.u-tokyo.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26545824

  9. 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

  10. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  11. An Accurate Method for Prediction of Protein-Ligand Binding Site on Protein Surface Using SVM and Statistical Depth Function

    PubMed Central

    Wang, Kui; Gao, Jianzhao; Shen, Shiyi; Tuszynski, Jack A.; Ruan, Jishou

    2013-01-01

    Since proteins carry out their functions through interactions with other molecules, accurately identifying the protein-ligand binding site plays an important role in protein functional annotation and rational drug discovery. In the past two decades, a lot of algorithms were present to predict the protein-ligand binding site. In this paper, we introduce statistical depth function to define negative samples and propose an SVM-based method which integrates sequence and structural information to predict binding site. The results show that the present method performs better than the existent ones. The accuracy, sensitivity, and specificity on training set are 77.55%, 56.15%, and 87.96%, respectively; on the independent test set, the accuracy, sensitivity, and specificity are 80.36%, 53.53%, and 92.38%, respectively. PMID:24195070

  12. Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET).

    PubMed

    Davydov, Dmitri R; Rumfeldt, Jessica A O; Sineva, Elena V; Fernando, Harshica; Davydova, Nadezhda Y; Halpert, James R

    2012-02-24

    The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.

  13. Computational design of an endo-1,4-[beta]-xylanase ligand binding site

    SciTech Connect

    Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie; Harp, Joel M.; Mizoue, Laura S.; Meiler, Jens

    2012-09-05

    The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-{beta}-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 {beta}-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the {beta}-xylanase proteins.

  14. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis

    PubMed Central

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  15. The utility of geometrical and chemical restraint information extracted from predicted ligand binding sites in protein structure refinement

    PubMed Central

    Brylinski, Michal; Lee, Seung Yup; Zhou, Hongyi

    2010-01-01

    Exhaustive exploration of molecular interactions at the level of complete proteomes requires efficient and reliable computational approaches to protein function inference. Ligand docking and ranking techniques show considerable promise in their ability to quantify the interactions between proteins and small molecules. Despite the advances in the development of docking approaches and scoring functions, the genome-wide application of many ligand docking/screening algorithms is limited by the quality of the binding sites in theoretical receptor models constructed by protein structure prediction. In this study, we describe a new template-based method for the local refinement of ligand-binding regions in protein models using remotely related templates identified by threading. We designed a Support Vector Regression (SVR) model that selects correct binding site geometries in a large ensemble of multiple receptor conformations. The SVR model employs several scoring functions that impose geometrical restraints on the Cα positions, account for the specific chemical environment within a binding site and optimize the interactions with putative ligands. The SVR score is well correlated with the RMSD from the native structure; in 47% (70%) of the cases, the Pearson’s correlation coefficient is >0.5 (>0.3). When applied to weakly homologous models, the average heavy atom, local RMSD from the native structure of the top-ranked (best of top five) binding site geometries is 3.1 Å (2.9 Å) for roughly half of the targets; this represents a 0.1 (0.3) Å average improvement over the original predicted structure. Focusing on the subset of strongly conserved residues, the average heavy atom RMSD is 2.6 Å (2.3 Å). Furthermore, we estimate the upper bound of template-based binding site refinement using only weakly related proteins to be ~2.6 Å RMSD. This value also corresponds to the plasticity of the ligand-binding regions in distant homologues. The Binding Site Refinement (BSR

  16. Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland.

    PubMed

    Abreu, P; Sugden, D

    1990-09-14

    Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study. PMID:2169739

  17. DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands.

    PubMed

    Abe, Yukiko S; Sasaki, Shigeki

    2016-02-15

    DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase-polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase-polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.

  18. Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle*

    PubMed Central

    Feinberg, Hadar; Rambaruth, Neela D. S.; Jégouzo, Sabine A. F.; Jacobsen, Kristian M.; Djurhuus, Rasmus; Poulsen, Thomas B.; Weis, William I.; Taylor, Maureen E.; Drickamer, Kurt

    2016-01-01

    The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages. PMID:27542410

  19. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

    PubMed Central

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S.; Olson, Arthur J.; Sanner, Michel F.

    2015-01-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that

  20. AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility.

    PubMed

    Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S; Olson, Arthur J; Sanner, Michel F

    2015-12-01

    Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR-AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 -a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 -a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down

  1. Characterization of the ligand binding site of the bovine IgA Fc receptor (bFc alpha R).

    PubMed

    Morton, H Craig; Pleass, Richard J; Woof, Jenny M; Brandtzaeg, Per

    2004-12-24

    Recently, we identified a bovine IgA Fc receptor (bFc alpha R), which shows high homology to the human myeloid Fc alpha R, CD89. IgA binding has previously been shown to depend on several specific residues located in the B-C and F-G loops of the membrane-distal extracellular domain 1 of CD89. To compare the ligand binding properties of these two Fc alpha Rs, we have mapped the IgA binding site of bFc alpha R. We show that, in common with CD89, Tyr-35 in the B-C loop is essential for IgA binding. However, in contrast to earlier observations on CD89, mutation of residues in the F-G loop did not significantly inhibit IgA binding.

  2. SMAP-WS: a parallel web service for structural proteome-wide ligand-binding site comparison.

    PubMed

    Ren, Jingyuan; Xie, Lei; Li, Wilfred W; Bourne, Philip E

    2010-07-01

    The proteome-wide characterization and analysis of protein ligand-binding sites and their interactions with ligands can provide pivotal information in understanding the structure, function and evolution of proteins and for designing safe and efficient therapeutics. The SMAP web service (SMAP-WS) meets this need through parallel computations designed for 3D ligand-binding site comparison and similarity searching on a structural proteome scale. SMAP-WS implements a shape descriptor (the Geometric Potential) that characterizes both local and global topological properties of the protein structure and which can be used to predict the likely ligand-binding pocket [Xie,L. and Bourne,P.E. (2007) A robust and efficient algorithm for the shape description of protein structures and its application in predicting ligand-binding sites. BMC bioinformatics, 8 (Suppl. 4.), S9.]. Subsequently a sequence order independent profile-profile alignment (SOIPPA) algorithm is used to detect and align similar pockets thereby finding protein functional and evolutionary relationships across fold space [Xie, L. and Bourne, P.E. (2008) Detecting evolutionary relationships across existing fold space, using sequence order-independent profile-profile alignments. Proc. Natl Acad. Sci. USA, 105, 5441-5446]. An extreme value distribution model estimates the statistical significance of the match [Xie, L., Xie, L. and Bourne, P.E. (2009) A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery. Bioinformatics, 25, i305-i312.]. These algorithms have been extensively benchmarked and shown to outperform most existing algorithms. Moreover, several predictions resulting from SMAP-WS have been validated experimentally. Thus far SMAP-WS has been applied to predict drug side effects, and to repurpose existing drugs for new indications. SMAP-WS provides both a user-friendly web interface and

  3. SMAP-WS: a parallel web service for structural proteome-wide ligand-binding site comparison.

    PubMed

    Ren, Jingyuan; Xie, Lei; Li, Wilfred W; Bourne, Philip E

    2010-07-01

    The proteome-wide characterization and analysis of protein ligand-binding sites and their interactions with ligands can provide pivotal information in understanding the structure, function and evolution of proteins and for designing safe and efficient therapeutics. The SMAP web service (SMAP-WS) meets this need through parallel computations designed for 3D ligand-binding site comparison and similarity searching on a structural proteome scale. SMAP-WS implements a shape descriptor (the Geometric Potential) that characterizes both local and global topological properties of the protein structure and which can be used to predict the likely ligand-binding pocket [Xie,L. and Bourne,P.E. (2007) A robust and efficient algorithm for the shape description of protein structures and its application in predicting ligand-binding sites. BMC bioinformatics, 8 (Suppl. 4.), S9.]. Subsequently a sequence order independent profile-profile alignment (SOIPPA) algorithm is used to detect and align similar pockets thereby finding protein functional and evolutionary relationships across fold space [Xie, L. and Bourne, P.E. (2008) Detecting evolutionary relationships across existing fold space, using sequence order-independent profile-profile alignments. Proc. Natl Acad. Sci. USA, 105, 5441-5446]. An extreme value distribution model estimates the statistical significance of the match [Xie, L., Xie, L. and Bourne, P.E. (2009) A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery. Bioinformatics, 25, i305-i312.]. These algorithms have been extensively benchmarked and shown to outperform most existing algorithms. Moreover, several predictions resulting from SMAP-WS have been validated experimentally. Thus far SMAP-WS has been applied to predict drug side effects, and to repurpose existing drugs for new indications. SMAP-WS provides both a user-friendly web interface and

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

    SciTech Connect

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan . E-mail: jonathan.whittaker@case.edu

    2006-08-18

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

  5. Validation of a computational docking methodology to identify the non-covalent binding site of ligands to DNA.

    PubMed

    Deligkaris, Christos; Ascone, Anthony Thomas; Sweeney, Kevin Joseph; Greene, Alan Jonathan Quentin

    2014-08-01

    Despite the biomedical consequences of carcinogen-DNA interactions and the potential of DNA as a drug target in medicinal chemistry, only a small number of studies have validated or used docking methods for the prediction of the physical binding of small molecules to DNA. Knowledge of the DNA-physically-bound ligand geometry can lead to the elucidation of the molecular-level mechanism of drugs as well as predicting the subsequent chemical interactions that lead to DNA damage from carcinogens. We sought to validate AutoDock 4.2, a docking method that includes a physics-based free energy function and a Lamarckian Genetic Algorithm, for the prediction of ligand geometries upon physical binding to DNA. We performed simulations by systematically changing the length of the search process for a comprehensive set of 32 ligand-DNA molecular systems with different physico-chemical properties, and we used a free-energy-based convergence criterion to terminate our simulations. For 11 out of 28 molecular systems for which convergence was achieved, the lowest binding free energy geometries were within 2 Å of the experimentally determined geometry. Considering all predicted sites with free energy changes within 20% of the lowest binding free energy site, we found a site within 2 Å of the experimentally determined geometry for 24 out of the 28 systems. However, the predicted hydrogen bonding interactions were different for most molecular systems compared to the same interactions in the experimentally determined geometry. We discuss reasons for the successes and failures, implications, and the importance of ensuring an adequate search in docking calculations. Overall, we concluded that AutoDock 4.2 can be used to predict the non-covalent binding geometry of a small molecule to DNA with some limitations.

  6. Binding site multiplicity with fatty acid ligands: implications for the regulation of PKR kinase autophosphorylation with palmitate.

    PubMed

    Fang, Liang; Cho, Hyun Ju; Chan, Christina; Feig, Michael

    2014-10-01

    Saturated long chain-free fatty acids (FFAs), especially palmitate, have been implicated in apoptosis by inhibiting the activity of PKR (double-stranded RNA-dependent protein kinase). We recently found evidence that palmitate interacts directly with the kinase domain of PKR, subsequently inhibiting the autophosphorylation of PKR. To investigate the interactions of palmitate with PKR and its effects on PKR autophosphorylation, we performed extensive unbiased MD simulations combined with biochemical and biophysical experiments. The simulations predict multiple putative binding sites of palmitate on both the phosphorylated and unphosphorylated PKR with similar binding affinities. Ligand-protein interactions involving a large variety of different binding modes challenge the conventional view of highly specific, single binding sites. Key interactions of palmitate involve the αC-helix of PKR, especially near residue R307. Experimental mutation of R307 was found to affect palmitate binding and reduce its inhibitory effect. Based on this study a new allosteric mechanism is proposed where palmitate binding to the αC-helix prevents the inactive-to-active transition of PKR and subsequently reduces its ability to autophosphorylate.

  7. Binding hotspots on K-ras: consensus ligand binding sites and other reactive regions from probe-based molecular dynamics analysis.

    PubMed

    Prakash, Priyanka; Hancock, John F; Gorfe, Alemayehu A

    2015-05-01

    We have used probe-based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K-Ras G12D. Combining the probe-based query with an ensemble-based pocket identification scheme and an analysis of existing Ras-ligand complexes, we show that (i) pMD is a robust and cost-effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure-based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in nonpocket-like regions with known protein- and membrane-interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein-biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu.

  8. Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state.

    PubMed

    Huang, Li-Shar; Shen, John T; Wang, Andy C; Berry, Edward A

    2006-01-01

    Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.

  9. Crystallographic Studies of the Binding of Ligands to theDicarboxylate Site of Complex II, and the Identity of the Ligand in the'Oxaloacetate-Inhibited' State

    SciTech Connect

    Huang, Li-Shar; Shen, John T.; Wang, Andy C.; Berry, Edward A.

    2006-07-01

    Mitochondrial Complex II (succinate:ubiquinoneoxidoreductase) is purified in a partially innactivated state, which canbe activated by removal of tightly bound oxaloacetate (Kearney, E.B. etal. Biochem Biophys Res Commun 49, 1115-1121). We crystallized Complex IIin the presence of oxaloacetate or with the endogenous inhibitor bound.The structure showed a ligand essentially identical to the "malate-likeintermediate" found in Shewanella Flavocytochrome c crystallized withfumarate (Taylor, P., et al. Nat Struct Biol 6, 1108-1112.)Crystallization of Complex II in the presence of excess fumarate alsogave the malate-like intermediate or a mixture of that and fumarate atthe active site. In order to more conveniently monitor the occupationstate of the dicarboxylate site, we are developing a library of UV/Visspectral effects induced by binding different ligands to the site.Treatment with fumarate results in rapid development of the fumaratedifference spectrum and then a very slow conversion into a speciesspectrally similar to the OAA liganded complex. Complex II is known to becapable of oxidizing malate to the enol form of oxaloacetate (Belikova,Y.O., et al. Biochim Biophys Acta 936, 1-9). The observations abovesuggest it may also be capable of interconverting fumarate and malate. Itmay be useful for understanding the mechanism and regulation of theenzyme to identify the malate-like intermediate and its pathway offormation from oxaloacetate or fumarate.

  10. Design of a Fluorescent Ligand Targeting the S-adenosylmethioine Binding Site of the Histone Methyltransferase MLL1

    PubMed Central

    Luan, Yepeng; Blazer, Levi L.; Hu, Hao; Hajian, Taraneh; Zhang, Jing; Wu, Hong; Houliston, Scott; Arrowsmith, Cheryl H.; Vedadi, Masoud; Zheng, Yujun George

    2015-01-01

    The histone methyltransferase MLL1 has been linked to translocation-associated gene fusion in childhood leukemias and is an attractive drug target. High-throughput biochemical analysis of MLL1 methyltransferase activity requires the production of at least a trimeric complex of MLL1, RbBP5 and WDR5 to elicit robust activity. Production of trimeric and higher order MLL1 complexes in the quantities and reproducibility required for high-throughput screening presents a significant impediment to MLL1 drug discovery efforts. We present here a small molecule fluorescent ligand (FL-NAH, 6) that is able to bind to the S-adenosylmethionine (SAM) binding site of MLL1 in a manner independent of the associated complex members. We have used FL-NAH to develop a fluorescence polarization-based SAM displacement assay in a 384-well format targeting the MLL1 SET domain in the absence of associated complex members. FL-NAH competes with SAM and is displaced from the MLL1 SET domain by other SAM-binding site ligands with Kdisp values similar to the higher-order complexes, but is unaffected by the H3 peptide substrate. This assay enables screening for SAM-competitive MLL1 inhibitors without requiring the use of trimeric or higher order MLL1 complexes, significantly reducing screening time and cost. PMID:26541578

  11. The high affinity melationin binding site probed with conformationally restricted ligand--I. Pharmacophore and minireceptor models.

    PubMed

    Jansen, J M; Copinga, S; Gruppen, G; Molinari, E J; Dubocovich, M L; Grol, C J

    1996-08-01

    The affinities of enantiomers of conformationally restricted melatonin analogues for the ML-1 and ML-2 putative melatonin receptor subtypes are reported. Most ligands exhibited reversed stereoselectivity when competing with 125I 2-iodomelatonin binding to chicken retinal (ML-1) and hamster brain (ML-2) membranes, further supporting the biochemical and pharmacological differences reported for these two sites. Based on the data for the ML-1 site and thorough conformational analyses of several ligands, two pharmacophore models were derived using the program APOLLO. The pharmacophoric elements included were putative receptor points from the amide NH, the amide CO, and the methoxy-O, together with the normal through the phenyl ring. The large drop in ML-1 affinity observed for 4-methoxy-2-acetamido-indan (6a) could not be explained from either of these models. Minireceptors were subsequently built around the two pharmacophores using Yak. Analysis of the resulting ligand-minireceptor interactions offered an explanation for the low affinity of 6a and allowed one of the pharmacophore models to be selected for use in future drug design. PMID:8879554

  12. Improving the performance of the PLB index for ligand-binding site prediction using dihedral angles and the solvent-accessible surface area

    PubMed Central

    Cao, Chen; Xu, Shutan

    2016-01-01

    Protein ligand-binding site prediction is highly important for protein function determination and structure-based drug design. Over the past twenty years, dozens of computational methods have been developed to address this problem. Soga et al. identified ligand cavities based on the preferences of amino acids for the ligand-binding site (RA) and proposed the propensity for ligand binding (PLB) index to rank the cavities on the protein surface. However, we found that residues exhibit different RAs in response to changes in solvent exposure. Furthermore, previous studies have suggested that some dihedral angles of amino acids in specific regions of the Ramachandran plot are preferred at the functional sites of proteins. Based on these discoveries, the amino acid solvent-accessible surface area and dihedral angles were combined with the RA and PLB to obtain two new indexes, multi-factor RA (MF-RA) and multi-factor PLB (MF-PLB). MF-PLB, PLB and other methods were tested using two benchmark databases and two particular ligand-binding sites. The results show that MF-PLB can improve the success rate of PLB for both ligand-bound and ligand-unbound structures, particularly for top choice prediction. PMID:27619067

  13. Improving the performance of the PLB index for ligand-binding site prediction using dihedral angles and the solvent-accessible surface area.

    PubMed

    Cao, Chen; Xu, Shutan

    2016-01-01

    Protein ligand-binding site prediction is highly important for protein function determination and structure-based drug design. Over the past twenty years, dozens of computational methods have been developed to address this problem. Soga et al. identified ligand cavities based on the preferences of amino acids for the ligand-binding site (RA) and proposed the propensity for ligand binding (PLB) index to rank the cavities on the protein surface. However, we found that residues exhibit different RAs in response to changes in solvent exposure. Furthermore, previous studies have suggested that some dihedral angles of amino acids in specific regions of the Ramachandran plot are preferred at the functional sites of proteins. Based on these discoveries, the amino acid solvent-accessible surface area and dihedral angles were combined with the RA and PLB to obtain two new indexes, multi-factor RA (MF-RA) and multi-factor PLB (MF-PLB). MF-PLB, PLB and other methods were tested using two benchmark databases and two particular ligand-binding sites. The results show that MF-PLB can improve the success rate of PLB for both ligand-bound and ligand-unbound structures, particularly for top choice prediction. PMID:27619067

  14. Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

    PubMed Central

    de Nobel, H; Lipke, P N; Kurjan, J

    1996-01-01

    The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin. Images PMID:8741846

  15. Hydration properties of ligands and drugs in protein binding sites: tightly-bound, bridging water molecules and their effects and consequences on molecular design strategies.

    PubMed

    García-Sosa, Alfonso T

    2013-06-24

    Some water molecules in binding sites are important for intermolecular interactions and stability. The way binding site explicit water molecules are dealt with affects the diversity and nature of designed ligand chemical structures and properties. The strategies commonly employed frequently assume that a gain in binding affinity will be achieved by their targeting or neglect. However, in the present work, 2332 high-resolution X-ray crystal structures of hydrated and nonhydrated, drug and nondrug compounds in biomolecular complexes with reported Ki or Kd show that compounds that use tightly bound, bridging water molecules are as potent as those that do not. The distribution of their energies, physicochemical properties, and ligand efficiency indices were compared for statistical significance, and the results were confirmed using 2000 permutation runs. Ligand cases were also split into agonists and antagonists, and crystal structure pairs with differing tightly bound water molecules were also compared. In addition, agonists and antagonists that use tightly bound water bridges are smaller, less lipophilic, and less planar; have deeper ligand efficiency indices; and in general, possess better physicochemical properties for further development. Therefore, tightly bound, bridging water molecules may in some cases be replaced and targeted as a strategy, though sometimes keeping them as bridges may be better from a pharmacodynamic perspective. The results suggest general indications on tightly hydrated and nontightly hydrated compounds in binding sites and practical considerations to adopt a strategy in drug and molecular design when faced with this special type of water molecules. There are also benefits of lower log P and better developability for tightly hydrated compounds, while stronger potency is not always required or beneficial. The hydrated binding site may be one of the many structure conformations available to the receptor, and different ligands will have a

  16. 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

  17. Molecular simulations of β-lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites.

    PubMed

    Evoli, Stefania; Guzzi, Rita; Rizzuti, Bruno

    2014-10-01

    The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β-lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head-group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α-helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands.

  18. High throughput screening of high-affinity ligands for proteins with anion-binding sites using desorption electrospray ionization (DESI) mass spectrometry.

    PubMed

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  19. High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  20. A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery

    PubMed Central

    Xie, Lei; Xie, Li; Bourne, Philip E.

    2009-01-01

    Functional relationships between proteins that do not share global structure similarity can be established by detecting their ligand-binding-site similarity. For a large-scale comparison, it is critical to accurately and efficiently assess the statistical significance of this similarity. Here, we report an efficient statistical model that supports local sequence order independent ligand–binding-site similarity searching. Most existing statistical models only take into account the matching vertices between two sites that are defined by a fixed number of points. In reality, the boundary of the binding site is not known or is dependent on the bound ligand making these approaches limited. To address these shortcomings and to perform binding-site mapping on a genome-wide scale, we developed a sequence-order independent profile–profile alignment (SOIPPA) algorithm that is able to detect local similarity between unknown binding sites a priori. The SOIPPA scoring integrates geometric, evolutionary and physical information into a unified framework. However, this imposes a significant challenge in assessing the statistical significance of the similarity because the conventional probability model that is based on fixed-point matching cannot be applied. Here we find that scores for binding-site matching by SOIPPA follow an extreme value distribution (EVD). Benchmark studies show that the EVD model performs at least two-orders faster and is more accurate than the non-parametric statistical method in the previous SOIPPA version. Efficient statistical analysis makes it possible to apply SOIPPA to genome-based drug discovery. Consequently, we have applied the approach to the structural genome of Mycobacterium tuberculosis to construct a protein–ligand interaction network. The network reveals highly connected proteins, which represent suitable targets for promiscuous drugs. Contact: lxie@sdsc.edu PMID:19478004

  1. Structural insights into a novel interkingdom signaling circuit by cartography of the ligand-binding sites of the homologous quorum sensing LuxR-family.

    PubMed

    Covaceuszach, Sonia; Degrassi, Giuliano; Venturi, Vittorio; Lamba, Doriano

    2013-01-01

    Recent studies have identified a novel interkingdom signaling circuit, via plant signaling molecules, and a bacterial sub-family of LuxR proteins, bridging eukaryotes and prokaryotes. Indeed pivotal plant-bacteria interactions are regulated by the so called Plant Associated Bacteria (PAB) LuxR solo regulators that, although closely related to the quorum sensing (QS) LuxR family, do not bind or respond to canonical quorum sensing N-acyl homoserine lactones (AHLs), but only to specific host plant signal molecules. The large body of structural data available for several members of the QS LuxR family complexed with different classes of ligands (AHLs and other compounds), has been exploited to dissect the cartography of their regulatory domains through structure-based multiple sequence alignments, structural superimposition and a comparative analysis of the contact residues involved in ligand binding. In the absence of experimentally determined structures of members of the PAB LuxR solos subfamily, an homology model of its prototype OryR is presented, aiming to elucidate the architecture of its ligand-binding site. The obtained model, in combination with the cartography of the regulatory domains of the homologous QS LuxRs, provides novel insights into the 3D structure of its ligand-binding site and unveils the probable molecular determinants responsible for differences in selectivity towards specific host plant signal molecules, rather than to canonical QS compounds.

  2. Differential Effects of Methoxy Group on the Interaction of Curcuminoids with Two Major Ligand Binding Sites of Human Serum Albumin

    PubMed Central

    Sato, Hiroki; Chuang, Victor Tuan Giam; Yamasaki, Keishi; Yamaotsu, Noriyuki; Watanabe, Hiroshi; Nagumo, Kohei; Anraku, Makoto; Kadowaki, Daisuke; Ishima, Yu; Hirono, Shuichi; Otagiri, Masaki; Maruyama, Toru

    2014-01-01

    Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds. PMID:24498401

  3. Mechanism of ligand-protein interaction in plant seed thiamin-binding proteins. Probing the binding site of protein isolated from buckwheat seeds with a series of thiamin-related compounds.

    PubMed

    Rapała-Kozik, M; Kozik, A

    1992-09-23

    Affinities of 14 thiamin derivatives or antagonists to a thiamin-binding protein isolated from buckwheat seeds were determined. A competitive displacement of radiolabeled thiamin by unlabeled ligand was analysed by a computerized model-fitting procedure. The dissociation constant of the thiamin-protein complex was 0.93 microM. Most modifications in ligand chemical structure weakened the ligand-protein interaction. A model of the thiamin-binding site is suggested. The hydroxyethyl-chain of thiamin while protein-bound appears to be excluded from the binding region. A positively charged quaternary nitrogen atom of the thiazolium ring probably interacts with some negative group(s) of protein. The rest of the thiazolium ring as well as the amino group of the pyrimidine fragment serve as additional anchors. The three structural features of the thiamin molecule accounting for binding contribute equally to overall binding energy by about 11-12 kJ/mol.

  4. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  5. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.

  6. Characterization of the interaction constants for the binding of two unlabelled ligands to the sites of a receptor. An experimental strategy.

    PubMed

    Swillens, S; Gourdin, A; Delahaut, O

    1989-05-01

    The model describing the binding of three ligands to the sites of a receptor is theoretically studied in order to characterize the binding interaction between two unlabelled ligands. For this, the binding of a third labelled ligand, the affinity of which is dependent on the presence of the unlabelled ligands, must be measured in different conditions. This paper describes an experimental strategy leading to an accurate determination of the equilibrium parameters of the model. This strategy, which assumes that the model is compatible with the data, rests on the determination of the optimal experimental conditions, i.e. the choice of the ligand concentrations which minimize the error of the parameter estimates. For this purpose, a computer program which runs on a microcomputer under the MS-DOS operating system has been elaborated on the basis of the D-optimization criterion. This work shows that such a computer analysis is essential to the determination of the optimal experimental design, even when very simple biochemical systems are considered. PMID:2770883

  7. Designing ligands to bind proteins.

    PubMed

    Whitesides, George M; Krishnamurthy, Vijay M

    2005-11-01

    The ability to design drugs (so-called 'rational drug design') has been one of the long-term objectives of chemistry for 50 years. It is an exceptionally difficult problem, and many of its parts lie outside the expertise of chemistry. The much more limited problem - how to design tight-binding ligands (rational ligand design) - would seem to be one that chemistry could solve, but has also proved remarkably recalcitrant. The question is 'Why is it so difficult?' and the answer is 'We still don't entirely know'. This perspective discusses some of the technical issues - potential functions, protein plasticity, enthalpy/entropy compensation, and others - that contribute, and suggests areas where fundamental understanding of protein-ligand interactions falls short of what is needed. It surveys recent technological developments (in particular, isothermal titration calorimetry) that will, hopefully, make now the time for serious progress in this area. It concludes with the calorimetric examination of the association of a series of systematically varied ligands with a model protein. The counterintuitive thermodynamic results observed serve to illustrate that, even in relatively simple systems, understanding protein-ligand association is challenging.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-11-01

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

  10. Ligand Binding Site of Tear Lipocalin: Contribution of a Trigonal Cluster of Charged Residues Probed by 8-Anilino-1-naphthalenesulfonic Acid†

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2010-01-01

    Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster–sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous–lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film. PMID:18179255

  11. Changes in ligand binding to GABAA receptor sites in pacific salmon (Oncorhynchus) brain during spawning migration and "aging".

    PubMed

    Erdö, S L; Meyer, D L; Malz, C R; Hofmann, M H; Ebbesson, S O

    1992-01-01

    When several years old, pacific salmon return to the site of birth, to spawn. At this time, a rapid aging process begins and the fish die within a few weeks after reproducing. Age-related changes of high and low affinity GABA binding sites were studied in salmon brains at three different phases of the spawning migration, i.e. shortly after returning to the natal stream, at the time of spawning, and thereafter. High affinity GABA binding slightly increased while the fish deteriorated. The low affinity component showed a remarkable decrease in density and a concomitant increase in affinity during this final episode of salmon life.

  12. Porphyrin binding to jacalin is facilitated by the inherent plasticity of the carbohydrate-binding site: novel mode of lectin-ligand interaction.

    PubMed

    Goel, M; Anuradha, P; Kaur, K J; Maiya, B G; Swamy, M J; Salunke, D M

    2004-02-01

    The crystal structure of the complex of meso-tetrasulfonatophenylporphyrin (H(2)TPPS) with jack fruit (Artocarpus integriflora) agglutinin (jacalin) has been determined at 1.8 A resolution. A porphyrin pair is sandwiched between two symmetry-related jacalin monomers in the crystal, leading to a cross-linking network of protein molecules. Apart from the stacking interactions, H(2)TPPS also forms hydrogen bonds, some involving water bridges, with jacalin at the carbohydrate-binding site. The residues that are involved in rendering galactopyranoside specificity to jacalin undergo conformational adjustments in order to accommodate the H(2)TPPS molecule. The water molecules at the carbohydrate-binding site of jacalin cement the jacalin-porphyrin interactions, optimizing their complementarity. Interactions of porphyrin with jacalin are relatively weak compared with those observed between galactopyranoside and jacalin, perhaps because the former largely involves water-mediated hydrogen bonds. While H(2)TPPS binds to jacalin at the carbohydrate-binding site as in the case of ConA, its mode of interaction with jacalin is very different. H(2)TPPS does not enter the carbohydrate-binding cavity of jacalin. Instead, it sits over the binding site. While the porphyrin binding is mediated by replicating the hydrogen-bonding network of mannopyranoside through the sulfonate atoms in the case of ConA, the plasticity associated with the carbohydrate-binding site accommodates the pluripotent porphyrin molecule in the case of jacalin through an entirely different set of interactions.

  13. Langerin-heparin interaction: two binding sites for small and large ligands as revealed by a combination of NMR spectroscopy and cross-linking mapping experiments.

    PubMed

    Muñoz-García, Juan C; Chabrol, Eric; Vivès, Romain R; Thomas, Aline; de Paz, José L; Rojo, Javier; Imberty, Anne; Fieschi, Franck; Nieto, Pedro M; Angulo, Jesús

    2015-04-01

    Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns

  14. Architectural repertoire of ligand-binding pockets on protein surfaces.

    PubMed

    Weisel, Martin; Kriegl, Jan M; Schneider, Gisbert

    2010-03-01

    Knowledge of the three-dimensional structure of ligand binding sites in proteins provides valuable information for computer-assisted drug design. We present a method for the automated extraction and classification of ligand binding site topologies, in which protein surface cavities are represented as branched frameworks. The procedure employs a growing neural gas approach for pocket topology assignment and pocket framework generation. We assessed the structural diversity of 623 known ligand binding site topologies based on framework cluster analysis. At a resolution of 5 A only 23 structurally distinct topology groups were formed; this suggests an overall limited structural diversity of ligand-accommodating protein cavities. Higher resolution allowed for identification of protein-family specific pocket features. Pocket frameworks highlight potentially preferred modes of ligand-receptor interactions and will help facilitate the identification of druggable subpockets suitable for ligand affinity and selectivity optimization. PMID:20069621

  15. Calculation of Vibrational Shifts of Nitrile Probes in the Active Site of Ketosteroid Isomerase upon Ligand Binding

    PubMed Central

    Layfield, Joshua P.

    2012-01-01

    The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analog equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active site water molecule that is directly hydrogen bonded to the nitrile probe, resulting in a more linear CNH angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis. PMID:23210919

  16. 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

  17. Prediction of ligand binding site by insilico approach in cold resistant protein isolated from cold resistant mutant of Pseudomonas fluorescens.

    PubMed

    Kumar, Amit; Khan, Mahejibin

    2012-09-01

    Cold shock proteins perform vital functions, such as mRNA masking, coupling of transcription to translation and developmental timing and regulation, which aids in survival of microbes in cold stress. Pseudomonas fluorescens is an ecologically important bacterium which helps in plant growth promotion. Since the cold tolerant mutant of the bacterium is able to grow at the temperature ranges from 30 to 4°C, it is of interest to study the process of its survival in the extreme temperatures. Therefore, this study is focused on the three dimensional structure and molecular modeling of cold resistant protein (CRP) from P. fluorescens to predict its molecular mechanism. Investigating the structure of CRP confirmed the presence of a conserved domain characteristic of the cold shock domain (CSD) family and a single nucleotide binding domain. When 3D structure of CRP was compared with the existing cold shock proteins, major deviations were found in the loop regions connecting the β2-β3, β3-β4 and β4-β5 sheets. Docking studies showed that CRP forms a significant clamp like structure at the substrate binding cleft which stabilizes the ligand. Therefore, it can be concluded that CRP has a strong affinity for the poly thymidine (poly T) stretch and can be considered a candidate for transcription regulation. PMID:23099776

  18. Evidence for a Transient Additional Ligand Binding Site in the TAS2R46 Bitter Taste Receptor.

    PubMed

    Sandal, Massimo; Behrens, Maik; Brockhoff, Anne; Musiani, Francesco; Giorgetti, Alejandro; Carloni, Paolo; Meyerhof, Wolfgang

    2015-09-01

    Most human G protein coupled receptors (GPCRs) are activated by small molecules binding to their 7-transmembrane (7-TM) helix bundle. They belong to basally diverging branches: the 25 bitter taste 2 receptors and most members of the very large rhodopsin-like/class A GPCRs subfamily. Some members of the class A branch have been suggested to feature not only an orthosteric agonist-binding site but also a more extracellular or "vestibular" site, involved in the binding process. Here we use a hybrid molecular mechanics/coarse-grained (MM/CG) molecular dynamics approach on a widely studied bitter taste receptor (TAS2R46) receptor in complex with its agonist strychnine. Three ∼1 μs molecular simulation trajectories find two sites hosting the agonist, which together elucidate experimental data measured previously and in this work. This mechanism shares similarities with the one suggested for the evolutionarily distant class A GPCRs. It might be instrumental for the remarkably broad but specific spectrum of agonists of these chemosensory receptors.

  19. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

    PubMed Central

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

    1992-01-01

    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  20. Plasmon resonance enhanced mechanical detection of ligand binding

    SciTech Connect

    Ariyaratne, Amila; Zocchi, Giovanni

    2015-01-05

    Small molecule binding to the active site of enzymes typically modifies the mechanical stiffness of the enzyme. We exploit this effect, in a setup which combines nano-mechanics and surface plasmon resonance (SPR) enhanced optics, for the label free detection of ligand binding to an enzyme. The large dynamic range of the signal allows to easily obtain binding curves for small ligands, in contrast to traditional SPR methods which rely on small changes in index of refraction. Enzyme mechanics, assessed by nano-rheology, thus emerges as an alternative to electronic and spin resonances, assessed by traditional spectroscopies, for detecting ligand binding.

  1. FRET analysis using sperm-activating peptides tagged with fluorescent proteins reveals that ligand-binding sites exist as clusters.

    PubMed

    Arcos-Hernández, César; Romero, Francisco; Sánchez-Guevara, Yoloxochitl; Beltrán, Carmen; Nishigaki, Takuya

    2016-02-01

    Long-range cellular communication between the sperm and egg is critical for external fertilization. Sperm-activating peptides (SAPs) are diffusible components of the outer layer of eggs in echinoderms, and function as chemoattractants for spermatozoa. The decapeptide named speract is the best-characterized sea urchin SAP. Biochemical and physiological actions of speract have been studied with purified or chemically synthesized peptides. In this work, we prepared recombinant speract fused to a fluorescent protein (FP; FP-speract) using three color variants: a cyan (eCFP), a yellow (mVenus) and a large Stokes shift yellow (mAmetrine) FP. Although these fluorescence tags are 20 times larger than speract, competitive binding experiments using mAmetrine-speract revealed that this FP-speract has binding affinity to the receptor that is comparable (7.6-fold less) to that of non-labeled speract. Indeed, 10 nmol l(-1) eCFP-speract induces physiological sperm responses such as membrane potential changes and increases in intracellular pH and Ca(2+) concentrations similar to those triggered by 10 nmol l(-1) speract. Furthermore, FP-speract maintains its fluorescence upon binding to its receptor. Using this property, we performed fluorescence resonance energy transfer (FRET) measurements with eCFP-speract and mVenus-speract as probes and obtained a positive FRET signal upon binding to the receptor, which suggests that the speract receptor exists as an oligomer, at least as a dimer, or alternatively that a single speract receptor protein possesses multiple binding sites. This property could partially account for the positive and/or negative cooperative binding of speract to the receptor.

  2. Critical role of the H6-H7 loop in the conformational adaptation of all-trans retinoic acid and synthetic retinoids within the ligand-binding site of RARalpha.

    PubMed

    Mailfait, S; Thoreau, E; Belaiche, D; Formstecher And B Sablonniè, P

    2000-06-01

    The pleiotropic effects of the natural and synthetic retinoids are mediated by the activation of the two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors (RXRs). At the molecular level, these events begin with the specific ligand recognition by a nuclear receptor subtype. The adaptation of ligands to the receptor binding site leads to an optimal number of interactions for binding and selectivity which justifies elucidation of the structural requirements of the ligand binding pocket. To explore the contribution of H6-H7 loop folding in the ligand-induced conformational changes explained by the mouse-trap model, four RARalpha mutants were constructed. Ligand binding and transactivation studies revealed that three residues from the H6-H7 loop (Gly(301), Phe(302) and Gly(303)) are critical for the conformational adaptation of both synthetic agonists and antagonists. Model building and analysis of both RARalpha-ATRA and RARalpha-CD367 complexes demonstrate that accommodation of CD367 results in a less tight contact of the saturated ring of this ligand with the amino acid side chains of the receptor ligand-binding pocket compared with that of ATRA. According to the flexibility of the agonists tested (ATRA>TTNPB=Am580> CD367), we observed a decrease in binding that was dependent on ligand structure rigidity. In contrast, the binding and transactivating activities of the L266A mutant confirmed the structural constraints imposed by synthetic ligands on binding affinity for the receptor and revealed that subtle local rearrangements induced by specific conformational adaptation changes result in different binding affinities. Our results illustrate the dynamic nature of the interaction between RARalpha and its ligands and demonstrate the critical role of the H6-H7 loop in the binding of both synthetic retinoid agonists and antagonists.

  3. Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

    PubMed

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R; Vaughan, Roxanne A; Henry, L Keith

    2014-10-24

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  4. Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site*

    PubMed Central

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D.; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R.; Vaughan, Roxanne A.; Henry, L. Keith

    2014-01-01

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([125I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [125I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  5. Cyclic mismatch binding ligand CMBL4 binds to the 5'-T-3'/5'-GG-3' site by inducing the flipping out of thymine base.

    PubMed

    Mukherjee, Sanjukta; Dohno, Chikara; Asano, Kaori; Nakatani, Kazuhiko

    2016-09-01

    A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4: with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4: were stacked on each other in aqueous solutions. The most efficient binding of CMBL4: to DNA was observed for the sequence 5'-T-3'/5'-GG-3' (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4: with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4: -bound duplex containing the T/GG site showed that the CMBL4: -binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4: compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site. PMID:27466390

  6. Cyclic mismatch binding ligand CMBL4 binds to the 5'-T-3'/5'-GG-3' site by inducing the flipping out of thymine base.

    PubMed

    Mukherjee, Sanjukta; Dohno, Chikara; Asano, Kaori; Nakatani, Kazuhiko

    2016-09-01

    A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4: with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4: were stacked on each other in aqueous solutions. The most efficient binding of CMBL4: to DNA was observed for the sequence 5'-T-3'/5'-GG-3' (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4: with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4: -bound duplex containing the T/GG site showed that the CMBL4: -binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4: compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site.

  7. Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: Pharmacological characterization of allosteric interactions with the sup 3 Hnitrobenzylthioinosine binding site

    SciTech Connect

    Hammond, J.R. )

    1991-06-01

    Kinetic analysis of the binding of {sup 3}Hnitrobenzylthioinosine ({sup 3}H NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of {sup 3}H NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the {sup 3}HNBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of {sup 3}HNBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of {sup 3}HNBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of {sup 3}HNBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of {sup 3}HNBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both {sup 3}HNBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of {sup 3}HNBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of {sup 3}HNBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of {sup 3}HNBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of {sup 3}HNBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam.

  8. Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

    PubMed Central

    Ruff, Karen M.

    2014-01-01

    The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform. PMID:25246650

  9. Structural insight into PPARgamma ligands binding.

    PubMed

    Farce, A; Renault, N; Chavatte, P

    2009-01-01

    Peroxisome Proliferator Activated Receptors (PPARs) are a family of three related nuclear receptors first cloned in 1990. Their involvement in glucidic and lipidic homeostasis quickly made them an attractive target for the treatment of metabolic syndrome, the most prevalent mortality factor in developed countries. They therefore attracted much synthetic efforts, more particularly PPARgamma. Supported by a large number of crystallographic studies, data derived from these compounds lead to a fairly clear view of the agonist binding mode into the Ligand Binding Domain (LBD). Nearly all the compounds conform to a three-module structure, with a binder group involved in a series of hydrogen bonds in front of the ligand-dependent Activation Function (AF2), a linker mostly arranged around a phenoxyethyl and an effector end occupying the large cavity of the binding site. Following the marketing of the glitazones and the observation of the hepatotoxicity of troglitazone, variations in the binder led to the glitazars, and then pharmacomodulations have been undertaken on the two other modules, leading to a large family of highly related chemical structures. Some compounds, while still adhering to the three-module structure, diverge from the mainstream, such as the phthalates. Curiously, these plasticizers were known to elicit biological effects that led to the discovery of PPARs but were not actively studied as PPARs agonists. As the biological effects of PPARs became clearer, new compounds were also found to exert at least a part of their actions by the activation of PPARgamma. PMID:19442144

  10. Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE*

    PubMed Central

    Syson, Karl; Stevenson, Clare E. M.; Miah, Farzana; Barclay, J. Elaine; Tang, Minhong; Gorelik, Andrii; Rashid, Abdul M.; Lawson, David M.; Bornemann, Stephen

    2016-01-01

    GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential. PMID:27531751

  11. The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins*

    PubMed Central

    Rui, Xianliang; Mehrbod, Mehrdad; Van Agthoven, Johannes F.; Anand, Saurabh; Xiong, Jian-Ping; Mofrad, Mohammad R. K.; Arnaout, M. Amin

    2014-01-01

    The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β3 integrins αIIbβ3 and αVβ3, but a potential role of this interaction on stability of the metal ion at LIMBS in β3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbβ3 versus αVβ3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVβ3. Replacing the αIIb-LIMBS interface residue Phe191 in αIIb (equivalent to Trp179 in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbβ3; a Trp179 to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbβ3 and a W179/F substitution in αVβ3 reduced and increased, respectively, the apparent affinity of Mn2+ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVβ3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation. PMID:24975416

  12. Anisotropic energy flow and allosteric ligand binding in albumin

    NASA Astrophysics Data System (ADS)

    Li, Guifeng; Magana, Donny; Dyer, R. Brian

    2014-01-01

    Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

  13. Topological Analyses of Protein-Ligand Binding: a Network Approach.

    PubMed

    Costanzi, Stefano

    2016-01-01

    Proteins can be conveniently represented as networks of interacting residues, thus allowing the study of several network parameters that can shed light onto several of their structural and functional aspects. With respect to the binding of ligands, which are central for the function of many proteins, network analysis may constitute a possible route to assist the identification of binding sites. As the bulk of this review illustrates, this has generally been easier for enzymes than for non-enzyme proteins, perhaps due to the different topological nature of the binding sites of the former over those of the latter. The article also illustrates how network representations of binding sites can be used to search PDB structures in order to identify proteins that bind similar molecules and, lastly, how codifying proteins as networks can assist the analysis of the conformational changes consequent to ligand binding.

  14. Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand (/sup 3/H)PK 14105

    SciTech Connect

    Benavides, J.; Dubois, A.; Dennis, T.; Hamel, E.; Scatton, B.

    1989-04-01

    The anatomical distribution of omega 3 (peripheral type benzodiazepine binding) sites in the immune system organs of the rat has been studied autoradiographically at both macroscopic and microscopic levels of resolution using either reversible or irreversible (UV irradiation) labeling with (/sup 3/H)PK 14105. In thymus sections, (/sup 3/H)PK 14105 labeled with high affinity (Kd, derived from saturation experiments = 10.8 nM) a single population of sites which possessed the pharmacological characteristics of omega 3 sites. In the thymus gland, higher omega 3 site densities were detected in the cortex than in the medulla; in these subregions, silver grains were associated to small (10-18 microns diameter) cells. In the spleen, omega 3 sites were more abundant in the white than in the red pulp. In the white pulp, silver grains were denser in the marginal zone than in the vicinity of the central artery and labeling was, as in the thymus, associated to small cytoplasm-poor cells. In the red pulp, omega 3 site associated silver grains were observed mainly in the Bilroth cords. In the lymph nodes, the medullary region showed a higher labeling than the surrounding follicles and paracortex. A significant accumulation of silver grains was observed in the lymph node medullary cords. In the intestine, Peyer patches were particularly enriched in omega 3 sites (especially in the periphery of the follicles). The distribution of omega 3 sites in the immune system organs suggests a preferential labeling of cells of T and monocytic lineages. This is consistent with the proposed immunoregulatory properties of some omega 3 site ligands.

  15. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    SciTech Connect

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. )

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  16. Coarse-grained molecular dynamics simulations of protein-ligand binding.

    PubMed

    Negami, Tatsuki; Shimizu, Kentaro; Terada, Tohru

    2014-09-30

    Coarse-grained molecular dynamics (CGMD) simulations with the MARTINI force field were performed to reproduce the protein-ligand binding processes. We chose two protein-ligand systems, the levansucrase-sugar (glucose or sucrose), and LinB-1,2-dichloroethane systems, as target systems that differ in terms of the size and shape of the ligand-binding pocket and the physicochemical properties of the pocket and the ligand. Spatial distributions of the Coarse-grained (CG) ligand molecules revealed potential ligand-binding sites on the protein surfaces other than the real ligand-binding sites. The ligands bound most strongly to the real ligand-binding sites. The binding and unbinding rate constants obtained from the CGMD simulation of the levansucrase-sucrose system were approximately 10 times greater than the experimental values; this is mainly due to faster diffusion of the CG ligand in the CG water model. We could obtain dissociation constants close to the experimental values for both systems. Analysis of the ligand fluxes demonstrated that the CG ligand molecules entered the ligand-binding pockets through specific pathways. The ligands tended to move through grooves on the protein surface. Thus, the CGMD simulations produced reasonable results for the two different systems overall and are useful for studying the protein-ligand binding processes.

  17. Mapping structural landmarks, ligand binding sites and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions and variation in phenotypes in inherited diseases affecting basement membranes

    PubMed Central

    Des Parkin, J.; San Antonio, James D.; Pedchenko, Vadim; Hudson, Billy; Jensen, Shane T.; Savige, Judy

    2016-01-01

    Collagen IV is the major protein found in basement membranes. It comprises 3 heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (‘interactomes’) that indicated major structural landmarks, known and predicted ligand-binding sites, and missense mutations, in order to identify functional and disease-associated domains, potential interactions between ligands, and genotype-phenotype relationships. The maps documented more than 30 known ligand-binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases and heparin. The maps indicated that mutations affecting major ligand-binding sites, for example for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms and muscle Cramps (HANAC) syndrome, and early onset Alport syndrome respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. PMID:21280145

  18. Ligand clouds around protein clouds: a scenario of ligand binding with intrinsically disordered proteins.

    PubMed

    Jin, Fan; Yu, Chen; Lai, Luhua; Liu, Zhirong

    2013-01-01

    Intrinsically disordered proteins (IDPs) were found to be widely associated with human diseases and may serve as potential drug design targets. However, drug design targeting IDPs is still in the very early stages. Progress in drug design is usually achieved using experimental screening; however, the structural disorder of IDPs makes it difficult to characterize their interaction with ligands using experiments alone. To better understand the structure of IDPs and their interactions with small molecule ligands, we performed extensive simulations on the c-Myc₃₇₀₋₄₀₉ peptide and its binding to a reported small molecule inhibitor, ligand 10074-A4. We found that the conformational space of the apo c-Myc₃₇₀₋₄₀₉ peptide was rather dispersed and that the conformations of the peptide were stabilized mainly by charge interactions and hydrogen bonds. Under the binding of the ligand, c-Myc₃₇₀₋₄₀₉ remained disordered. The ligand was found to bind to c-Myc₃₇₀₋₄₀₉ at different sites along the chain and behaved like a 'ligand cloud'. In contrast to ligand binding to more rigid target proteins that usually results in a dominant bound structure, ligand binding to IDPs may better be described as ligand clouds around protein clouds. Nevertheless, the binding of the ligand and a non-ligand to the c-Myc₃₇₀₋₄₀₉ target could be clearly distinguished. The present study provides insights that will help improve rational drug design that targets IDPs.

  19. Interaction of (D-Ser/sup 2/,Leu/sup 5/)enkephalin-Thr/sup 6/ (DSLET), a relatively selective delta ligand, with mu/sub 1/ opioid binding sites

    SciTech Connect

    Itzhak, Y.; Pasternak, G.W.

    1987-01-19

    Using binding approaches, the high selectivity of (D-Ser/sup 2/,Leu/sup 5/)enkephalin-Thr/sup 6/ (DSLET) to delta, as opposed to morphine-preferring (mu/sub 2/) sites in rat brain has been confirmed. However, detailed experiments studies indicate that this ligand also labels mu/sub 1/ sites with very high affinity. Saturation studies of /sup 3/H-DSLET binding reveal curvilinear plots. Treating tissue with naloxonazine to block mu/sub 1/ sites, eliminates the higher affinity binding component. Competition studies of the other peptides against /sup 3/H-DSLET and /sup 3/H(D-Ala/sup 2/, MePhe/sup 4/, Gly(o1)/sup 5/)enkephalin (/sup 3/H-DAMPGO) binding also implied high affinity binding of these peptides to mu/sub 1/ sites. The ability of these peptides to interact with mu/sub 1/ sites may help explain some of their pharmacological actions.

  20. Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment.

    PubMed

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L; Ogorzalek Loo, Rachel R; Loo, Joseph A

    2014-12-01

    "Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

  1. Revealing Ligand Binding Sites and Quantifying Subunit Variants of Non-Covalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

    PubMed Central

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Loo, Rachel R. Ogorzalek; Loo, Joseph A.

    2015-01-01

    “Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains. PMID:24912433

  2. Response of SCP-2L domain of human MFE-2 to ligand removal: binding site closure and burial of peroxisomal targeting signal.

    PubMed

    Lensink, M F; Haapalainen, A M; Hiltunen, J K; Glumoff, T; Juffer, A H

    2002-10-11

    In the study of the structure and function relationship of human MFE-2, we have investigated the dynamics of human MFE-2SCP-2L (hSCP-2L) and its response to ligand removal. A comparison was made with homologous rabbit SCP-2. Breathing and a closing motion are found, identifiable with an adjustment in size and a closing off of the binding pocket. Crucial residues for structural integrity have been identified. Particularly mobile areas of the protein are loop 1 that is connecting helices A and C in space, and helix D, next to the entrance of the pocket. In hSCP-2L, the binding pocket gets occupied by Phe93, which is making a tight hydrophobic contact with Trp36. In addition, it is found that the C-terminal peroxisomal targeting signal (PTS1) that is solvent exposed in the complexed structure becomes buried when no ligand is present. Moreover, an anti-correlation exists between burial of PTS1 and the size of the binding pocket. The results are in accordance with plant nsLTPs, where a similar accommodation of binding pocket size was found after ligand binding/removal. Furthermore, the calculations support the suggestion of a ligand-assisted targeting mechanism.

  3. Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity

    PubMed Central

    Fleetwood, Filippa; Klint, Susanne; Hanze, Martin; Gunneriusson, Elin; Frejd, Fredrik Y.; Ståhl, Stefan; Löfblom, John

    2014-01-01

    Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases. PMID:25515662

  4. The IntFOLD server: an integrated web resource for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction.

    PubMed

    Roche, Daniel B; Buenavista, Maria T; Tetchner, Stuart J; McGuffin, Liam J

    2011-07-01

    The IntFOLD server is a novel independent server that integrates several cutting edge methods for the prediction of structure and function from sequence. Our guiding principles behind the server development were as follows: (i) to provide a simple unified resource that makes our prediction software accessible to all and (ii) to produce integrated output for predictions that can be easily interpreted. The output for predictions is presented as a simple table that summarizes all results graphically via plots and annotated 3D models. The raw machine readable data files for each set of predictions are also provided for developers, which comply with the Critical Assessment of Methods for Protein Structure Prediction (CASP) data standards. The server comprises an integrated suite of five novel methods: nFOLD4, for tertiary structure prediction; ModFOLD 3.0, for model quality assessment; DISOclust 2.0, for disorder prediction; DomFOLD 2.0 for domain prediction; and FunFOLD 1.0, for ligand binding site prediction. Predictions from the IntFOLD server were found to be competitive in several categories in the recent CASP9 experiment. The IntFOLD server is available at the following web site: http://www.reading.ac.uk/bioinf/IntFOLD/.

  5. Structural Basis of Cooperative Ligand Binding by the Glycine Riboswitch

    SciTech Connect

    E Butler; J Wang; Y Xiong; S Strobel

    2011-12-31

    The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 {angstrom} crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.

  6. Highly selective ligand binding by Methylophilus methylotrophus cytochrome c''.

    PubMed

    Quintas, Pedro O; Catarino, Teresa; Todorovic, Smilja; Turner, David L

    2011-06-28

    Cytochrome c'' (cyt c'') from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c'' having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c''. Binding of CO, as well as CN(-), on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c'' for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe(2+)-NO adduct by stopped-flow and resonance Raman spectroscopy, respectively.

  7. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    PubMed Central

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  8. Characterization of [35S]-ATPαS and [3H]-α,β-MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations

    PubMed Central

    Schäfer, Rainer; Reiser, Georg

    1997-01-01

    We made a comparative analysis of the binding characteristics of the radioligands [35S]-ATPαS and [3H]-α,β-MeATP in order to test whether these ligands can be used to analyse P2-purinoceptors in synaptosomal membranes from rat brain cortex. Synaptosomes possess sites with high affinity for [35S]-ATPαS (Kd=22.2±9.1 nM, Bmax=14.8 pmol mg−1 protein). The rank order of the competition potency of the different compounds (ATPαS, ATP, ATPγS>ADPβS, 2-MeSATP>deoxyATP, ADP>>UTP, α,β-MeATP, AMP, Reactive Blue-2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y-purinoceptors. Under identical conditions [35S]-ATPαS and [3H]-α,β-MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]-α,β-MeATP binding sites (Kd=13.7±1.8 nM, Bmax=6.34±0.28 pmol mg−1 protein) was 38 fold higher than the potency of α,β-MeATP to displace [35S]-ATPαS binding (Ki=0.52 μM). ATP and ADPβS competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2-MeSATP, UTP, GTP) did not affect specific [35H]-α,β-MeATP binding at concentrations up to 100 μM. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites. Binding of [35S]-ATPαS on synaptosomes was regulated by GTP, which is indicative for G-protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 nM to 8.6 nM). In the presence of Mg2+ the affinity was increased (Kd 1.8 nM versus 22 nM in the absence of Mg2+). The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]-α,β-MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration from 10 μM to 1 mM, whereas the addition of Mg2+ in the same concentration range resulted in an 80

  9. The CD11a binding site of efalizumab in psoriatic skin tissue as analyzed by Multi-Epitope Ligand Cartography robot technology. Introduction of a novel biological drug-binding biochip assay.

    PubMed

    Bonnekoh, B; Böckelmann, R; Pommer, A J; Malykh, Y; Philipsen, L; Gollnick, H

    2007-01-01

    Efalizumab (Raptiva) is an immunomodulating recombinant humanized IgG1 monoclonal antibody that binds to CD11a, the alpha-subunit of leukocyte function antigen-1 (LFA-1). By blocking the binding of LFA-1 to ICAM-1, efalizumab inhibits the adhesion of leukocytes to other cell types and interferes with the migration of T lymphocytes to sites of inflammation (including psoriatic skin plaques). Analysis of the response in patients treated with efalizumab to date shows that distinct groups of responders and nonresponders to the drug exist. It would therefore be of great practical value to be able to predict which patients are most likely to respond to treatment, by identifying key parameters in the mechanism of action of efalizumab. Detailed investigation and detection of multiple epitopes in microcompartments of skin tissue has until recently been restricted by the available technology. However, the newly developed technique of Multi-Epitope Ligand Cartography (MELC) robot technology combines proteomics and biomathematical tools to visualize protein networks at the cellular and subcellular levels in situ, and to decipher cell functions. The MELC technique, which is outlined in this paper, was used to help characterize the binding of efalizumab to affected and unaffected psoriatic skin as compared to normal control skin under ex vivomodel conditions. Efalizumab was labeled with fluorescein isothiocyanate and integrated into a MELC library of more than 40 antibodies. These antibodies were selected for their potential to detect epitopes which may be indicative of (a) various cell types, (b) structural components of the extracellular matrix, or (c) the processes of cell proliferation, activation and adhesion. Efalizumab bound to CD11a in affected psoriatic skin by a factor 15x and 32x higher than in unaffected psoriatic skin and normal control skin, respectively. CD11a and the efalizumab binding site were primarily expressed in the extravascular dermis, whereas CD54 (ICAM

  10. Identification of the bile salt binding site on ipad from Shigella flexneri and the influence of ligand binding on IpaD structure

    SciTech Connect

    Barta, Michael L.; Guragain, Manita; Adam, Philip; Dickenson, Nicholas E.; Patil, Mrinalini; Geisbrecht, Brian V.; Picking, Wendy L.; Picking, William D.

    2012-10-25

    Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nano-machine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices {alpha}3 and {alpha}7, with concomitant movement in the orientation of helix {alpha}7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC-bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.

  11. Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure.

    PubMed

    Barta, Michael L; Guragain, Manita; Adam, Philip; Dickenson, Nicholas E; Patil, Mrinalini; Geisbrecht, Brian V; Picking, Wendy L; Picking, William D

    2012-03-01

    Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.

  12. Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure

    PubMed Central

    Barta, Michael L.; Guragain, Manita; Adam, Philip; Dickenson, Nicholas E.; Patil, Mrinalini; Geisbrecht, Brian V.; Picking, Wendy L.; Picking, William D.

    2011-01-01

    Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nano-machine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC-bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation. PMID:22423359

  13. The Binding Mode Prediction and Similar Ligand Potency in the Active Site of Vitamin D Receptor with QM/MM Interaction, MESP, and MD Simulation.

    PubMed

    Selvaraman, Nagamani; Selvam, Saravana Kumar; Muthusamy, Karthikeyan

    2016-08-01

    Non-secosteroidal ligands are well-known vitamin D receptor (VDR) agonists. In this study, we described a combined QM/MM to define the protein-ligand interaction energy a strong positive correlation in both QM-MM interaction energy and binding free energy against the biological activity. The molecular dynamics simulation study was performed, and specific interactions were extensively studied. The molecular docking results and surface analysis shed light on steric and electrostatic complementarities of these non-secosteroidal ligands to VDR. Finally, the drug likeness properties were also calculated and found within the acceptable range. The results show that bulky group substitutions in side chain decrease the VDR activity, whereas a small substitution increased it. Functional analyses of H393A and H301A mutations substantiate their roles in the VDR agonistic and antagonistic activities. Apart from the His393 and His301, two other amino acids in the hinge region viz. Ser233 and Arg270 acted as an electron donor/acceptor specific to the agonist in the distinct ligand potency. The results from this study disclose the binding mechanism of VDR agonists and structural modifications required to improve the selectivity.

  14. Ligand deconstruction: Why some fragment binding positions are conserved and others are not.

    PubMed

    Kozakov, Dima; Hall, David R; Jehle, Stefan; Jehle, Sefan; Luo, Lingqi; Ochiana, Stefan O; Jones, Elizabeth V; Pollastri, Michael; Allen, Karen N; Whitty, Adrian; Vajda, Sandor

    2015-05-19

    Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots--regions of the protein where interactions with a ligand contribute substantial binding free energy--the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand. PMID:25918377

  15. Ligand deconstruction: Why some fragment binding positions are conserved and others are not

    PubMed Central

    Kozakov, Dima; Hall, David R.; Jehle, Stefan; Luo, Lingqi; Ochiana, Stefan O.; Jones, Elizabeth V.; Pollastri, Michael; Allen, Karen N.; Whitty, Adrian; Vajda, Sandor

    2015-01-01

    Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots—regions of the protein where interactions with a ligand contribute substantial binding free energy—the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand. PMID:25918377

  16. Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet. alpha. sub 2 -adrenergic receptor

    SciTech Connect

    Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W. )

    1989-05-02

    The human platelet {alpha}{sub 2}-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: ({sup 3}H)SKF 102229 (an antagonist) or p-azido({sup 3}H)clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of ({sup 3}H)SKF 102229 labeled receptor yielded one peptide of M{sub r} 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M{sub r} 4000, which was further digested to the M{sub r} 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido({sup 3}H)clonidine-labeled receptor, a similar M{sub r} 2400 peptide was obtained by lysylendopeptidase cleavage. This M{sub r} 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet {alpha}{sub 2}-adrenergic receptor.

  17. Time, the Forgotten Dimension of Ligand Binding Teaching

    ERIC Educational Resources Information Center

    Corzo, Javier

    2006-01-01

    Ligand binding is generally explained in terms of the equilibrium constant K[subscript d] for the protein-ligand complex dissociation. However, both theoretical considerations and experimental data point to the life span of the protein-ligand complex as an important, but generally overlooked, aspect of ligand binding by macromolecules. Short-lived…

  18. Receptor-binding sites: bioinformatic approaches.

    PubMed

    Flower, Darren R

    2006-01-01

    It is increasingly clear that both transient and long-lasting interactions between biomacromolecules and their molecular partners are the most fundamental of all biological mechanisms and lie at the conceptual heart of protein function. In particular, the protein-binding site is the most fascinating and important mechanistic arbiter of protein function. In this review, I examine the nature of protein-binding sites found in both ligand-binding receptors and substrate-binding enzymes. I highlight two important concepts underlying the identification and analysis of binding sites. The first is based on knowledge: when one knows the location of a binding site in one protein, one can "inherit" the site from one protein to another. The second approach involves the a priori prediction of a binding site from a sequence or a structure. The full and complete analysis of binding sites will necessarily involve the full range of informatic techniques ranging from sequence-based bioinformatic analysis through structural bioinformatics to computational chemistry and molecular physics. Integration of both diverse experimental and diverse theoretical approaches is thus a mandatory requirement in the evaluation of binding sites and the binding events that occur within them. PMID:16671408

  19. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  20. Allosteric Ligand Binding and Anisotropic Energy Flow in Albumin

    NASA Astrophysics Data System (ADS)

    Dyer, Brian

    2014-03-01

    Protein allostery usually involves propagation of local structural changes through the protein to a remote site. Coupling of structural changes at remote sites is thought to occur through anisotropic energy transport, but the nature of this process is poorly understood. We have studied the relationship between allosteric interactions of remote ligand binding sites of the protein and energy flow through the structure of bovine serum albumin (BSA). We applied ultrafast infrared spectroscopy to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic flow through the protein structure following input of thermal energy into the flexible ligand binding sites. We also observe anisotropic heat flow through the structure, without local heating of the rigid helix bundles that connect these sites. We will discuss the implications of this efficient energy transport mechanism with regard to the allosteric propagation of binding energy through the connecting helix structures.

  1. (/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.

  2. Effect of the Active Site D25N Mutation on the Structure, Stability and Ligand Binding of the Mature HIV-1 Protease

    SciTech Connect

    Sayer, Jane M.; Liu, Fengling; Ishima, Rieko; Weber, Irene T.; Louis, John M.

    2008-09-03

    All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR{sub D25N}) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 {+-} 0.09 {mu}m) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR{sub D25N} in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 C, respectively. Only minimal differences are observed in high resolution crystal structures of PR{sub D25N} complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH{sub 2} (RPB), as compared with PR{center_dot}DRV and PR{center_dot}RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T{sub m} is smaller for PR{sub D25N} (6.2 C) than for PR (8.7 C). The T{sub m} of PR{sub D25N}{center_dot}DRV increases by only 3 C relative to free PR{sub D25N}, as compared with a 22 C increase for PR{center_dot}DRV, and the mutation increases the ligand dissociation constant of PR{sub D25N}{center_dot}DRV by a factor of {approx}10{sup 6} relative to PR{center_dot}DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

  3. The dynamics of ligands binding to proteins

    NASA Astrophysics Data System (ADS)

    Callender, Robert

    2001-03-01

    The static structures of many proteins have been solved, and this has revealed much about how they function. On the other hand, although the importance of atomic motion to how proteins function has been conjectured for several decades, the characterization of protein dynamics on multiple time scales is scant. This is because of severe experimental and theoretical difficulties, particularly characterizing the nanosecond to millisecond time scales. Recently, several new techniques have been introduced that make it possible to initiate chemical reactions on fast time scales. We have applied advanced laser induced temperature jump relaxation spectroscopy with nanosecond resolution to examine the binding kinetics of ligands to several enzymes. The observed kinetics take place over multiple time scales. The results reveal the dynamical nature of the binding process and show that there are substantial populations of many structures that are in a constant dynamic equilibrium in some cases. Some of these structures lie quite far from the static structure defined in crystallographic studies, which suggest that the conventional thermodynamical picture of binding (an equilibrium between ligand free in solution and bound) is far off the mark. Moreover, the results suggest that the dynamics can certainly play a crucial role in kinetic control of protein function as in, for example, affecting the rates of enzymatic catalysis. This work is a collaborative project with Hong Deng and Nick Zhadin, also at Albert Einstein. Work supported by the NSF and NIH.

  4. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  5. Preparation and characterization of half-apo dopamine-[beta]-hydroxylase by selective removal of Cu[sub A]. Identification of a sulfur ligand at the dioxygen binding site by EXAFS and FTIR spectroscopy

    SciTech Connect

    Reedy, B.J.; Blackburn, N.J. )

    1994-03-09

    Progress has been made in determining the individual coordination of each of the copper sites (Cu[sub A] and Cu[sub B]) which comprise the active center in dopamine-[beta]-hydroxylase. Previous studies have determined the average ligand environment per copper in the fully metalated enzyme as two to three histidines and one to two O/N donors in the Cu(II) form changing to 2-3 histidines and 0.5 sulfur donors upon reduction to the Cu(I) form. Derivatives of the Cu(I) form of DBH have been made in which Cu[sub A] has been selectively removed, allowing Cu[sub B], the O[sub 2] binding center to be studied by EXAFS and FTIR. Cu[sub B] has been found to be coordinated to two histidines (Cu-N = 1.99 [+-] 0.03 [angstrom]), a S-donor ligand (Cu-S = 2.25 [+-] 0.03 [angstrom]), and a fourth, as yet unidentified ligand X (Cu-X = 2.53 [+-] 0.03 [angstrom]). The FTIR spectrum of the carbonyl derivative of Cu[sub B] indicates that V[sub CO] (2089 cm[sup [minus]1]) is identical to that found for the fully metalated enzyme, providing strong evidence that the Cu[sub B] site is not perturbed by Cu[sub A] removal. EXAFS results on Cu[sub B]-CO indicate that CO binding does not displace the S ligand but appears to displace the weakly bound ligand X. 44 refs., 4 figs., 3 tabs.

  6. Molecular modulators of benzodiazepine receptor ligand binding

    SciTech Connect

    Villar, H.O.; Loew, G.H. )

    1989-01-01

    Ten derivatives of {beta}-carbolines with known affinities to the GABA{sub A}/BDZ (benzodiazepine) receptor were studied using the Am 1 and MNDO/H Semiempirical techniques to identify and characterize molecular modulators of receptor recognition. Steric, lipophilic, and electrostatic properties of these compounds were calculated and examined for their possible role in recognition. Particular attention was paid to the regions around the two most favorable proton-accepting sites, the ON and the substituent at the C{sub 3} position, already implicated in recognition, as well as to the acidic N9H group that could be a proton donating center. To probe further the role of these three ligand sites in receptor interactions, a model of the receptor using three methanol molecules was made and optimum interactions of these three sites with them characterized. The results indicate some similarity in the shape of these ligands, which could reflect a steric requirement. The receptor affinity appears to be modulated to some extent by the ratio of lipophilic to hydrophilic surface, the negative potential at the {beta}N, provided there is also one at the C{sub 3} substituent confirming the importance of two accepting sites in recognition. The acidic N9H does not appear to be a modulator of affinity or does it form a stable H-bond with methanol as acceptor. The two proton donating molecules do form such a stable complex, and both are needed for high affinity.

  7. Farnesyl Diphosphate Synthase Inhibitors With Unique Ligand-Binding Geometries

    PubMed Central

    2015-01-01

    Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis. PMID:25815158

  8. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  9. Detection of persistent organic pollutants binding modes with androgen receptor ligand binding domain by docking and molecular dynamics

    PubMed Central

    2013-01-01

    Background Persistent organic pollutants (POPs) are persistent in the environment after release from industrial compounds, combustion productions or pesticides. The exposure of POPs has been related to various reproductive disturbances, such as reduced semen quality, testicular cancer, and imbalanced sex ratio. Among POPs, dichlorodiphenyldichloroethylene (4,4’-DDE) and polychlorinated biphenyls (PCBs) are the most widespread and well-studied compounds. Recent studies have revealed that 4,4’-DDE is an antagonist of androgen receptor (AR). However, the mechanism of the inhibition remains elusive. CB-153 is the most common congener of PCBs, while the action of CB-153 on AR is still under debate. Results Molecular docking and molecular dynamics (MD) approaches have been employed to study binding modes and inhibition mechanism of 4,4’-DDE and CB-153 against AR ligand binding domain (LBD). Several potential binding sites have been detected and analyzed. One possible binding site is the same binding site of AR natural ligand androgen 5α-dihydrotestosterone (DHT). Another one is on the ligand-dependent transcriptional activation function (AF2) region, which is crucial for the co-activators recruitment. Besides, a novel possible binding site was observed for POPs with low binding free energy with the receptor. Detailed interactions between ligands and the receptor have been represented. The disrupting mechanism of POPs against AR has also been discussed. Conclusions POPs disrupt the function of AR through binding to three possible biding sites on AR/LBD. One of them shares the same binding site of natural ligand of AR. Another one is on AF2 region. The third one is in a cleft near N-terminal of the receptor. Significantly, values of binding free energy of POPs with AR/LBD are comparable to that of natural ligand androgen DHT. PMID:24053684

  10. Amino acids of the Torpedo marmorata acetylcholine receptor. cap alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

    SciTech Connect

    Dennis, M.; Giraudat, J.; Kotzyba-Hibert, F.; Goeldner, M.; Hirth, C.; Chang, J.Y.; Lazure, C.; Chretien, M.; Changeux, J.P.

    1988-04-05

    The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent (/sup 3/H)-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The ..cap alpha..-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the ..cap alpha..-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment ..cap alpha.. 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the ..cap alpha..-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the ..cap alpha..-chain that assign the amino-terminal segment ..cap alpha.. 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified (/sup 3/H)DDF-labeled resides, which are conserved in muscle and neuronal ..cap alpha..-chains but not in the other subunits, may be directly involved in agonist binding.

  11. Modeling the cis-oxo-labile binding site motif of non-heme iron oxygenases: water exchange and oxidation reactivity of a non-heme iron(IV)-oxo compound bearing a tripodal tetradentate ligand.

    PubMed

    Company, Anna; Prat, Irene; Frisch, Jonathan R; Mas-Ballesté, Ruben; Güell, Mireia; Juhász, Gergely; Ribas, Xavi; Münck, Eckard; Luis, Josep M; Que, Lawrence; Costas, Miquel

    2011-02-01

    The spectroscopic and chemical characterization of a new synthetic non-heme iron(IV)-oxo species [Fe(IV)(O)((Me,H) Pytacn)(S)](2+) (2, (Me,H)Pytacn=1-(2'-pyridylmethyl)-4,7-dimethyl-1,4,7-triazacyclononane, S=CH(3)CN or H(2)O) is described. Complex 2 was prepared by reaction of [Fe(II)(CF(3)SO(3))(2)((Me,H) Pytacn)] (1) with peracetic acid. Complex 2 bears a tetradentate N(4) ligand that leaves two cis sites available for binding an oxo group and a second external ligand but, unlike the related iron(IV)-oxo species with tetradentate ligands, it is remarkably stable at room temperature (t(1/2)>2 h at 288 K). Its ability to exchange the oxygen atom of the oxo ligand with water has been analyzed in detail by means of kinetic studies, and a mechanism is proposed on the basis of DFT calculations. Hydrogen-atom abstraction from C-H bonds and oxygen-atom transfer to sulfides by 2 have also been studied. Despite its thermal stability, 2 proves to be a very powerful oxidant that is capable of breaking the strong C-H bond of cyclohexane (bond dissociation energy=99.3 kcal mol(-1)).

  12. Solution Structure of the PAS Domain of a Thermophilic YybT Protein Homolog Reveals a Potential Ligand-binding Site*

    PubMed Central

    Tan, Edward; Rao, Feng; Pasunooti, Swathi; Pham, Thi Huong; Soehano, Ishin; Turner, Mark S.; Liew, Chong Wai; Lescar, Julien; Pervushin, Konstantin; Liang, Zhao-Xun

    2013-01-01

    The Bacillus subtilis protein YybT (or GdpP) and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP. YybT homologs contain a small Per-ARNT-Sim (PAS) domain (∼80 amino acids) that can bind b-type heme with 1:1 stoichiometry despite the small size of the domain and the lack of a conserved heme iron-coordinating residue. We determined the solution structure of the PAS domain of GtYybT from Geobacillus thermodenitrificans by NMR spectroscopy to further probe its function. The solution structure confirms that PASGtYybT adopts the characteristic PAS fold composed of a five-stranded antiparallel β sheet and a few short α-helices. One α-helix and three central β-strands of PASGtYybT are noticeably shorter than those of the typical PAS domains. Despite the small size of the protein domain, a hydrophobic pocket is formed by the side chains of nonpolar residues stemming from the β-strands and α-helices. A set of residues in the vicinity of the pocket and in the C-terminal region at the dimeric interface exhibits perturbed NMR parameters in the presence of heme or zinc protoporphyrin. Together, the results unveil a compact PAS domain with a potential ligand-binding pocket and reinforce the view that the PASYybT domains function as regulatory domains in the modulation of cellular cyclic di-AMP concentration. PMID:23504327

  13. Uncoupling the Structure-Activity Relationships of β2 Adrenergic Receptor Ligands from Membrane Binding.

    PubMed

    Dickson, Callum J; Hornak, Viktor; Velez-Vega, Camilo; McKay, Daniel J J; Reilly, John; Sandham, David A; Shaw, Duncan; Fairhurst, Robin A; Charlton, Steven J; Sykes, David A; Pearlstein, Robert A; Duca, Jose S

    2016-06-23

    Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the β2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of β2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions. PMID:27239696

  14. Time, the forgotten dimension of ligand binding teaching.

    PubMed

    Corzo, Javier

    2006-11-01

    Ligand binding is generally explained in terms of the equilibrium constant K(d) for the protein-ligand complex dissociation. However, both theoretical considerations and experimental data point to the life span of the protein-ligand complex as an important, but generally overlooked, aspect of ligand binding by macromolecules. Short-lived protein-ligand complexes may be unable to trigger further biological processes as signal transduction or internalization if such processes are relatively slow with respect to dissociation of the complex that initiated them. Protein-ligand complex life span depends on the first-order rate constant for the dissociation of the complex, K(off) , but this constant and its implications are generally not treated in textbooks. This report presents a brief discussion and some examples useful for teaching the importance of time in ligand binding by macromolecules in the context of a general biochemistry course.

  15. Identification of a new hormone-binding site on the surface of thyroid hormone receptor.

    PubMed

    Souza, P C T; Puhl, A C; Martínez, L; Aparício, R; Nascimento, A S; Figueira, A C M; Nguyen, P; Webb, P; Skaf, M S; Polikarpov, I

    2014-04-01

    Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors involved in cell differentiation, growth, and homeostasis. Although X-ray structures of many nuclear receptor ligand-binding domains (LBDs) reveal that the ligand binds within the hydrophobic core of the ligand-binding pocket, a few studies suggest the possibility of ligands binding to other sites. Here, we report a new x-ray crystallographic structure of TR-LBD that shows a second binding site for T3 and T4 located between H9, H10, and H11 of the TRα LBD surface. Statistical multiple sequence analysis, site-directed mutagenesis, and cell transactivation assays indicate that residues of the second binding site could be important for the TR function. We also conducted molecular dynamics simulations to investigate ligand mobility and ligand-protein interaction for T3 and T4 bound to this new TR surface-binding site. Extensive molecular dynamics simulations designed to compute ligand-protein dissociation constant indicate that the binding affinities to this surface site are of the order of the plasma and intracellular concentrations of the thyroid hormones, suggesting that ligands may bind to this new binding site under physiological conditions. Therefore, the second binding site could be useful as a new target site for drug design and could modulate selectively TR functions.

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

    PubMed Central

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

    2015-01-01

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets. PMID:26655740

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

    PubMed

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

    2015-12-29

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

  18. Water networks contribute to enthalpy/entropy compensation in protein-ligand binding.

    PubMed

    Breiten, Benjamin; Lockett, Matthew R; Sherman, Woody; Fujita, Shuji; Al-Sayah, Mohammad; Lange, Heiko; Bowers, Carleen M; Heroux, Annie; Krilov, Goran; Whitesides, George M

    2013-10-16

    The mechanism (or mechanisms) of enthalpy-entropy (H/S) compensation in protein-ligand binding remains controversial, and there are still no predictive models (theoretical or experimental) in which hypotheses of ligand binding can be readily tested. Here we describe a particularly well-defined system of protein and ligands--human carbonic anhydrase (HCA) and a series of benzothiazole sulfonamide ligands with different patterns of fluorination--that we use to define enthalpy/entropy (H/S) compensation in this system thermodynamically and structurally. The binding affinities of these ligands (with the exception of one ligand, in which the deviation is understood) to HCA are, despite differences in fluorination pattern, indistinguishable; they nonetheless reflect significant and compensating changes in enthalpy and entropy of binding. Analysis reveals that differences in the structure and thermodynamic properties of the waters surrounding the bound ligands are an important contributor to the observed H/S compensation. These results support the hypothesis that the molecules of water filling the active site of a protein, and surrounding the ligand, are as important as the contact interactions between the protein and the ligand for biomolecular recognition, and in determining the thermodynamics of binding.

  19. Ligand-Binding Pocket Bridges DNA-Binding and Dimerization Domains of the Urate-Responsive MarR Homologue MftR from Burkholderia thailandensis

    PubMed Central

    2015-01-01

    Members of the multiple antibiotic resistance regulator (MarR) family often regulate gene activity by responding to a specific ligand. In the absence of ligand, most MarR proteins function as repressors, while ligand binding causes attenuated DNA binding and therefore increased gene expression. Previously, we have shown that urate is a ligand for MftR (major facilitator transport regulator), which is encoded by the soil bacterium Burkholderia thailandensis. We show here that both mftR and the divergently oriented gene mftP encoding a major facilitator transport protein are upregulated in the presence of urate. MftR binds two cognate sites in the mftR-mftP intergenic region with equivalent affinity and sensitivity to urate. Mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter PecS significantly reduced protein stability and DNA binding affinity but not ligand binding. These data suggest that residues equivalent to those implicated in ligand binding to HucR and PecS serve structural roles and that MftR relies on distinct residues for ligand binding. MftR exhibits a two-step melting transition suggesting independent unfolding of the dimerization and DNA-binding regions; urate binding or mutations in the predicted ligand-binding sites result in one-step unfolding transitions. We suggest that MftR binds the ligand in a cleft between the DNA-binding lobes and the dimer interface but that the mechanism of ligand-mediated attenuation of DNA binding differs from that proposed for other urate-responsive MarR homologues. Since DNA binding by MftR is attenuated at 37 °C, our data also suggest that MftR responds to both ligand and a thermal upshift by attenuated DNA binding and upregulation of the genes under its control. PMID:24955985

  20. Protoglobin: structure and ligand-binding properties.

    PubMed

    Pesce, Alessandra; Bolognesi, Martino; Nardini, Marco

    2013-01-01

    Protoglobin is the first globin identified in Archaea; its biological role is still unknown, although it can bind O2, CO and NO reversibly in vitro. The X-ray structure of Methanosarcina acetivorans protoglobin revealed several peculiar structural features. Its tertiary structure can be considered as an expanded version of the canonical globin fold, characterised by the presence of a pre-A helix (named Z) and a 20-residue N-terminal extension. Other unusual trends are a large distortion of the haem moiety, and its complete burial in the protein matrix due to the extended CE and FG loops and the 20-residue N-terminal loop. Access of diatomic ligands to the haem has been proposed to be granted by two tunnels, which are mainly defined by helices B/G (tunnel 1) and B/E (tunnel 2), and whose spatial orientation and topology give rise to an almost orthogonal two-tunnel system unprecedented in other globins. At a quaternary level, protoglobin forms a tight dimer, mostly based on the inter-molecular four-helix bundle built by the G- and H-helices, similar to that found in globin-coupled sensor proteins, which share with protoglobin a common phylogenetic origin. Such unique structural properties, together with an unusually low O2 dissociation rate and a selectivity ratio for O2/CO binding that favours O2 ligation, make protoglobin a peculiar case for gaining insight into structure to function relationships within the globin superfamily. While recent structural and biochemical data have given answers to important questions, the functional issue is still unclear and it is expected to represent the major focus of future investigations. PMID:24054795

  1. Mu opioid receptor binding sites in human brain

    SciTech Connect

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

    1986-01-01

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

  2. Comparative Analysis of Homology Models of the Ah Receptor Ligand Binding Domain: Verification of Structure-Function Predictions by Site-Directed Mutagenesis of a Non-Functional AHR†

    PubMed Central

    Fraccalvieri, Domenico; Soshilov, Anatoly A.; Karchner, Sibel I.; Franks, Diana G.; Pandini, Alessandro; Bonati, Laura; Hahn, Mark E.; Denison, Michael S.

    2013-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity and response have been observed, the structural determinants responsible have not been determined and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of sixteen AHRs from twelve mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, His388) that reduce the internal space available to TCDD. Mutagenesis of two of these residues in zfAhR1a to those present in zfAHR2 (Y296H, T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue to examine species specific differences in AHR responsiveness. PMID:23286227

  3. Homology model of human retinoic acid metabolising enzyme cytochrome P450 26A1 (CYP26A1): active site architecture and ligand binding.

    PubMed

    Gomaa, Mohamed Sayed; Yee, Sook Wah; Milbourne, Ceri Elizabeth; Barbera, Maria Chiara; Simons, Claire; Brancale, Andrea

    2006-08-01

    Homology models of cytochrome P450 RA1 (CYP26A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using MOE software the lowest energy CYP26A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP26A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP26A1 model. The natural substrate, all-trans-retinoic acid (atRA), and inhibitor R 15866, were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure. Multiple hydrophobic interactions, including the side chains of TRP112, PHE299, PHE222, PHE84, PHE374 and PRO371, are important for binding of atRA and R115866. Additional hydrogen bonding interactions were noted as follows: atRA-- C==O of the atRA carboxylate group and ARG86; R115866--benzothiazole nitrogen and the backbone NH of SER115.

  4. Multivalent Ligand-Receptor Binding on Supported Lipid Bilayers

    PubMed Central

    Jung, Hyunsook; Robison, Aaron D.; Cremer, Paul S.

    2009-01-01

    Fluid supported lipid bilayers provide an excellent platform for studying multivalent protein-ligand interactions because the two-dimensional fluidity of the membrane allows for lateral rearrangement of ligands in order to optimize binding. Our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy (TIRFM) to obtain equilibrium dissociation constant (KD) data for these systems. This high throughput, on-chip approach provides highly accurate thermodynamic information about multivalent binding events while requiring only very small sample volumes. Herein, we review some of the most salient findings from these studies. In particular, increasing ligand density on the membrane surface can provide a modest enhancement or attenuation of ligand-receptor binding depending upon whether the surface ligands interact strongly with each other. Such effects, however, lead to little more than one order of magnitude change in the apparent KD values. On the other hand, the lipophilicity and presentation of lipid bilayer-conjugated ligands can have a much greater impact. Indeed, changing the way a particular ligand is conjugated to the membrane can alter the apparent KD value by at least three orders of magnitude. Such a result speaks strongly to the role of ligand availability for multivalent ligand-receptor binding. PMID:19508894

  5. NMR studies of DNA oligomers and their interactions with minor groove binding ligands

    SciTech Connect

    Fagan, P A

    1996-05-01

    The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I{center_dot}C base pairs are functional analogs of A{center_dot}T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1 ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.

  6. Exchange Kinetics of a Hydrophobic Ligand Binding Protein

    NASA Astrophysics Data System (ADS)

    Vaughn, Jeff; Stone, Martin

    2002-03-01

    Conformational fluctuations of proteins are thought to be important for determining the functional roles in biological activity. In some cases, the rates of these conformational changes may be directly correlated to, for example, the rates of catalysis or ligand binding. We are studying the role of conformational fluctuations in the binding of small volatile hydrophobic pheromones by the mouse major urinary proteins (MUPs). Communication among mice occurs, in part, with the MUP-1 protein. This urinary protein binds pheromones as a way to increase the longevity of the pheromone in an extracellular environment. Of interest is that the crystal structure of MUP-1 with a pheromone ligand shows the ligand to be completely occluded from the solvent with no obvious pathway to enter or exit. This suggests that conformational exchange of the protein may be required for ligand binding and release to occur. We hypothesize that the rate of conformational exchange may be a limiting factor determining the rate of ligand association and dissociation. By careful measurement of the on- and off-rates of ligand binding and the rates of conformational changes of the protein, a more defined picture of the interplay between protein structure and function can be obtained. To this end, heteronuclear saturation transfer, ^15N-exchange and ^15N dynamics experiments have been employed to probe the kinetics of ligand binding to MUP-1.

  7. Cyclic mismatch binding ligand CMBL4 binds to the 5′-T-3′/5′-GG-3′ site by inducing the flipping out of thymine base

    PubMed Central

    Mukherjee, Sanjukta; Dohno, Chikara; Asano, Kaori; Nakatani, Kazuhiko

    2016-01-01

    A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4 with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4 were stacked on each other in aqueous solutions. The most efficient binding of CMBL4 to DNA was observed for the sequence 5′-T-3′/5′-GG-3′ (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4 with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4-bound duplex containing the T/GG site showed that the CMBL4-binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a π-stack. Preferential binding was observed for CMBL4 compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site. PMID:27466390

  8. Characterization of zinc-binding sites in human stromelysin-1: stoichiometry of the catalytic domain and identification of a cysteine ligand in the proenzyme.

    PubMed

    Salowe, S P; Marcy, A I; Cuca, G C; Smith, C K; Kopka, I E; Hagmann, W K; Hermes, J D

    1992-05-19

    A determination of the zinc stoichiometry of the catalytic domain of the human matrix metalloproteinase stromelysin-1 has been carried out using enzyme purified from recombinant Escherichia coli that express C-terminally truncated protein. Atomic absorption spectrometry revealed that both the proenzyme (prostrom255) and the mature active form (strom255) contained nearly 2 mol of Zn/mol of protein. Full-length prostromelysin purified from a mammalian cell culture line also contained zinc in excess of 1 equiv. While zinc in prostrom255 could not be removed by dialysis against o-phenanthroline, similar treatment of mature strom255 resulted in the loss of one-half of the original zinc content. The peptidase activity of the zinc-depleted protein was reduced by greater than 85% but could be restored upon addition of Zn2+ or Co2+. Addition of a thiol-containing inhibitor to a CoZn hybrid enzyme resulted in marked spectral changes in both the visible and ultraviolet regions characteristic of sulfur ligation to Co2+. This direct evidence for an integral role in catalysis and inhibitor binding confirms the location of the exchangeable metal at the active site. To examine the environment of zinc in the proenzyme, a fully cobalt-substituted proenzyme was prepared by in vivo metal replacement. The absorbance features of dicobalt prostrom255 were consistent with metal coordination by the single cysteine present in the propeptide, although the data do not allow assignment to a particular zinc site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1581308

  9. Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes.

    PubMed

    Jain, Tarun; Jayaram, B

    2007-06-01

    Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).

  10. Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.

    PubMed

    Kim, Yaewon; Hilty, Christian

    2015-04-13

    Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.

  11. Time-dynamic imaging of individual cell ligand binding kinetics

    NASA Astrophysics Data System (ADS)

    Gross, David; Chung, Johnson

    1997-05-01

    Ligand-binding assays are commonly applied to large numbers of cells in culture; the binding parameters derived from such assays reflect the ensemble average behavior of many cells. Equilibrium binding assays of epidermal growth factor (EGF) binding to the EGF receptor (EGFR) indicate that the EGFR exhibits two affinity states for EGF, one low affinity with Kd about 10 nM and one high affinity with Kd < 1 nM. Bulk binding studies cannot determined if such multiple ligand binding classes are due to cell population heterogeneity or are due to heterogeneity at the individual cell level. Here is described a technique based on single cell imaging of fluorescein-EGF (f-EGF) binding to individual human epidermoid carcinoma A431 cells that demonstrates that both classes of EGFR are found on all A431 cells, that the time course of f-EGF binding to individual cells shows two kinetic on-rates and two off-rates, that cell-to-cell heterogeneity of EGF binding is significant and that ligand binding kinetics vary across an individual cell. Contributions of cell autofluorescence photobleaching and f- EGF photobleaching in the measurement of fluorescent ligand binding are shown to be significant.

  12. Copper binding ligands: production by marine plankton and characterization by ESI-MS

    NASA Astrophysics Data System (ADS)

    Orians, K.; Ross, A.; Lawrence, M.; Ikonomou, M.

    2003-04-01

    Organic complexation affects the bioavailability and distribution of copper in the surface ocean. The cyanobacterium Synechococcus sp. PCC 7002 was cultured in the lab and subjected to near-toxic Cu concentrations. Strong Cu-binding ligands were produced under these conditions, as found for other species of Synechococcus. The copper-binding ligand produced had a log K'cond. (log conditional stability constant) of 12.2, similar to the natural ligands found in the surface ocean. The amount of ligand produced was proportional to the amount of copper present. Isolation and concentration of these compounds for characterization by electrospray mass spectrometry (ESI-MS) provides information about the structure of the organic ligands and their metal-ion complexes. Using model ligands, we'll show that ligands can be characterized by ESI-MS and that the location of the copper binding site can be determined in complex molecules. We'll also present results of copper-complexing ligands extracted from the coastal waters of British Columbia. Ligand concentrations are higher at low salinity and in surface waters, indicating that these ligands are produced in surface waters and/or delivered to the region via the Fraser River. Analysis of the extracts with highest UV absorbance identified two Cu2+ ligands of molecular weight 259 and 264. The mass and isotopic distributions are consistent with dipeptides and tripeptides containing two metal-binding amino groups. This result is consistent with the findings of other studies attempting to characterize Cu2+ ligands in seawater. The structure of the identified ligand is similar to that of rhodotorulic acid (a microbial siderophore), glutathione, and phytochelatins, indicating that small peptides and related compounds can act as strong, specific metal chelators in natural waters

  13. A general framework improving teaching ligand binding to a macromolecule.

    PubMed

    Haiech, Jacques; Gendrault, Yves; Kilhoffer, Marie-Claude; Ranjeva, Raoul; Madec, Morgan; Lallement, Christophe

    2014-10-01

    The interaction of a ligand with a macromolecule has been modeled following different theories. The tenants of the induced fit model consider that upon ligand binding, the protein-ligand complex undergoes a conformational change. In contrast, the allosteric model assumes that only one among different coexisting conformers of a given protein is suitable to bind the ligand optimally. In the present paper, we propose a general framework to model the binding of ligands to a macromolecule. Such framework built on the binding polynomial allows opening new ways to teach in a unified manner ligand binding, enzymology and receptor binding in pharmacology. Moreover, we have developed simple software that allows building the binding polynomial from the schematic description of the biological system under study. Taking calmodulin as a canonical example, we show here that the proposed tool allows the easy retrieval of previously experimental and computational reports. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

  14. Calculation of cooperativity and equilibrium constants of ligands binding to G-quadruplex DNA in solution.

    PubMed

    Kudrev, A G

    2013-11-15

    Equilibrium model of a ligand binding with DNA oligomer has been considered as a process of small molecule adsorption onto a lattice of multiple binding sites. An experimental example has been used to verify the assertion that during saturation of the macromolecule by a ligand should expect effect of cooperativity due to changes in DNA conformation or the mutual influence between bound ligands. Such phenomenon cannot be entirely described by the classical stepwise complex formation model. To evaluate a ligand binding affinity and cooperativity of ligand-oligomer complex formation the statistical approach has been proposed. This new computational approach used to re-examine previously studded ligand binding towards DNA quadruplexes targets with multiple binding sites. The intrinsic equilibrium constants K1-3 of the mesotetrakis-(N-methyl-4-pyridyl)-porphyrin (TMPyP4) binding with the [d(T4G4)]4 and with the [AG3(T2AG3)3] quadruplexes and the correction for the mutual influence between bound ligands (cooperativity parameters ω) was determined from the Job plots based upon the nonlinear least-squares fitting procedure. The re-examination of experimental curves reveals that the equilibrium is affected by the positive cooperative (ω>1) binding of the TMPyP4 ligand with tetramolecular [d(T4G4)]4. However for an intramolecular antiparallel-parallel hybrid structure [AG3(T2AG3)3] the weak anti-cooperativity of TMPyP4 accommodation (ω<1) onto two from three nonidentical sites was detected. PMID:24148442

  15. Predicting Ca2+ -binding sites using refined carbon clusters.

    PubMed

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

    2012-12-01

    Identifying Ca(2+) -binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca(2+) -binding proteins. Currently, these sites are identified in structures either through X-ray crystallography or NMR analysis. However, Ca(2+) -binding sites are not always visible in X-ray structures due to flexibility in the binding region or low occupancy in a Ca(2+) -binding site. Similarly, both Ca(2+) and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca(2+) -binding sites in both X-ray and NMR structures, we report a new graph theory algorithm (MUG(C) ) to predict Ca(2+) -binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side-chain oxygen ligand co-ordinates, MUG(C) is able to achieve 94% sensitivity with 76% selectivity on a dataset of X-ray structures composed of 43 Ca(2+) -binding proteins. Additionally, prediction of Ca(2+) -binding sites in NMR structures was obtained by MUG(C) using a different set of parameters, which were determined by the analysis of both Ca(2+) -constrained and unconstrained Ca(2+) -loaded structures derived from NMR data. MUG(C) identified 20 of 21 Ca(2+) -binding sites in NMR structures inferred without the use of Ca(2+) constraints. MUG(C) predictions are also highly selective for Ca(2+) -binding sites as analyses of binding sites for Mg(2+) , Zn(2+) , and Pb(2+) were not identified as Ca(2+) -binding sites. These results indicate that the geometric arrangement of the second-shell carbon cluster is sufficient not only for accurate identification of Ca(2+) -binding sites in NMR and X-ray structures but also for selective differentiation between Ca(2+) and other relevant divalent cations.

  16. Ligand Binding and Structural Changes Associated with Allostery in Yeast NAD+-specific Isocitrate Dehydrogenase

    PubMed Central

    Lee, McAlister-Henn

    2011-01-01

    Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four each of regulatory IDH1 and catalytic IDH2 subunits that share 42% sequence identity. IDH2 contains catalytic isocitrate/Mg2+ and NAD+ binding sites whereas IDH1 contains homologous binding sites, respectively, for cooperative binding of isocitrate and for allosteric binding of AMP. Ligand binding is highly ordered in vitro, and IDH exhibits the unusual property of half-site binding for all ligands. The structures of IDH solved in the absence or presence of ligands have shown: (a) a heterodimer to be the basic structural/functional unit of the enzyme, (b) the organization of heterodimers to form tetramer and octamer structures, (c) structural differences that may underlie cooperative and allosteric regulatory mechanisms, and (d) the possibility for formation of a disulfide bond that could reduce catalytic activity. In vivo analyses of mutant enzymes have elucidated the physiological importance of catalytic activity and allosteric regulation of this tricarboxylic acid cycle enzyme. Other studies have established the importance of a disulfide bond in regulation of IDH activity in vivo, as well as contributions of this bond to the property of half-site ligand binding exhibited by the wild-type enzyme. PMID:22008468

  17. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    PubMed

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates. PMID:26275106

  18. 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.

  19. 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.

  20. Combined Effects of Ankylosing Spondylitis-associated ERAP1 Polymorphisms Outside the Catalytic and Peptide-binding Sites on the Processing of Natural HLA-B27 Ligands*

    PubMed Central

    Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A.

    2014-01-01

    ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis. PMID:24352655

  1. How to Illustrate Ligand-Protein Binding in a Class Experiment: An Elementary Fluorescent Assay.

    ERIC Educational Resources Information Center

    Marty, Alain; And Others

    1986-01-01

    Describes an experiment (taking approximately five hours) which illustrates the binding of a small molecule to a protein. By using an appropriate fluorescent ligand and a given protein, the fluorescent probe technique is applied to measure the number of bonding sites, and number of site classes, and their association constants. (JN)

  2. Molecular dynamics simulation of ligand dissociation from liver fatty acid binding protein.

    PubMed

    Long, Dong; Mu, Yuguang; Yang, Daiwen

    2009-06-30

    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized "portal region" could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques.

  3. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    PubMed

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  4. Essential role of conformational selection in ligand binding

    PubMed Central

    Vogt, Austin D.; Pozzi, Nicola; Chen, Zhiwei; Di Cera, Enrico

    2013-01-01

    Two competing and mutually exclusive mechanisms of ligand recognition – conformational selection and induced fit - have dominated our interpretation of ligand binding in biological macromolecules for almost six decades. Conformational selection posits the pre-existence of multiple conformations of the macromolecule from which the ligand selects the optimal one. Induced fit, on the other hand, postulates the existence of conformational rearrangements of the original conformation into an optimal one that is induced by binding of the ligand. In the former case, conformational transitions precede the binding event; in the latter, conformational changes follow the binding step. Kineticists have used a facile criterion to distinguish between the two mechanisms based on the dependence of the rate of relaxation to equilibrium, kobs, on the ligand concentration, [L]. A value of kobs decreasing hyperbolically with [L] is seen as diagnostic of conformational selection, while a value of kobs increasing hyperbolically with [L] is considered diagnostic of induced fit. However, this simple conclusion is only valid in the rather unrealistic assumption of conformational transitions being much slower than binding and dissociation events. In general, induced fit only produces values of kobs that increase with [L] but conformational selection is more versatile and is associated with values of kobs that increase, decrease with or are independent of [L]. The richer repertoire of kinetic properties of conformational selection applies to kinetic mechanisms with single or multiple saturable relaxations and explains the behavior of nearly all experimental systems reported in the literature thus far. Conformational selection is always sufficient and often necessary to account for the relaxation kinetics of ligand binding to a biological macromolecule and is therefore an essential component of any binding mechanism. On the other hand, induced fit is never necessary and only sufficient in a

  5. Elucidation of Nonadditive Effects in Protein-Ligand Binding Energies: Thrombin as a Case Study.

    PubMed

    Calabrò, Gaetano; Woods, Christopher J; Powlesland, Francis; Mey, Antonia S J S; Mulholland, Adrian J; Michel, Julien

    2016-06-23

    Accurate predictions of free energies of binding of ligands to proteins are challenging partly because of the nonadditivity of protein-ligand interactions; i.e., the free energy of binding is the sum of numerous enthalpic and entropic contributions that cannot be separated into functional group contributions. In principle, molecular simulations methodologies that compute free energies of binding do capture nonadditivity of protein-ligand interactions, but efficient protocols are necessary to compute well-converged free energies of binding that clearly resolve nonadditive effects. To this end, an efficient GPU-accelerated implementation of alchemical free energy calculations has been developed and applied to two congeneric series of ligands of the enzyme thrombin. The results show that accurate binding affinities are computed across the two congeneric series and positive coupling between nonpolar R(1) substituents and a X = NH3(+) substituent is reproduced, albeit with a weaker trend than experimentally observed. By contrast, a docking methodology completely fails to capture nonadditive effects. Further analysis shows that the nonadditive effects are partly due to variations in the strength of a hydrogen-bond between the X = NH3(+) ligands family and thrombin residue Gly216. However, other partially compensating interactions occur across the entire binding site, and no single interaction dictates the magnitude of the nonadditive effects for all the analyzed protein-ligand complexes. PMID:27248478

  6. The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

    PubMed Central

    Rajendra, S; Vandenberg, R J; Pierce, K D; Cunningham, A M; French, P W; Barry, P H; Schofield, P R

    1995-01-01

    A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily. Images PMID:7621814

  7. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  8. Persistent Binding of Ligands to the Aryl Hydrocarbon Receptor

    PubMed Central

    Bohonowych, Jessica E.; Denison, Michael S.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([3H] β-naphthoflavone [βNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [3H]TCDD and 26% to 85% for [3H] βNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [3H]TCDD, shifted the apparent IC50 of these compounds as competitive AhR ligands by ∼10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs. PMID:17431010

  9. Analyzing Ligand Depletion in a Saturation Equilibrium Binding Experiment

    ERIC Educational Resources Information Center

    Claro, Enrique

    2006-01-01

    I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [[superscript 3]H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high…

  10. Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands.

    PubMed

    Taylor, Paul; Takeuchi, Hideyuki; Sheppard, Devon; Chillakuri, Chandramouli; Lea, Susan M; Haltiwanger, Robert S; Handford, Penny A

    2014-05-20

    The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.

  11. Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

    PubMed

    Jeyakumar, M; Carlson, Kathryn E; Gunther, Jillian R; Katzenellenbogen, John A

    2011-04-15

    The estrogen receptors, ERα and ERβ, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERβ. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERβ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity. PMID:21321128

  12. Calculation of Mg(+)-ligand relative binding energies

    NASA Technical Reports Server (NTRS)

    Partridge, Harry; Bauschlicher, Charles W., Jr.

    1992-01-01

    The calculated relative binding energies of 16 organic molecules to Mg(+) are compared with experimental results where available. The geometries of the ligands and the Mg(+)-ligand complexes arc optimized at the self-consistent field level using a 6-31G* basis set. The Mg(+) binding energies are evaluated using second-order perturbation theory and basis sets of triple-sigma quality augmented with two sets of polarization functions. This level of theory is calibrated against higher levels of theory for selected systems. The computed binding energies are accurate to about 2 kcal/mol.

  13. Chelate effects in sulfate binding by amide/urea-based ligands.

    PubMed

    Jia, Chuandong; Wang, Qi-Qiang; Begum, Rowshan Ara; Day, Victor W; Bowman-James, Kristin

    2015-07-01

    The influence of chelate and mini-chelate effects on sulfate binding was explored for six amide-, amide/amine-, urea-, and urea/amine-based ligands. Two of the urea-based hosts were selective for SO4(2-) in water-mixed DMSO-d6 systems. Results indicated that the mini-chelate effect provided by a single urea group with two NH binding sites appears to provide enhanced binding over two amide groups. Furthermore, additional urea binding sites incorporated into the host framework appeared to overcome to some extent competing hydration effects with increasing water content.

  14. Ligand-induced conformational changes in a thermophilic ribose-binding protein

    SciTech Connect

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2009-05-21

    Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (appTm value is 108 C) than the mesophilic Escherichia coli homolog (ecRBP) (appTm value is 56 C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different

  15. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  16. Quantification of the Effects of Ionic Strength, Viscosity, and Hydrophobicity on Protein–Ligand Binding Affinity

    PubMed Central

    2014-01-01

    In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (Kd) is essential. Estimation of the binding affinity in this way is routinely performed in “favorable” conditions for macromolecules. Crucial data for ligand–protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the Kd of three previously characterized protein–ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity. PMID:25147617

  17. Critical ligand binding reagent preparation/selection: when specificity depends on reagents.

    PubMed

    Rup, Bonita; O'Hara, Denise

    2007-05-11

    Throughout the life cycle of biopharmaceutical products, bioanalytical support is provided using ligand binding assays to measure the drug product for pharmacokinetic, pharmacodynamic, and immunogenicity studies. The specificity and selectivity of these ligand binding assays are highly dependent on the ligand binding reagents. Thus the selection, characterization, and management processes for ligand binding reagents are crucial to successful assay development and application. This report describes process considerations for selection and characterization of ligand binding reagents that are integral parts of the different phases of assay development. Changes in expression, purification, modification, and storage of the ligand binding reagents may have a profound effect on the ligand binding assay performance. Thus long-term management of the critical ligand binding assay reagents is addressed including suggested characterization criteria that allow ligand binding reagents to be used in as consistent a manner as possible. Examples of challenges related to the selection, modification, and characterization of ligand binding reagents are included.

  18. Metalloprotein-inhibitor binding: human carbonic anhydrase II as a model for probing metal-ligand interactions in a metalloprotein active site.

    PubMed

    Martin, David P; Hann, Zachary S; Cohen, Seth M

    2013-11-01

    An ever-increasing number of metalloproteins are being discovered that play essential roles in physiological processes. Inhibitors of these proteins have significant potential for the treatment of human disease, but clinical success of these compounds has been limited. Herein, zinc(II)-dependent metalloprotein inhibitors in clinical use are reviewed, and the potential for using novel metal-binding groups (MBGs) in the design of these inhibitors is discussed. By using human carbonic anhydrase II as a model system, the nuances of MBG-metal interactions in the context of a protein environment can be probed. Understanding how metal coordination influences inhibitor binding may help in the design of new therapeutics targeting metalloproteins.

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

    PubMed

    Hochachka, P W

    1974-12-01

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

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

    PubMed Central

    Hochachka, Peter W.

    1974-01-01

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

  1. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity

    PubMed Central

    Ayres, Cory M.; Scott, Daniel R.; Corcelli, Steven A.; Baker, Brian M.

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  2. Characterisation of iron binding ligands in seawater by reverse titration.

    PubMed

    Hawkes, Jeffrey A; Gledhill, Martha; Connelly, Douglas P; Achterberg, Eric P

    2013-03-01

    Here we demonstrate the use of reverse titration - competitive ligand exchange-adsorptive cathodic stripping voltammetry (RT-CLE-ACSV) for the analysis of iron (Fe) binding ligands in seawater. In contrast to the forward titration, which examines excess ligands in solution, RT-CLE-ACSV examines the existing Fe-ligand complexes by increasing the concentration of added (electroactive) ligand (1-nitroso-2-naphthol) and analysis of the proportion of Fe bound to the added ligand. The data manipulation allows the accurate characterisation of ligands at equal or lower concentrations than Fe in seawater, and disregards electrochemically inert dissolved Fe such as some colloidal phases. The method is thus superior to the forward titration in environments with high Fe and low ligand concentrations or high concentrations of inert Fe. We validated the technique using the siderophore ligand ferrioxamine B, and observed a stability constant [Formula: see text] of 0.74-4.37×10(21) mol(-1), in agreement with previous results. We also successfully analysed samples from coastal waters and a deep ocean hydrothermal plume. Samples from these environments could not be analysed with confidence using the forward titration, highlighting the effectiveness of the RT-CLE-ACSV technique in waters with high concentrations of inert Fe.

  3. Differentiation between ligand trapping into intact cells and binding on muscarinic receptors.

    PubMed

    Gossuin, A; Maloteaux, J M; Trouet, A; Laduron, P

    1984-05-22

    Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells. PMID:6722181

  4. Ligand Binding Thermodynamics in Drug Discovery: Still a Hot Tip?

    PubMed

    Geschwindner, Stefan; Ulander, Johan; Johansson, Patrik

    2015-08-27

    The use of ligand binding thermodynamics has been proposed as a potential success factor to accelerate drug discovery. However, despite the intuitive appeal of optimizing binding enthalpy, a number of factors complicate routine use of thermodynamic data. On a macroscopic level, a range of experimental parameters including temperature and buffer choice significantly influence the observed thermodynamic signatures. On a microscopic level, solute effects, structural flexibility, and cooperativity lead to nonlinear changes in enthalpy. This multifactorial character hides essential enthalpy contributions of intermolecular contacts, making them experimentally nonobservable. In this perspective, we present three case studies, reflect on some key factors affecting thermodynamic signatures, and investigate their relation to the hydrophobic effect, enthalpy-entropy compensation, lipophilic ligand efficiency, and promiscuity. The studies highlight that enthalpy and entropy cannot be used as direct end points but can together with calculations increase our understanding of ligand binding and identify interesting outliers that do not behave as expected.

  5. Novel computational methodologies for structural modeling of spacious ligand binding sites of G-protein-coupled receptors: development and application to human leukotriene B4 receptor.

    PubMed

    Ishino, Yoko; Harada, Takanori

    2012-01-01

    This paper describes a novel method to predict the activated structures of G-protein-coupled receptors (GPCRs) with high accuracy, while aiming for the use of the predicted 3D structures in in silico virtual screening in the future. We propose a new method for modeling GPCR thermal fluctuations, where conformation changes of the proteins are modeled by combining fluctuations on multiple time scales. The core idea of the method is that a molecular dynamics simulation is used to calculate average 3D coordinates of all atoms of a GPCR protein against heat fluctuation on the picosecond or nanosecond time scale, and then evolutionary computation including receptor-ligand docking simulations functions to determine the rotation angle of each helix of a GPCR protein as a movement on a longer time scale. The method was validated using human leukotriene B4 receptor BLT1 as a sample GPCR. Our study demonstrated that the proposed method was able to derive the appropriate 3D structure of the active-state GPCR which docks with its agonists.

  6. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    SciTech Connect

    Zhang, Bing; Liu, Caini; Qian, Wen; Han, Yue; Li, Xiaoxia; Deng, Junpeng

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  7. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    SciTech Connect

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  8. N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

    PubMed

    Hood, W F; Compton, R P; Monahan, J B

    1990-03-01

    In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

  9. VEGFR-2 conformational switch in response to ligand binding

    PubMed Central

    Sarabipour, Sarvenaz; Ballmer-Hofer, Kurt; Hristova, Kalina

    2016-01-01

    VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer. DOI: http://dx.doi.org/10.7554/eLife.13876.001 PMID:27052508

  10. Relation between the change in DNA elasticity on ligand binding and the binding energetics.

    PubMed

    Kostjukov, Viktor V; Evstigneev, Maxim P

    2012-09-01

    The widespread use of tweezers for measurement of ligand-DNA binding parameters is based on the McGhee-von Hippel treatment of the DNA contour and persistence length as a function of concentration. The McGhee-von Hippel approach contains the basic assumption that the binding constant K is independent of the number of already bound ligands. However, the change in elasticity of DNA on binding affects the entropic part of the Gibbs free energy and, hence, the K value in a concentration-dependent manner, making the whole approach inconsistent. In the present work we show that the energetic effect of DNA stiffening on noncovalent binding of small ligands is negligible with respect to the net energy of reaction, whereas the DNA stiffening on binding of large ligands must always be considered in each particular case.

  11. Relation between the change in DNA elasticity on ligand binding and the binding energetics

    NASA Astrophysics Data System (ADS)

    Kostjukov, Viktor V.; Evstigneev, Maxim P.

    2012-09-01

    The widespread use of tweezers for measurement of ligand-DNA binding parameters is based on the McGhee-von Hippel treatment of the DNA contour and persistence length as a function of concentration. The McGhee-von Hippel approach contains the basic assumption that the binding constant K is independent of the number of already bound ligands. However, the change in elasticity of DNA on binding affects the entropic part of the Gibbs free energy and, hence, the K value in a concentration-dependent manner, making the whole approach inconsistent. In the present work we show that the energetic effect of DNA stiffening on noncovalent binding of small ligands is negligible with respect to the net energy of reaction, whereas the DNA stiffening on binding of large ligands must always be considered in each particular case.

  12. Ion binding sites and their representations by reduced models.

    PubMed

    Roux, Benoît

    2012-06-14

    The binding of small metal ions to complex macromolecular structures is typically dominated by strong local interactions of the ion with its nearest ligands. Progress in understanding the molecular determinants of ion selectivity can often be achieved by considering simplified reduced models comprised of only the most important ion-coordinating ligands. Although the main ingredients underlying simplified reduced models are intuitively clear, a formal statistical mechanical treatment is nonetheless necessary in order to draw meaningful conclusions about complex macromolecular systems. By construction, reduced models only treat the ion and the nearest coordinating ligands explicitly. The influence of the missing atoms from the protein or the solvent is incorporated indirectly. Quasi-chemical theory offers one example of how to carry out such a separation in the case of ion solvation in bulk liquids, and in several ways, a statistical mechanical formulation of reduced binding site models for macromolecules is expected to follow a similar route. However, there are also important differences when the ion-coordinating moieties are not solvent molecules from a bulk phase but are molecular ligands covalently bonded to a macromolecular structure. Here, a statistical mechanical formulation of reduced binding site models is elaborated to address these issues. The formulation provides a useful framework to construct reduced binding site models, and define the average effect from the surroundings on the ion and the nearest coordinating ligands.

  13. An Experiment Illustrating the Change in Ligand p"K"[subscript a] upon Protein Binding

    ERIC Educational Resources Information Center

    Chenprakhon, Pirom; Panijpan, Bhinyo; Chaiyen, Pimchai

    2012-01-01

    The modulation of ligand p"K"[subscript a] due to its surrounding environment is a crucial feature that controls many biological phenomena. For example, the shift in the p"K"[subscript a] of substrates or catalytic residues at enzyme active sites upon substrate binding often triggers and controls enzymatic reactions. In this work, we developed an…

  14. Fluorescent ligands to investigate GPCR binding properties and oligomerization.

    PubMed

    Cottet, Martin; Faklaris, Orestis; Falco, Amadine; Trinquet, Eric; Pin, Jean-Philippe; Mouillac, Bernard; Durroux, Thierry

    2013-02-01

    Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.

  15. Novel peptide ligand with high binding capacity for antibody purification.

    PubMed

    Lund, Line Naomi; Gustavsson, Per-Erik; Michael, Roice; Lindgren, Johan; Nørskov-Lauritsen, Leif; Lund, Martin; Houen, Gunnar; Staby, Arne; St Hilaire, Phaedria M

    2012-02-17

    Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1M NaOH. Here, we present a novel synthetic peptide ligand for purification of human IgG. Immobilized on WorkBeads, an agarose-based base matrix from Bio-Works, the ligand has a dynamic binding capacity of up to 48 mg/mL and purifies IgG from harvest cell culture fluid with purities and recovery of >93%. The binding affinity is ∼10⁵ M⁻¹ and the interaction is favorable and entropy-driven with an enthalpy penalty. Our results show that the binding of the Fc fragment of IgG is mediated by hydrophobic interactions and that elution at low pH is most likely due to electrostatic repulsion. Furthermore, we have separated aggregated IgG from non-aggregated IgG, indicating that the ligand could be used both as a primary purification step of IgG as well as a subsequent polishing step.

  16. Insights into the Interaction Mechanism of Ligands with Aβ42 Based on Molecular Dynamics Simulations and Mechanics: Implications of Role of Common Binding Site in Drug Design for Alzheimer's Disease.

    PubMed

    Kundaikar, Harish S; Degani, Mariam S

    2015-10-01

    Aggregation of β-amyloid (Aβ) into oligomers and further into fibrils is hypothesized to be a key factor in pathology of Alzheimer's disease (AD). In this study, mapping and docking were used to study the binding of ligands to protofibrils. It was followed by molecular simulations to understand the differences in interactions of known therapeutic agents such as curcumin, fluorescence-based amyloid staining agents such as thioflavin T, and diagnostic agents such as florbetapir (AV45), with Aβ protofibrils. We show that therapeutic agents bind to and distort the protofibrils, thus causing destabilization or prevention of oligomerization, in contrast to diagnostic agents which bind to but do not distort such structures. This has implications in the rational design of ligands, both for diagnostics and therapeutics of AD.

  17. SIENA: Efficient Compilation of Selective Protein Binding Site Ensembles.

    PubMed

    Bietz, Stefan; Rarey, Matthias

    2016-01-25

    Structural flexibility of proteins has an important influence on molecular recognition and enzymatic function. In modeling, structure ensembles are therefore often applied as a valuable source of alternative protein conformations. However, their usage is often complicated by structural artifacts and inconsistent data annotation. Here, we present SIENA, a new computational approach for the automated assembly and preprocessing of protein binding site ensembles. Starting with an arbitrarily defined binding site in a single protein structure, SIENA searches for alternative conformations of the same or sequentially closely related binding sites. The method is based on an indexed database for identifying perfect k-mer matches and a recently published algorithm for the alignment of protein binding site conformations. Furthermore, SIENA provides a new algorithm for the interaction-based selection of binding site conformations which aims at covering all known ligand-binding geometries. Various experiments highlight that SIENA is able to generate comprehensive and well selected binding site ensembles improving the compatibility to both known and unconsidered ligand molecules. Starting with the whole PDB as data source, the computation time of the whole ensemble generation takes only a few seconds. SIENA is available via a Web service at www.zbh.uni-hamburg.de/siena .

  18. Regulation of neurexin 1beta tertiary structure and ligand binding through alternative splicing.

    PubMed

    Shen, Kaiser C; Kuczynska, Dorota A; Wu, Irene J; Murray, Beverly H; Sheckler, Lauren R; Rudenko, Gabby

    2008-03-01

    Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a "splice-insert signaling code." In particular, neurexin 1beta carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1beta+SS#4 reveals dramatic rearrangements to the "hypervariable surface," the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop beta10-beta11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca(2+)-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1beta isoforms acquire neuroligin splice isoform selectivity.

  19. A model for the study of ligand binding to the ribosomal RNA helix h44

    SciTech Connect

    Dibrov, Sergey M.; Parsons, Jerod; Hermann, Thomas

    2010-09-02

    Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.

  20. The FTMap family of web servers for determining and characterizing ligand binding hot spots of proteins

    PubMed Central

    Kozakov, Dima; Grove, Laurie E.; Hall, David R.; Bohnuud, Tanggis; Mottarella, Scott; Luo, Lingqi; Xia, Bing; Beglov, Dmitri; Vajda, Sandor

    2016-01-01

    FTMap is a computational mapping server that identifies binding hot spots of macromolecules, i.e., regions of the surface with major contributions to the ligand binding free energy. To use FTMap, users submit a protein, DNA, or RNA structure in PDB format. FTMap samples billions of positions of small organic molecules used as probes and scores the probe poses using a detailed energy expression. Regions that bind clusters of multiple probe types identify the binding hot spots, in good agreement with experimental data. FTMap serves as basis for other servers, namely FTSite to predict ligand binding sites, FTFlex to account for side chain flexibility, FTMap/param to parameterize additional probes, and FTDyn to map ensembles of protein structures. Applications include determining druggability of proteins, identifying ligand moieties that are most important for binding, finding the most bound-like conformation in ensembles of unliganded protein structures, and providing input for fragment based drug design. FTMap is more accurate than classical mapping methods such as GRID and MCSS, and is much faster than the more recent approaches to protein mapping based on mixed molecular dynamics. Using 16 probe molecules, the FTMap server finds the hot spots of an average size protein in less than an hour. Since FTFlex performs mapping for all low energy conformers of side chains in the binding site, its completion time is proportionately longer. PMID:25855957

  1. Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction.

    PubMed

    Molina, H; Brenner, C; Jacobi, S; Gorka, J; Carel, J C; Kinoshita, T; Holers, V M

    1991-07-01

    The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7.

  2. Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy.

    PubMed

    Hegener, Oliver; Jordan, Randolf; Häberlein, Hanns

    2002-11-01

    We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.

  3. RXR function requires binding to an endogenous terpenoid ligand

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The issue of whether the nuclear receptor RXR must bind to an endogenous, nanomolar affinity ligand in order to perform its natural function is still unsettled (1). On the basis of our previous studies establishing that the Drosophilamelanogaster ortholog of the retinoid X receptor ("ultraspiracle,"...

  4. Ligand recognition by influenza virus. The binding of bivalent sialosides.

    PubMed

    Glick, G D; Toogood, P L; Wiley, D C; Skehel, J J; Knowles, J R

    1991-12-15

    Infection by influenza virus is initiated by a cellular adhesion event that is mediated by the viral protein, hemagglutinin, which is exposed on the surface of the virion. Hemagglutinin recognizes and binds to cell surface sialic acid residues. Although each individual ligand binding interaction is weak, the high affinity of influenza virus for cells that bear sialic acid residues is thought to result from a multivalent attachment process involving many similar recognition events. To evaluate such binding we have synthesized three series of compounds, each containing two sialic acid residues separated by spacers of different length, and have tested them as ligands for influenza hemagglutinin. No increased binding to the bromelain-released hemagglutinin ectodomain was seen for any of the bivalent compounds as determined by 1H NMR titration. In contrast, however, a spacer length between sialic acid residues of approximately 55 A sharply increases the binding of these bidentate species to whole virus as determined by hemagglutination inhibition assays. The most effective compound containing glycines in the linking chain displayed 100-fold increased affinity for whole virus over the paradigm monovalent ligand, Neu5Ac alpha 2Me.

  5. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    PubMed Central

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  6. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  7. Synthesis and binding profile of haloperidol-based bivalent ligands targeting dopamine D(2)-like receptors.

    PubMed

    Salama, Ismail; Löber, Stefan; Hübner, Harald; Gmeiner, Peter

    2014-08-15

    Homodimers of dopamine D2-like receptors are suggested to be of particular importance in the pathophysiology of schizophrenia and, thus, serve as promising targets for the discovery of atypical antipsychotics. This study describes the development of a series of novel bivalent molecules with a pharmacophore derived from the dopamine receptor antagonist haloperidol. These dimers were investigated in comparison to their monomeric analogues for their D2long, D2short, D3, and D4 receptor binding and the ability to bridge two neighboring receptor protomers. Radioligand binding studies provided diagnostic insights when Hill slopes close to two for the bivalent ligand 13 incorporating 22 spacer atoms and a comparative analysis with monovalent control ligands indicated a bivalent binding mode with a simultaneous occupancy of two neighboring binding sites. PMID:25047579

  8. Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

    PubMed Central

    Li, Yan; Zhang, Jingxiao; Gao, Weimin; Zhang, Lilei; Pan, Yanqiu; Zhang, Shuwei; Wang, Yonghua

    2015-01-01

    Cyclin-dependent kinase 2 (CDK2) is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP) binding site (Site I) and two non-competitive binding sites (Site II and III). In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV). All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate). In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2. PMID:25918937

  9. Solvent fluctuations in hydrophobic cavity–ligand binding kinetics

    PubMed Central

    Setny, Piotr; Baron, Riccardo; Michael Kekenes-Huskey, Peter; McCammon, J. Andrew; Dzubiella, Joachim

    2013-01-01

    Water plays a crucial part in virtually all protein–ligand binding processes in and out of equilibrium. Here, we investigate the role of water in the binding kinetics of a ligand to a prototypical hydrophobic pocket by explicit-water molecular dynamics (MD) simulations and implicit diffusional approaches. The concave pocket in the unbound state exhibits wet/dry hydration oscillations whose magnitude and time scale are significantly amplified by the approaching ligand. In turn, the ligand’s stochastic motion intimately couples to the slow hydration fluctuations, leading to a sixfold-enhanced friction in the vicinity of the pocket entrance. The increased friction considerably decelerates association in the otherwise barrierless system, indicating the importance of molecular-scale hydrodynamic effects in cavity–ligand binding arising due to capillary fluctuations. We derive and analyze the diffusivity profile and show that the mean first passage time distribution from the MD simulation can be accurately reproduced by a standard Brownian dynamics simulation if the appropriate position-dependent friction profile is included. However, long-time decays in the water–ligand (random) force autocorrelation demonstrate violation of the Markovian assumption, challenging standard diffusive approaches for rate prediction. Remarkably, the static friction profile derived from the force correlations strongly resembles the profile derived on the Markovian assumption apart from a simple shift in space, which can be rationalized by a time–space retardation in the ligand’s downhill dynamics toward the pocket. The observed spatiotemporal hydrodynamic coupling may be of biological importance providing the time needed for conformational receptor–ligand adjustments, typical of the induced-fit paradigm. PMID:23297241

  10. A β-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

    PubMed

    Byrne, Cillian; Henen, Morkos A; Belnou, Mathilde; Cantrelle, François-Xavier; Kamah, Amina; Qi, Haoling; Giustiniani, Julien; Chambraud, Béatrice; Baulieu, Etienne-Emile; Lippens, Guy; Landrieu, Isabelle; Jacquot, Yves

    2016-09-27

    The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II β-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein. PMID:27641460

  11. A β-Turn Motif in the Steroid Hormone Receptor's Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52.

    PubMed

    Byrne, Cillian; Henen, Morkos A; Belnou, Mathilde; Cantrelle, François-Xavier; Kamah, Amina; Qi, Haoling; Giustiniani, Julien; Chambraud, Béatrice; Baulieu, Etienne-Emile; Lippens, Guy; Landrieu, Isabelle; Jacquot, Yves

    2016-09-27

    The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II β-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein.

  12. STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism

    PubMed Central

    Létourneau, Danny; Bédard, Mikaël; Cabana, Jérôme; Lefebvre, Andrée; LeHoux, Jean-Guy; Lavigne, Pierre

    2016-01-01

    START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6. PMID:27340016

  13. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  14. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  16. 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.

  17. Ligands Binding and Molecular Simulation: the Potential Investigation of a Biosensor Based on an Insect Odorant Binding Protein

    PubMed Central

    Yi, Xin; Zhang, Yanbo; Wang, Peidan; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua

    2015-01-01

    Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications. PMID:25552932

  18. Ligands binding and molecular simulation: the potential investigation of a biosensor based on an insect odorant binding protein.

    PubMed

    Yi, Xin; Zhang, Yanbo; Wang, Peidan; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua

    2015-01-01

    Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications. PMID:25552932

  19. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    NASA Astrophysics Data System (ADS)

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-06-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

  20. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    PubMed Central

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-01-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach. PMID:24903953

  1. Does the ligand-biopolymer equilibrium binding constant depend on the number of bound ligands?

    PubMed

    Beshnova, Daria A; Lantushenko, Anastasia O; Evstigneev, Maxim P

    2010-11-01

    Conventional methods, such as Scatchard or McGhee-von Hippel analyses, used to treat ligand-biopolymer interactions, indirectly make the assumption that the microscopic binding constant is independent of the number of ligands, i, already bound to the biopolymer. Recent results on the aggregation of aromatic molecules (Beshnova et al., J Chem Phys 2009, 130, 165105) indicated that the equilibrium constant of self-association depends intrinsically on the number of molecules in an aggregate due to loss of translational and rotational degrees of freedom on formation of the complex. The influence of these factors on the equilibrium binding constant for ligand-biopolymer complexation was analyzed in this work. It was shown that under the conditions of binding of "small" molecules, these factors can effectively be ignored and, hence, do not provide any hidden systematic error in such widely-used approaches, such as the Scatchard or McGhee-von Hippel methods for analyzing ligand-biopolymer complexation. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 932-935, 2010.

  2. Recent improvements to Binding MOAD: a resource for protein–ligand binding affinities and structures

    PubMed Central

    Ahmed, Aqeel; Smith, Richard D.; Clark, Jordan J.; Dunbar, James B.; Carlson, Heather A.

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein–ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23 269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

  3. Protein Unfolding Coupled to Ligand Binding: Differential Scanning Calorimetry Simulation Approach

    NASA Astrophysics Data System (ADS)

    Soledad Celej, María; Fidelio, Gerardo Daniel; Dassie, Sergio Alberto

    2005-01-01

    The aim of this work is to present the physicochemical basis underlying the changes in protein thermostability upon ligand binding. The article is addressed to advanced undergraduate and postgraduate chemistry students with an interest in protein biophysics. In addition, this article provides a useful tool for both learning and teaching biophysics because it links fundamental concepts: thermodynamics, chemical equilibrium, and protein stability. The influence of protein ligand interactions on thermally-induced protein denaturation was monitored by differential scanning calorimetry (DSC). The changes in DSC output (thermogram) emerge by linking binding equilibrium with reversible protein unfolding thermodynamics. We derive the formalism for the description of protein unfolding in the presence of ligand that can bind to a single site on either native, unfolded, or both protein states. In addition to a rigorous mathematical description of the involved equilibria, the model provides the general formulation for simulating thermograms and calculating the changes in protein species during heating. First, we describe ligand interaction and emphasize the relationship between protein stability parameters and redistribution of species in equilibrium. After that, we describe the origin of bimodal thermograms, and finally, the effect on thermogram shape of protein concentration at constant ligand/protein mole ratio.

  4. Directed evolution of estrogen receptor proteins with altered ligand-binding specificities.

    PubMed

    Islam, Kazi Mohammed Didarul; Dilcher, Meik; Thurow, Corinna; Vock, Carsten; Krimmelbein, Ilga Kristine; Tietze, Lutz Friedjan; Gonzalez, Victor; Zhao, Huimin; Gatz, Christiane

    2009-01-01

    Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor alpha (hER alpha) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER alpha and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 microM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 microM) and a 10-fold decreased activity to 17beta-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.

  5. PSCDB: a database for protein structural change upon ligand binding.

    PubMed

    Amemiya, Takayuki; Koike, Ryotaro; Kidera, Akinori; Ota, Motonori

    2012-01-01

    Proteins are flexible molecules that undergo structural changes to function. The Protein Data Bank contains multiple entries for identical proteins determined under different conditions, e.g. with and without a ligand molecule, which provides important information for understanding the structural changes related to protein functions. We gathered 839 protein structural pairs of ligand-free and ligand-bound states from monomeric or homo-dimeric proteins, and constructed the Protein Structural Change DataBase (PSCDB). In the database, we focused on whether the motions were coupled with ligand binding. As a result, the protein structural changes were classified into seven classes, i.e. coupled domain motion (59 structural changes), independent domain motion (70), coupled local motion (125), independent local motion (135), burying ligand motion (104), no significant motion (311) and other type motion (35). PSCDB provides lists of each class. On each entry page, users can view detailed information about the motion, accompanied by a morphing animation of the structural changes. PSCDB is available at http://idp1.force.cs.is.nagoya-u.ac.jp/pscdb/. PMID:22080505

  6. 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.

  7. In silico identification of anthropogenic chemicals as ligands of zebrafish sex hormone binding globulin

    SciTech Connect

    Thorsteinson, Nels; Ban, Fuqiang; Santos-Filho, Osvaldo; Tabaei, Seyed M.H.; Miguel-Queralt, Solange; Underhill, Caroline; Cherkasov, Artem Hammond, Geoffrey L.

    2009-01-01

    Anthropogenic compounds with the capacity to interact with the steroid-binding site of sex hormone binding globulin (SHBG) pose health risks to humans and other vertebrates including fish. Building on studies of human SHBG, we have applied in silico drug discovery methods to identify potential binders for SHBG in zebrafish (Danio rerio) as a model aquatic organism. Computational methods, including; homology modeling, molecular dynamics simulations, virtual screening, and 3D QSAR analysis, successfully identified 6 non-steroidal substances from the ZINC chemical database that bind to zebrafish SHBG (zfSHBG) with low-micromolar to nanomolar affinities, as determined by a competitive ligand-binding assay. We also screened 80,000 commercial substances listed by the European Chemicals Bureau and Environment Canada, and 6 non-steroidal hits from this in silico screen were tested experimentally for zfSHBG binding. All 6 of these compounds displaced the [{sup 3}H]5{alpha}-dihydrotestosterone used as labeled ligand in the zfSHBG screening assay when tested at a 33 {mu}M concentration, and 3 of them (hexestrol, 4-tert-octylcatechol, and dihydrobenzo(a)pyren-7(8H)-one) bind to zfSHBG in the micromolar range. The study demonstrates the feasibility of large-scale in silico screening of anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Such studies could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.

  8. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    PubMed

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design. PMID:27399277

  9. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    PubMed

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design.

  10. Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

    PubMed

    Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

    2015-09-11

    Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions.

  11. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    PubMed

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  12. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    PubMed

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  13. Energetic Coupling between Ligand Binding and Dimerization in Escherichia coli Phosphoglycerate Mutase.

    PubMed

    Gardner, Nathan W; Monroe, Lyman K; Kihara, Daisuke; Park, Chiwook

    2016-03-29

    Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding of an exceptional quality. The homodimeric Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. We found that, in the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization.

  14. Energetic Coupling between Ligand Binding and Dimerization in E. coli Phosphoglycerate Mutase

    PubMed Central

    Gardner, Nathan W.; Monroe, Lyman K.; Kihara, Daisuke; Park, Chiwook

    2016-01-01

    Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding in an exceptional quality. The homodimeric E. coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. In the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active-site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization. PMID:26919584

  15. Dopaminergic receptor-ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors.

    PubMed

    Naklua, Wanpen; Suedee, Roongnapa; Lieberzeit, Peter A

    2016-07-15

    Molecularly imprinted polymers (MIPs) have been successfully applied as selective materials for assessing the binding activity of agonist and antagonist of dopamine D1 receptor (D1R) by using quartz crystal microbalance (QCM). In this study, D1R derived from rat hypothalamus was used as a template and thus self-organized on stamps. Those were pressed into an oligomer film consisting of acrylic acid: N-vinylpyrrolidone: N,N'-(1,2-dihydroxyethylene) bis-acrylamide in a ratio of 2:3:12 spin coated onto a dual electrode QCM. Such we obtained one D1R-MIP-QCM electrode, whereas the other electrode carried the non-imprinted control polymer (NIP) that had remained untreated. Successful imprinting of D1R was confirmed by AFM. The polymer can re-incorporate D1R leading to frequency responses of 100-1200Hz in a concentration range of 5.9-47.2µM. In a further step such frequency changes proved inherently useful for examining the binding properties of test ligands to D1R. The resulting mass-sensitive measurements revealed Kd of dopamine∙HCl, haloperidol, and (+)-SCH23390 at 0.874, 25.6, and 0.004nM, respectively. These results correlate well with the values determined in radio ligand binding assays. Our experiments revealed that D1R-MIP sensors are useful for estimating the strength of ligand binding to the active single site. Therefore, we have developed a biomimetic surface imprinting strategy for QCM studies of D1R-ligand binding and presented a new method to ligand binding assay for D1R. PMID:26926593

  16. A model for the study of ligand binding to the ribosomal RNA helix h44

    PubMed Central

    Dibrov, Sergey M.; Parsons, Jerod; Hermann, Thomas

    2010-01-01

    Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand–RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region. PMID:20215440

  17. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

  18. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

  19. Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters.

    PubMed

    Kowalczykowski, S C; Paul, L S; Lonberg, N; Newport, J W; McSwiggen, J A; von Hippel, P H

    1986-03-25

    Many biologically important proteins bind nonspecifically, and often cooperatively, to single-or double-stranded nucleic acid lattices in discharging their physiological functions. This binding can generally be described in thermodynamic terms by three parameters: n, the binding site size; K, the intrinsic binding constant; omega, the binding cooperativity parameter. The experimental determination of these parameters often appears to be straightforward but can be fraught with conceptual and methodological difficulties that may not be readily apparent. In this paper we describe and analyze a number of approaches that can be used to measure these protein-nucleic acid interaction parameters and illustrate these methods with experiments on the binding of T4-coded gene 32 (single-stranded DNA binding) protein to various nucleic acid lattices. We consider the following procedures: (i) the titration of a fixed amount of lattice (nucleic acid) with added ligand (protein); (ii) the titration of a fixed amount of ligand with added lattice; (iii) the determination of ligand binding affinities at very low levels of lattice saturation; (iv) the analysis of ligand cluster size distribution on the lattice; (v) the analysis of ligand binding to lattices of finite length. The applicability and limitations of each approach are considered and discussed, and potential pitfalls are explicitly pointed out.

  20. Selectivity in ligand binding to uranyl compounds: A synthetic, structural, thermodynamic and computational study

    SciTech Connect

    Arnold, John

    2015-01-21

    The uranyl cation (UO₂²⁺) is the most abundant form of uranium on the planet. It is estimated that 4.5 billion tons of uranium in this form exist in sea water. The ability to bind and extract the uranyl cation from aqueous solution while separating it from other elements would provide a limitless source of nuclear fuel. A large body of research concerns the selective recognition and extraction of uranyl. A stable molecule, the cation has a linear O=U=O geometry. The short U-O bonds (1.78 Å) arise from the combination of uranium 5f/6d and oxygen 2p orbitals. Due to the oxygen moieties being multiply bonded, these sites were not thought to be basic enough for Lewis acidic coordination to be a viable approach to sequestration. The goal of this research is thus to broaden the coordination chemistry of the uranyl ion by studying new ligand systems via synthetic, structural, thermodynamic and computational methods. It is anticipated that this fundamental science will find use beyond actinide separation technologies in areas such as nuclear waste remediation and nuclear materials. The focus of this study is to synthesize uranyl complexes incorporating amidinate and guanidinate ligands. Both synthetic and computational methods are used to investigate novel equatorial ligand coordination and how this affects the basicity of the oxo ligands. Such an understanding will later apply to designing ligands incorporating functionalities that can bind uranyl both equatorially and axially for highly selective sequestration. Efficient and durable chromatography supports for lanthanide separation will be generated by (1) identifying robust peptoid-based ligands capable of binding different lanthanides with variable affinities, and (2) developing practical synthetic methods for the attachment of these ligands to Dowex ion exchange resins.

  1. Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the alphaVbeta3 integrin.

    PubMed

    Maile, Laura A; Aday, Ariel W; Busby, Walker H; Sanghani, Ravi; Veluvolu, Umadevi; Clemmons, David R

    2008-10-01

    The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.

  2. 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.

  3. 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)

  4. Structure and ligand binding of the extended Tudor domain of D. melanogaster Tudor-SN.

    PubMed

    Friberg, Anders; Corsini, Lorenzo; Mourão, André; Sattler, Michael

    2009-04-10

    The Tudor-SN protein (p100, SND1) has been implicated in a variety of cellular processes, such as transcription, processing of edited double-stranded RNA, and splicing regulation. Molecular details of these functions are not yet understood. Tudor domains have previously been shown to bind methylated ligands, such as methylated lysines and arginines. It has been suggested that the role of Tudor-SN in splicing may involve binding to such methylated ligands or to the methylated 5' cap of spliceosomal snRNAs. Here, we report the crystal structure of the extended Tudor domain of Tudor-SN from Drosophila melanogaster to a resolution of 2.1 A. NMR secondary chemical shifts, relaxation data, and residual dipolar couplings indicate that the solution and crystal structures are similar. Binding of various ligands was investigated by NMR. Binding sites and affinities were characterized by chemical shift perturbations. We show that the aromatic cage of the Tudor domain specifically binds a peptide containing symmetrically dimethylated arginines (sDMA) with micromolar affinity, while the same peptide comprising nonmethylated arginines does not show significant chemical shift perturbations. Tudor-SN preferentially recognizes sDMA over asymmetrically dimethylated arginine (aDMA). In contrast, two 5' cap analogues with different methylation patterns, as well as mono-, di-, and trimethyllysines, show no binding. Our data demonstrate that the Tudor domain of Tudor-SN specifically recognizes sDMA-containing ligands. The aromatic cage of Tudor-SN is very similar to the one in the Tudor domain of the survival of motor neuron protein, which also recognizes sDMA peptides, indicating a conserved binding motif for this methylation mark. Recognition of sDMA in the C-terminal tails of spliceosomal Sm proteins suggests how Tudor-SN may interact with small nuclear ribonucleoprotein particles during the regulation of splicing. PMID:19232356

  5. Specific [(3)H]tryptophan binding sites in rat brain.

    PubMed

    Wong, P T; Lee, H S; Tan, C H; Teo, W L

    1989-01-01

    [(3)H]Tryptophan binds to a single population of sites in the rat cortical synaptosomal membranes. The binding is reversible and follows the law of mass action. By saturation studies using increasing concentration of [(3)H]tryptophan with decreasing specific radioactivity, the apparent K(d) obtained was approx. 0.8 ?M and the B(max) 110 pmol/mg protein. However, the IC(50) obtained for unlabelled tryptophan in displacing [(3)H]tryptophan binding (3.5 nM) was 0.26 ?M. All six naturally occurring aromatic amino acids studied displaced [(3)H]tryptophan binding with tryptophan and phenylalanine showing higher apparent affinity than histidine, tyrosine, dihydroxyphenylalanine and 5-hydroxytryptophan. These binding sites are proteins in nature as they are sensitive to trypsin and ?-chymotrypsin. It is observed that about 37% of the sites seem to be protected from the proteolytic enzymes by the membrane structure. Furthermore, they are extremely sensitive to phospholipase A(2) presumably because altered membrane phospholipids conduce a conformational change in the binding protein. A considerable degree of stereospecificity was demonstrated with the affinity for l-tryptophan about 90 times higher than that for d-tryptophan. The affinity for l-phenylalanine was 8 times higher than that for d-phenylalanine. Ligand specificity for the aromatic amino acids is remarkable as hydrocinnamic acid, 2-phenylethylamine, 5-hydroxytryptamine, histamine, dopamine, ?-aminobutyric acid, glutamic acid and taurine did not displace [(3)H]tryptophan binding. Therefore, these sites are termed aromatic amino acid binding sites (AABS). Whether or not AABS are involved in neuromodulation at the synapse awaits clarification. If so, the endogenous ligand for the AABS may well be tryptophan.

  6. A determination of Mg(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry

    1991-01-01

    Theoretical calculations employing large basis sets and including correlation are carried out for Mg(+) with methanol, water, and formaldehyde. For Mg(+) with ethanol and acetaldehyde, the trends in the binding energies are studied at the self-consistent-field level. The predictions for the binding energy of Mg(+) to methanol and water of 41 + or - 5 and 36 + or - 5 kcal/mol, respectively, are much less than the experimental upper bounds, of 61 + or - 5 and 60 + or - 5 kcal mol, determined by using photodissociation techniques. The theoretical results are inconsistent with the onset of Mg(+) production observed in the photodissociation experiments, as the smallest absorptions are calculated at about 80 kcal/mol for both Mg(+)-CH3OH and Mg(+)-H2O, and these transitions are to bound excited states. The binding energy for Mg(+) with formaldehyde is predicted to be similar to Mg(+)-H2O. The relative binding energies are in reasonable agreement with experiment. The binding energy of a second water molecule to Mg(+) is predicted to be similar to the first. This suggests that the reduced reaction rate observed for the second ligand is not a consequence of a significantly smaller binding energy, at least for the smaller ligards such as those considered in this work.

  7. Computational design of enzyme-ligand binding using a combined energy function and deterministic sequence optimization algorithm.

    PubMed

    Tian, Ye; Huang, Xiaoqiang; Zhu, Yushan

    2015-08-01

    Enzyme amino-acid sequences at ligand-binding interfaces are evolutionarily optimized for reactions, and the natural conformation of an enzyme-ligand complex must have a low free energy relative to alternative conformations in native-like or non-native sequences. Based on this assumption, a combined energy function was developed for enzyme design and then evaluated by recapitulating native enzyme sequences at ligand-binding interfaces for 10 enzyme-ligand complexes. In this energy function, the electrostatic interaction between polar or charged atoms at buried interfaces is described by an explicitly orientation-dependent hydrogen-bonding potential and a pairwise-decomposable generalized Born model based on the general side chain in the protein design framework. The energy function is augmented with a pairwise surface-area based hydrophobic contribution for nonpolar atom burial. Using this function, on average, 78% of the amino acids at ligand-binding sites were predicted correctly in the minimum-energy sequences, whereas 84% were predicted correctly in the most-similar sequences, which were selected from the top 20 sequences for each enzyme-ligand complex. Hydrogen bonds at the enzyme-ligand binding interfaces in the 10 complexes were usually recovered with the correct geometries. The binding energies calculated using the combined energy function helped to discriminate the active sequences from a pool of alternative sequences that were generated by repeatedly solving a series of mixed-integer linear programming problems for sequence selection with increasing integer cuts.

  8. Fragment-Based Design of Ligands Targeting a Novel Site on the Integrase Enzyme of Human Immunodeficiency Virus;#8197;1

    SciTech Connect

    Wielens, Jerome; Headey, Stephen J.; Deadman, John J.; Rhodes, David I.; Parker, Michael W.; Chalmers, David K.; Scanlon, Martin J.

    2011-08-17

    Fragment-based screening has been used to identify a novel ligand binding site on HIV-1 integrase. Crystal structures of fragments bound at this site (shown) have been used to design elaborated second-generation compounds that bind with higher affinity and good ligand efficiency.

  9. 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.

  10. Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets.

    PubMed

    Chen, Wan-Na; Nitsche, Christoph; Pilla, Kala Bharath; Graham, Bim; Huber, Thomas; Klein, Christian D; Otting, Gottfried

    2016-04-01

    Structure-guided drug design relies on detailed structural knowledge of protein-ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall (1)H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group. PMID:26974502

  11. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    SciTech Connect

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  12. Ligand-binding study of Anopheles gambiae chemosensory proteins.

    PubMed

    Iovinella, Immacolata; Bozza, Francesco; Caputo, Beniamino; Della Torre, Alessandra; Pelosi, Paolo

    2013-06-01

    Chemosensory proteins (CSPs) are a class of small proteins expressed only in arthropods and endowed with heterogeneous functions. Some of them are involved in chemical communications, others in development or other physiological roles. The numbers of CSPs in different species of insects range from 4 in Drosophila to at least 70 in locusts, whereas in other arthropods such as crustaceans and millipedes, only 2-3 very similar sequences have been reported in each species. We have expressed, in a bacterial system, 5 of the 8 CSPs predicted by the genome of the malaria mosquito Anopheles gambiae, 4 identified at the protein level (SAP1, SAP2, SAP3, and CSP3) and a fifth annotated as part of this work, obtaining the proteins with high yields and in their soluble forms. Purified CSPs have been used to study their ligand-binding properties, both using competitive binding assays and quenching of intrinsic tryptophan fluorescence, in order to get insights into their physiological functions. The agreement between the 2 sets of data supports the assumptions that the ligands, including the fluorescent reporter, bind within the core of the proteins. Their different affinities toward a set of pure chemicals suggest specific roles in chemical communication.

  13. Exploring Hydrophobic Binding Surfaces Using Comfa and Flexible Hydrophobic Ligands

    NASA Astrophysics Data System (ADS)

    Thakkar, Shraddha; Sanchez, Rosa. I.; Bhuveneswaran, Chidambaram; Compadre, Cesar M.

    2011-06-01

    Cysteine proteinases are a very important group of enzymes involved in a variety of physiological and pathological processes including cancer metastasis and rheumatoid arthritis. In this investigation we used 3D-Quantitative Structure Activity Relationships (3D-QSAR) techniques to model the binding of a variety of substrates to two cysteine proteinases, papain, and cathepsin B. The analysis was performed using Comparative Molecular Field Analysis (CoMFA). The molecules were constructed using standard bond angles and lengths, minimized and aligned. Charges were calculated using the PM3 method in MOPAC. The CoMFA models derived for the binding of the studied substrates to the two proteinases were compared with the expected results from the experimental X-ray crystal structures of the same proteinases. The results showed the value of CoMFA modeling of flexible hydrophobic ligands to analyze ligand binding to protein receptors, and could also serve as the basis to design specific inhibitors of cysteine proteinases with potential therapeutic value.

  14. Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

    PubMed

    Istyastono, Enade P; Nijmeijer, Saskia; Lim, Herman D; van de Stolpe, Andrea; Roumen, Luc; Kooistra, Albert J; Vischer, Henry F; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

    2011-12-01

    The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

  15. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  16. Ligand Binding to the Androgen Receptor Induces Conformational Changes That Regulate Phosphatase Interactions▿

    PubMed Central

    Yang, Chun-Song; Xin, Hong-Wu; Kelley, Joshua B.; Spencer, Adam; Brautigan, David L.; Paschal, Bryce M.

    2007-01-01

    We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation. PMID:17325038

  17. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

  18. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates. PMID:27497172

  19. Relationships of ligand binding, redox properties, and protonation in Coprinus cinereus peroxidase.

    PubMed

    Ciaccio, Chiara; Rosati, Antonella; De Sanctis, Giampiero; Sinibaldi, Federica; Marini, Stefano; Santucci, Roberto; Ascenzi, Paolo; Welinder, Karen G; Coletta, Massimo

    2003-05-23

    The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25 degrees C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp245, which is H-bonded to N delta of the imidazole of the proximal His183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox- and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.

  20. Molecular Properties of Globin Channels and Pores: Role of Cholesterol in Ligand Binding and Movement.

    PubMed

    Morrill, Gene A; Kostellow, Adele B

    2016-01-01

    Globins contain one or more cavities that control or affect such functions as ligand movement and ligand binding. Here we report that the extended globin family [cytoglobin (Cygb); neuroglobin (Ngb); myoglobin (Mb); hemoglobin (Hb) subunits Hba(α); and Hbb(β)] contain either a transmembrane (TM) helix or pore-lining region as well as internal cavities. Protein motif/domain analyses indicate that Ngb and Hbb each contain 5 cholesterol- binding (CRAC/CARC) domains and 1 caveolin binding motif, whereas the Cygb dimer has 6 cholesterol-binding domains but lacks caveolin-binding motifs. Mb and Hba each exhibit 2 cholesterol-binding domains and also lack caveolin-binding motifs. The Hb αβ-tetramer contains 14 cholesterol-binding domains. Computer algorithms indicate that Cygb and Ngb cavities display multiple partitions and C-terminal pore-lining regions, whereas Mb has three major cavities plus a C-terminal pore-lining region. The Hb tetramer exhibits a large internal cavity but the subunits differ in that they contain a C-terminal TM helix (Hba) and pore-lining region (Hbb). The cavities include 43 of 190 Cygb residues, 38 of 151 of Ngb residues, 55 of 154 Mb residues, and 137 of 688 residues in the Hb tetramer. Each cavity complex includes 6 to 8 residues of the TM helix or pore-lining region and CRAC/CARC domains exist within all cavities. Erythrocyte Hb αβ-tetramers are largely cytosolic but also bind to a membrane anion exchange protein, "band 3," which contains a large internal cavity and 12 TM helices (5 being pore-lining regions). The Hba TM helix may be the erythrocyte membrane "band 3" attachment site. "Band 3" contributes 4 caveolin binding motifs and 10 CRAC/CARC domains. Cholesterol binding may create lipid-disordered phases that alter globin cavities and facilitate ligand movement, permitting ion channel formation and conformational changes that orchestrate anion and ligand (O2, CO2, NO) movement within the large internal cavities and channels of the

  1. Molecular Properties of Globin Channels and Pores: Role of Cholesterol in Ligand Binding and Movement

    PubMed Central

    Morrill, Gene A.; Kostellow, Adele B.

    2016-01-01

    Globins contain one or more cavities that control or affect such functions as ligand movement and ligand binding. Here we report that the extended globin family [cytoglobin (Cygb); neuroglobin (Ngb); myoglobin (Mb); hemoglobin (Hb) subunits Hba(α); and Hbb(β)] contain either a transmembrane (TM) helix or pore-lining region as well as internal cavities. Protein motif/domain analyses indicate that Ngb and Hbb each contain 5 cholesterol- binding (CRAC/CARC) domains and 1 caveolin binding motif, whereas the Cygb dimer has 6 cholesterol-binding domains but lacks caveolin-binding motifs. Mb and Hba each exhibit 2 cholesterol-binding domains and also lack caveolin-binding motifs. The Hb αβ-tetramer contains 14 cholesterol-binding domains. Computer algorithms indicate that Cygb and Ngb cavities display multiple partitions and C-terminal pore-lining regions, whereas Mb has three major cavities plus a C-terminal pore-lining region. The Hb tetramer exhibits a large internal cavity but the subunits differ in that they contain a C-terminal TM helix (Hba) and pore-lining region (Hbb). The cavities include 43 of 190 Cygb residues, 38 of 151 of Ngb residues, 55 of 154 Mb residues, and 137 of 688 residues in the Hb tetramer. Each cavity complex includes 6 to 8 residues of the TM helix or pore-lining region and CRAC/CARC domains exist within all cavities. Erythrocyte Hb αβ-tetramers are largely cytosolic but also bind to a membrane anion exchange protein, “band 3,” which contains a large internal cavity and 12 TM helices (5 being pore-lining regions). The Hba TM helix may be the erythrocyte membrane “band 3” attachment site. “Band 3” contributes 4 caveolin binding motifs and 10 CRAC/CARC domains. Cholesterol binding may create lipid-disordered phases that alter globin cavities and facilitate ligand movement, permitting ion channel formation and conformational changes that orchestrate anion and ligand (O2, CO2, NO) movement within the large internal cavities and

  2. Molecular Properties of Globin Channels and Pores: Role of Cholesterol in Ligand Binding and Movement

    PubMed Central

    Morrill, Gene A.; Kostellow, Adele B.

    2016-01-01

    Globins contain one or more cavities that control or affect such functions as ligand movement and ligand binding. Here we report that the extended globin family [cytoglobin (Cygb); neuroglobin (Ngb); myoglobin (Mb); hemoglobin (Hb) subunits Hba(α); and Hbb(β)] contain either a transmembrane (TM) helix or pore-lining region as well as internal cavities. Protein motif/domain analyses indicate that Ngb and Hbb each contain 5 cholesterol- binding (CRAC/CARC) domains and 1 caveolin binding motif, whereas the Cygb dimer has 6 cholesterol-binding domains but lacks caveolin-binding motifs. Mb and Hba each exhibit 2 cholesterol-binding domains and also lack caveolin-binding motifs. The Hb αβ-tetramer contains 14 cholesterol-binding domains. Computer algorithms indicate that Cygb and Ngb cavities display multiple partitions and C-terminal pore-lining regions, whereas Mb has three major cavities plus a C-terminal pore-lining region. The Hb tetramer exhibits a large internal cavity but the subunits differ in that they contain a C-terminal TM helix (Hba) and pore-lining region (Hbb). The cavities include 43 of 190 Cygb residues, 38 of 151 of Ngb residues, 55 of 154 Mb residues, and 137 of 688 residues in the Hb tetramer. Each cavity complex includes 6 to 8 residues of the TM helix or pore-lining region and CRAC/CARC domains exist within all cavities. Erythrocyte Hb αβ-tetramers are largely cytosolic but also bind to a membrane anion exchange protein, “band 3,” which contains a large internal cavity and 12 TM helices (5 being pore-lining regions). The Hba TM helix may be the erythrocyte membrane “band 3” attachment site. “Band 3” contributes 4 caveolin binding motifs and 10 CRAC/CARC domains. Cholesterol binding may create lipid-disordered phases that alter globin cavities and facilitate ligand movement, permitting ion channel formation and conformational changes that orchestrate anion and ligand (O2, CO2, NO) movement within the large internal cavities and

  3. Mechanism and thermodynamics of ligand binding to auxin amidohydrolase.

    PubMed

    Simunovic, Mijo; Zagrovic, Bojan; Tomić, Sanja

    2011-01-01

    BrILL2 is catalytically the most efficient auxin amidohydrolase from Brassica rapa, playing a key role in auxin metabolism by catalyzing its release from amino acid conjugates. Auxins, with the most abundant representative indole-acetic acid ([1H-indol-3-yl]-acetic acid, IAA), are a group of plant hormones that in very small concentrations regulate ubiquitin-mediated degradation of transcription regulators. Kinetic studies on BrILL2 showed that it hydrolyzes alanine conjugates of IAA and of its larger analogues, indole-propionic acid (3-[1H-indol-3-yl]-propionic acid, IPA) and indole-butyric acid (4-[1H-indol-3-yl]-butyric acid, IBA). Structurally, BrILL2 belongs to the largest known family of metallopeptidases (M20) that share a recognizable 3D structure, characterized by two perpendicular domains. Its members have been implicated in numerous biochemical processes and have been found across all species sequenced to date. Here, molecular dynamics simulations were carried out to study structural and thermodynamic properties of ligand binding to BrILL2. A conformational change was captured in multiple copies of 10 ns long simulations, described by a rigid body movement of the two domains, and its associated key interactions between residues were examined. For the three substrates, complexes in two possible binding modes were recreated, along with a single binding mode for the putative substrate tryptophanyl-alanine (Trp-Ala), which were subsequently simulated in multiple copies of 10 ns long simulations. Thermodynamic calculations were used to assess their binding affinities and explain the selectivity toward the longer ligands. Based on the results, a possible route for the reaction is proposed.

  4. MODELING THE BINDING OF THE METABOLITES OF SOME POLYCYCLIC AROMTIC HYDROCARBONS TO THE LIGAND BINDING DOMAIN OF THE ESTROGEN RECEPTOR

    EPA Science Inventory

    Modeling the binding of the metabolites of some Polycyclic Aromatic Hydrocarbons to the ligand binding domain of the estrogen receptor
    James Rabinowitz, Stephen Little, Katrina Brown, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC; Un...

  5. Study on effects of molecular crowding on G-quadruplex-ligand binding and ligand-mediated telomerase inhibition.

    PubMed

    Yaku, Hidenobu; Murashima, Takashi; Tateishi-Karimata, Hisae; Nakano, Shu-ichi; Miyoshi, Daisuke; Sugimoto, Naoki

    2013-11-01

    The telomere G-quadruplex-binding and telomerase-inhibiting capacity of two cationic (TMPyP4 and PIPER) and two anionic (phthalocyanine and Hemin) G-quadruplex-ligands were examined under conditions of molecular crowding (MC). Osmotic experiments showed that binding of the anionic ligands, which bind to G-quadruplex DNA via π-π stacking interactions, caused some water molecules to be released from the G-quadruplex/ligand complex; in contrast, a substantial number of water molecules were taken up upon electrostatic binding of the cationic ligands to G-quadruplex DNA. These behaviors of water molecules maintained or reduced the binding affinity of the anionic and the cationic ligands, respectively, under MC conditions. Consequently, the anionic ligands (phthalocyanine and Hemin) robustly inhibited telomerase activity even with MC; in contrast, the inhibition of telomerase caused by cationic TMPyP4 was drastically reduced by MC. These results allow us to conclude that the binding of G-quadruplex-ligands to G-quadruplex via non-electrostatic interactions is preferable for telomerase inhibition under physiological conditions.

  6. Study on effects of molecular crowding on G-quadruplex-ligand binding and ligand-mediated telomerase inhibition.

    PubMed

    Yaku, Hidenobu; Murashima, Takashi; Tateishi-Karimata, Hisae; Nakano, Shu-ichi; Miyoshi, Daisuke; Sugimoto, Naoki

    2013-11-01

    The telomere G-quadruplex-binding and telomerase-inhibiting capacity of two cationic (TMPyP4 and PIPER) and two anionic (phthalocyanine and Hemin) G-quadruplex-ligands were examined under conditions of molecular crowding (MC). Osmotic experiments showed that binding of the anionic ligands, which bind to G-quadruplex DNA via π-π stacking interactions, caused some water molecules to be released from the G-quadruplex/ligand complex; in contrast, a substantial number of water molecules were taken up upon electrostatic binding of the cationic ligands to G-quadruplex DNA. These behaviors of water molecules maintained or reduced the binding affinity of the anionic and the cationic ligands, respectively, under MC conditions. Consequently, the anionic ligands (phthalocyanine and Hemin) robustly inhibited telomerase activity even with MC; in contrast, the inhibition of telomerase caused by cationic TMPyP4 was drastically reduced by MC. These results allow us to conclude that the binding of G-quadruplex-ligands to G-quadruplex via non-electrostatic interactions is preferable for telomerase inhibition under physiological conditions. PMID:23562626

  7. Protein-ligand binding affinity by nonequilibrium free energy methods.

    PubMed

    Cossins, Benjamin P; Foucher, Sebastien; Edge, Colin M; Essex, Jonathan W

    2008-11-27

    Nonequilibrium (NE) free energy methods are embarrassingly parallel and may be very conveniently run on desktop computers using distributed computing software. In recent years there has been a proliferation of NE methods, but these approaches have barely, if at all, been used in the context of calculating protein-ligand binding free energies. In a recent study by these authors, different combinations of NE methods with various test systems were compared and protocols identified which yielded results as accurate as replica exchange thermodynamic integration (RETI). The NE approaches, however, lend themselves to extensive parallelization through the use of distributed computing. Here the best performing of those NE protocols, a replica exchange method using Bennett's acceptance ratio as the free energy estimator (RENE), is applied to two sets of congeneric inhibitors bound to neuraminidase and cyclooxygenase-2. These protein-ligand systems were originally studied with RETI, giving results to which NE and RENE simulations are compared. These NE calculations were carried out on a large, highly distributed group of low-performance desktop computers which are part of a Condor pool. RENE was found to produce results of a predictive quality at least as good as RETI in less than half the wall clock time. However, non-RE NE results were found to be far less predictive. In addition, the RENE method successfully identified a localized region of rapidly changing free energy gradients without the need for prior investigation. These results suggest that the RENE protocol is appropriate for use in the context of predicting protein-ligand binding free energies and that it can offer advantages over conventional, equilibrium approaches. PMID:18973369

  8. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

    PubMed Central

    Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  9. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

    PubMed

    Kržan, Mojca; Vianello, Robert; Maršavelski, Aleksandra; Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N-H and O-H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  10. Binding balls: fast detection of binding sites using a property of spherical Fourier transform.

    PubMed

    Comin, Matteo; Guerra, Concettina; Dellaert, Frank

    2009-11-01

    The functional prediction of proteins is one of the most challenging problems in modern biology. An established computational technique involves the identification of three-dimensional local similarities in proteins. In this article, we present a novel method to quickly identify promising binding sites. Our aim is to efficiently detect putative binding sites without explicitly aligning them. Using the theory of Spherical Harmonics, a candidate binding site is modeled as a Binding Ball. The Binding Ball signature, offered by the Spherical Fourier coefficients, can be efficiently used for a fast detection of putative regions. Our contribution includes the Binding Ball modeling and the definition of a scoring function that does not require aligning candidate regions. Our scoring function can be computed efficiently using a property of Spherical Fourier transform (SFT) that avoids the evaluation of all alignments. Experiments on different ligands show good discrimination power when searching for known binding sites. Moreover, we prove that this method can save up to 40% in time compared with traditional approaches.

  11. Automated Site Preparation in Physics-Based Rescoring of Receptor Ligand Complexes

    PubMed Central

    Rapp, Chaya S.; Schonbrun, Cheryl; Jacobson, Matthew P.; Kalyanaraman, Chakrapani; Huang, Niu

    2009-01-01

    Hydrogen atoms are not typically observable in xray crystal structures but inferring their locations is often important in structure-based drug design. In addition, protonation states of the protein can change in response to ligand binding, as can the orientations of OH groups, a subtle form of “induced fit”. We implement and evaluate an automated procedure for optimizing polar hydrogens in protein binding sites in complex with ligands. Specifically, we apply the previously described ICDA algorithm (Proteins 66: 824–837), which assigns the ionization states of titratable residues, the amide orientations of Asn/Gln side chains, the imidazole ring orientation in His, and the orientations of OH/SH groups, in a unified algorithm. We test the utility of this method for identifying native-like ligand poses using 247 protein-ligand complexes from an established database of docked decoys. Pose selection is performed with a physics-based scoring function based on a molecular mechanics energy function and a Generalized Born implicit solvent model. The use of the ICDA receptor preparation protocol, implemented with no knowledge of the native ligand pose, increases the accuracy of pose selection significantly, with the average RMSD over all complexes decreasing from 2.7 to 1.5 Å when applying ICDA. Large improvements are seen for specific classes of binding sites with titratable groups, such as aspartyl proteases. PMID:19382204

  12. Automated site preparation in physics-based rescoring of receptor ligand complexes.

    PubMed

    Rapp, Chaya S; Schonbrun, Cheryl; Jacobson, Matthew P; Kalyanaraman, Chakrapani; Huang, Niu

    2009-10-01

    Hydrogen atoms are not typically observable in X-ray crystal structures, but inferring their locations is often important in structure-based drug design. In addition, protonation states of the protein can change in response to ligand binding, as can the orientations of OH groups, a subtle form of "induced fit." We implement and evaluate an automated procedure for optimizing polar hydrogens in protein-binding sites in complex with ligands. Specifically, we apply the previously described Independent Cluster Decomposition Algorithm (ICDA) algorithm (Li et al., Proteins 2007;66:824-837), which assigns the ionization states of titratable residues, the amide orientations of Asn/Gln side chains, the imidazole ring orientation in His, and the orientations of OH/SH groups, in a unified algorithm. We test the utility of this method for identifying nativelike ligand poses using 247 protein-ligand complexes from an established database of docked decoys. Pose selection is performed with a physics-based scoring function based on a molecular mechanics energy function and a Generalized Born implicit solvent model. The use of the ICDA receptor preparation protocol, implemented with no knowledge of the native ligand pose, increases the accuracy of pose selection significantly, with the average RMSD over all complexes decreasing from 2.7 to 1.5 A when applying ICDA. Large improvements are seen for specific classes of binding sites with titratable groups, such as aspartyl proteases.

  13. 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.

  14. Elimination of a ligand gating site generates a supersensitive olfactory receptor

    PubMed Central

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

    2016-01-01

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

  15. Evolution of off-lattice model proteins under ligand binding constraints.

    PubMed

    Nelson, Erik D; Grishin, Nick V

    2016-08-01

    We investigate protein evolution using an off-lattice polymer model evolved to imitate the behavior of small enzymes. Model proteins evolve through mutations to nucleotide sequences (including insertions and deletions) and are selected to fold and maintain a specific binding site compatible with a model ligand. We show that this requirement is, in itself, sufficient to maintain an ordered folding domain, and we compare it to the requirement of folding an ordered (but otherwise unrestricted) domain. We measure rates of amino acid change as a function of local environment properties such as solvent exposure, packing density, and distance from the active site, as well as overall rates of sequence and structure change, both along and among model lineages in star phylogenies. The model recapitulates essentially all of the behavior found in protein phylogenetic analyses, and predicts that amino acid substitution rates vary linearly with distance from the binding site. PMID:27627338

  16. Evolution of off-lattice model proteins under ligand binding constraints

    NASA Astrophysics Data System (ADS)

    Nelson, Erik D.; Grishin, Nick V.

    2016-08-01

    We investigate protein evolution using an off-lattice polymer model evolved to imitate the behavior of small enzymes. Model proteins evolve through mutations to nucleotide sequences (including insertions and deletions) and are selected to fold and maintain a specific binding site compatible with a model ligand. We show that this requirement is, in itself, sufficient to maintain an ordered folding domain, and we compare it to the requirement of folding an ordered (but otherwise unrestricted) domain. We measure rates of amino acid change as a function of local environment properties such as solvent exposure, packing density, and distance from the active site, as well as overall rates of sequence and structure change, both along and among model lineages in star phylogenies. The model recapitulates essentially all of the behavior found in protein phylogenetic analyses, and predicts that amino acid substitution rates vary linearly with distance from the binding site.

  17. 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.

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

    SciTech Connect

    Sergeev, P.V.; Kalinin, G.V.; Dukhanin, A.S.

    1987-01-01

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

  19. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    PubMed

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  20. A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

    PubMed

    Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

    2014-12-01

    We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

  1. Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding

    PubMed Central

    Young, Tom; Abel, Robert; Kim, Byungchan; Berne, Bruce J.; Friesner, Richard A.

    2007-01-01

    The thermodynamic properties and phase behavior of water in confined regions can vary significantly from that observed in the bulk. This is particularly true for systems in which the confinement is on the molecular-length scale. In this study, we use molecular dynamics simulations and a powerful solvent analysis technique based on inhomogenous solvation theory to investigate the properties of water molecules that solvate the confined regions of protein active sites. Our simulations and analysis indicate that the solvation of protein active sites that are characterized by hydrophobic enclosure and correlated hydrogen bonds induce atypical entropic and enthalpic penalties of hydration. These penalties apparently stabilize the protein–ligand complex with respect to the independently solvated ligand and protein, which leads to enhanced binding affinities. Our analysis elucidates several challenging cases, including the super affinity of the streptavidin–biotin system. PMID:17204562

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

    PubMed Central

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

    2013-01-01

    Many proteins function by interacting with other small molecules (ligands). Identification of ligand-binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To enhance our understanding of LBSs, it is worth performing such comparisons among patches and classifying them based on similarities of their surface configurations and electrostatic potentials. In this study, we first developed a rapid method to compare two patches. We then clustered patches corresponding to the same PDB chemical component identifier for a ligand, and selected a representative patch from each cluster. We subsequently exhaustively as compared the representative patches and clustered them using similarity score, PatSim. Finally, the resultant PatSim scores were compared with similarities of atomic structures of the LBSs and those of the ligand-binding protein sequences and functions. Consequently, we classified the patches into ∼2000 well-characterized clusters. We found that about 63% of these clusters are used in identical protein folds, although about 25% of the clusters are conserved in distantly related proteins and even in proteins with cross-fold similarity. Furthermore, we showed that patches with higher PatSim score have potential to be involved in similar biological processes. PMID:23934772

  3. Interaction with membrane lipids and heme ligand binding properties of Escherichia coli flavohemoglobin.

    PubMed

    Bonamore, Alessandra; Farina, Anna; Gattoni, Maurizio; Schininà, M Eugenia; Bellelli, Andrea; Boffi, Alberto

    2003-05-20

    Escherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity. Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron. In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule. Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E. coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains. The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide. The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket. PMID:12741837

  4. Mechanism for attenuation of DNA binding by MarR family transcriptional regulators by small molecule ligands.

    PubMed

    Perera, Inoka C; Lee, Yong-Hwan; Wilkinson, Steven P; Grove, Anne

    2009-07-31

    Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands. PMID:19501097

  5. A Natural Mutation in Helix 5 of the Ligand Binding Domain of Glucocorticoid Receptor Enhances Receptor-Ligand Interaction

    PubMed Central

    Reyer, Henry; Ponsuksili, Siriluck; Kanitz, Ellen; Pöhland, Ralf; Wimmers, Klaus; Murani, Eduard

    2016-01-01

    The glucocorticoid receptor (GR) is a central player in the neuroendocrine stress response; it mediates feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis and physiological actions of glucocorticoids in the periphery. Despite intensive investigations of GR in the context of receptor-ligand interaction, only recently the first naturally occurring gain-of-function substitution, Ala610Val, of the ligand binding domain was identified in mammals. We showed that this mutation underlies a major quantitative trait locus for HPA axis activity in pigs, reducing cortisol production by about 40–50 percent. To unravel the molecular mechanisms behind this gain of function, receptor-ligand interactions were evaluated in silico, in vitro and in vivo. In accordance with previously observed phenotypic effects, the mutant Val610 GR showed significantly increased activation in response to glucocorticoid and non-glucocorticoid steroids, and, as revealed by GR-binding studies in vitro and in pituitary glands, enhanced ligand binding. Concordantly, the protein structure prediction depicted reduced binding distances between the receptor and ligand, and altered interactions in the ligand binding pocket. Consequently, the Ala610Val substitution opens up new structural information for the design of potent GR ligands and to examine effects of the enhanced GR responsiveness to glucocorticoids on the entire organism. PMID:27736993

  6. Ligand binding-dependent functions of the lipocalin NLaz: an in vivo study in Drosophila.

    PubMed

    Ruiz, Mario; Ganfornina, Maria D; Correnti, Colin; Strong, Roland K; Sanchez, Diego

    2014-04-01

    Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket. PMID:24361577

  7. Ligand binding-dependent functions of the lipocalin NLaz: an in vivo study in Drosophila.

    PubMed

    Ruiz, Mario; Ganfornina, Maria D; Correnti, Colin; Strong, Roland K; Sanchez, Diego

    2014-04-01

    Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket.

  8. An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.

    PubMed

    Syedbasha, Mohameedyaseen; Linnik, Janina; Santer, Deanna; O'Shea, Daire; Barakat, Khaled; Joyce, Michael; Khanna, Nina; Tyrrell, D Lorne; Houghton, Michael; Egli, Adrian

    2016-01-01

    A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

  9. Regulation of Neurexin 1[beta] Tertiary Structure and Ligand Binding through Alternative Splicing

    SciTech Connect

    Shen, Kaiser C.; Kuczynska, Dorota A.; Wu, Irene J.; Murray, Beverly H.; Sheckler, Lauren R.; Rudenko, Gabby

    2008-08-04

    Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a splice-insert signaling code. In particular, neurexin 1{beta} carrying an alternative splice insert at site SS{number_sign}4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1{beta}+SS{number_sign}4 reveals dramatic rearrangements to the 'hypervariable surface', the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop {beta}10-{beta}11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca{sup 2+}-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1{beta} isoforms acquire neuroligin splice isoform selectivity.

  10. Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase

    SciTech Connect

    Gruber, Todd D.; Borrok, M. Jack; Westler, William M.; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5{prime}-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 {angstrom} resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the

  11. Unspecific Cooperative Ligand Binding to One-Dimensional Lattice-like Macromolecules

    NASA Astrophysics Data System (ADS)

    Velazquez-Campoy, Adrian

    2006-08-01

    Unspecific ligand binding to one-dimensional lattice-like macromolecules, a common event in nature (e.g. ligand/protein binding to DNA and certain carbohydrates), presents distinctive features when compared to specific ligand binding to macromolecules. McGhee and von Hippel developed a mathematical formalism in the form of the Scatchard representation. This article presents the application of the theory for unspecific cooperative ligand binding to linear lattice-like macromolecules in isothermal titration calorimetry following an exact and accurate method, without the limitations and deficiencies of the Scatchard formalism.

  12. Muscarine binding sites in bovine adrenal medulla.

    PubMed

    Barron, B A; Murrin, L C; Hexum, T D

    1986-03-18

    The presence of muscarinic binding sites in the bovine adrenal medulla was investigated using [3H]QNB and the bovine adrenal medulla. Scatchard analysis combined with computer analysis yielded data consistent with a two binding site configuration. KDs of 0.15 and 14 nM and Bmax s of 29 and 210 fmol/mg protein, respectively, were observed. Displacement of [3H]QNB by various cholinergic agents is, in order of decreasing potency: QNB, dexetimide, atropine, scopolamine, imipramine, desipramine, oxotremorine, pilocarpine, acetylcholine, methacholine and carbachol. These results demonstrate the presence of more than one muscarine binding site in the bovine adrenal gland. PMID:3709656

  13. Multiple instance learning of Calmodulin binding sites

    PubMed Central

    Minhas, Fayyaz ul Amir Afsar; Ben-Hur, Asa

    2012-01-01

    Motivation: Calmodulin (CaM) is a ubiquitously conserved protein that acts as a calcium sensor, and interacts with a large number of proteins. Detection of CaM binding proteins and their interaction sites experimentally requires a significant effort, so accurate methods for their prediction are important. Results: We present a novel algorithm (MI-1 SVM) for binding site prediction and evaluate its performance on a set of CaM-binding proteins extracted from the Calmodulin Target Database. Our approach directly models the problem of binding site prediction as a large-margin classification problem, and is able to take into account uncertainty in binding site location. We show that the proposed algorithm performs better than the standard SVM formulation, and illustrate its ability to recover known CaM binding motifs. A highly accurate cascaded classification approach using the proposed binding site prediction method to predict CaM binding proteins in Arabidopsis thaliana is also presented. Availability: Matlab code for training MI-1 SVM and the cascaded classification approach is available on request. Contact: fayyazafsar@gmail.com or asa@cs.colostate.edu PMID:22962461

  14. Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

    SciTech Connect

    Zhang, Hongmin; Liu, Jin-huan; Yang, Wei; Springer, Timothy; Shimaoka, Motomu; Wang, Jia-huai

    2010-09-21

    The activity of integrin LFA-1 ({alpha}{sub L}{beta}{sub 2}) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of {alpha}{sub L} chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.

  15. Mac-2 binding protein is a novel E-selectin ligand expressed by breast cancer cells.

    PubMed

    Shirure, Venktesh S; Reynolds, Nathan M; Burdick, Monica M

    2012-01-01

    Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by

  16. A New Method for Navigating Optimal Direction for Pulling Ligand from Binding Pocket: Application to Ranking Binding Affinity by Steered Molecular Dynamics.

    PubMed

    Vuong, Quan Van; Nguyen, Tin Trung; Li, Mai Suan

    2015-12-28

    In this paper we present a new method for finding the optimal path for pulling a ligand from the binding pocket using steered molecular dynamics (SMD). Scoring function is defined as the steric hindrance caused by a receptor to ligand movement. Then the optimal path corresponds to the minimum of this scoring function. We call the new method MSH (Minimal Steric Hindrance). Contrary to existing navigation methods, our approach takes into account the geometry of the ligand while other methods including CAVER only consider the ligand as a sphere with a given radius. Using three different target + receptor sets, we have shown that the rupture force Fmax and nonequilibrium work Wpull obtained based on the MSH method show a much higher correlation with experimental data on binding free energies compared to CAVER. Furthermore, Wpull was found to be a better indicator for binding affinity than Fmax. Thus, the new MSH method is a reliable tool for obtaining the best direction for ligand exiting from the binding site. Its combination with the standard SMD technique can provide reasonable results for ranking binding affinities using Wpull as a scoring function. PMID:26595261

  17. Two bradykinin binding sites with picomolar affinities

    SciTech Connect

    Manning, D.C.; Vavrek, R.; Stewart, J.M.; Snyder, S.H.

    1986-05-01

    Bradykinin (BK) and related peptides exert a wide range of effects on several organ systems. We have attempted to sort out these effects by studying the binding interaction of (/sup 3/H)BK at the membrane level with in vitro receptor binding techniques. High specific activity (/sup 3/H)BK and an enzyme inhibitor cocktail has enabled us to label two BK binding sites with different affinity and peptide specificity in several guinea-pig tissues. In the guinea-pig ileum the high-affinity site has an equilibrium dissociation constant (Kd) for (/sup 3/H)BK of 13 pM and a maximal number of binding sites of 8.3 pmol/g of tissue wet weight. The low-affinity guinea-pig ileum site displays a Kd of 910 pM, a maximum number of binding sites of 14 pmol/g of tissue wet weight and shows a greater selectivity for BK analogs over Lysyl-BK analogs. Two similar sites can also be discriminated in kidney and heart. The potencies of a series of BK analogs at the high-affinity guinea-pig ileum site correlate well with their potencies in contracting ileal smooth muscle. The binding of (/sup 3/H)BK in the guinea-pig ileum is inhibited by physiological concentrations of monovalent and divalent cations.

  18. A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand

    PubMed Central

    Sun, Qi; Phan, Jason; Friberg, Anders R.; Camper, DeMarco V.; Olejniczak, Edward T.; Fesik, Stephen W.

    2015-01-01

    K-Ras is a well-validated cancer target but is considered to be “undruggable” due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets. PMID:25087006

  19. Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors.

    PubMed

    Aráoz, Rómulo; Vilariño, Natalia; Botana, Luis M; Molgó, Jordi

    2010-07-01

    Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents.

  20. CryptoSite: Expanding the Druggable Proteome by Characterization and Prediction of Cryptic Binding Sites.

    PubMed

    Cimermancic, Peter; Weinkam, Patrick; Rettenmaier, T Justin; Bichmann, Leon; Keedy, Daniel A; Woldeyes, Rahel A; Schneidman-Duhovny, Dina; Demerdash, Omar N; Mitchell, Julie C; Wells, James A; Fraser, James S; Sali, Andrej

    2016-02-22

    Many proteins have small-molecule binding pockets that are not easily detectable in the ligand-free structures. These cryptic sites require a conformational change to become apparent; a cryptic site can therefore be defined as a site that forms a pocket in a holo structure, but not in the apo structure. Because many proteins appear to lack druggable pockets, understanding and accurately identifying cryptic sites could expand the set of drug targets. Previously, cryptic sites were identified experimentally by fragment-based ligand discovery and computationally by long molecular dynamics simulations and fragment docking. Here, we begin by constructing a set of structurally defined apo-holo pairs with cryptic sites. Next, we comprehensively characterize the cryptic sites in terms of their sequence, structure, and dynamics attributes. We find that cryptic sites tend to be as conserved in evolution as traditional binding pockets but are less hydrophobic and more flexible. Relying on this characterization, we use machine learning to predict cryptic sites with relatively high accuracy (for our benchmark, the true positive and false positive rates are 73% and 29%, respectively). We then predict cryptic sites in the entire structurally characterized human proteome (11,201 structures, covering 23% of all residues in the proteome). CryptoSite increases the size of the potentially "druggable" human proteome from ~40% to ~78% of disease-associated proteins. Finally, to demonstrate the utility of our approach in practice, we experimentally validate a cryptic site in protein tyrosine phosphatase 1B using a covalent ligand and NMR spectroscopy. The CryptoSite Web server is available at http://salilab.org/cryptosite.

  1. Protein Structural Memory Influences Ligand Binding Mode(s) and Unbinding Rates.

    PubMed

    Xu, Min; Caflisch, Amedeo; Hamm, Peter

    2016-03-01

    The binding of small molecules (e.g., natural ligands, metabolites, and drugs) to proteins governs most biochemical pathways and physiological processes. Here, we use molecular dynamics to investigate the unbinding of dimethyl sulfoxide (DMSO) from two distinct states of a small rotamase enzyme, the FK506-binding protein (FKBP). These states correspond to the FKBP protein relaxed with and without DMSO in the active site. Since the time scale of ligand unbinding (2-20 ns) is faster than protein relaxation (100 ns), a novel methodology is introduced to relax the protein without having to introduce an artificial constraint. The simulation results show that the unbinding time is an order of magnitude longer for dissociation from the DMSO-bound state (holo-relaxed). That is, the actual rate of unbinding depends on the state of the protein, with the protein having a long-lived memory. The rate thus depends on the concentration of the ligand as the apo and holo states reflect low and high concentrations of DMSO, respectively. Moreover, there are multiple binding modes in the apo-relaxed state, while a single binding mode dominates the holo-relaxed state in which DMSO acts as hydrogen bond acceptor from the backbone NH of Ile56, as in the crystal structure of the DMSO/FKBP complex. The solvent relaxes very fast (∼1 ns) close to the NH of Ile56 and with the same time scale of the protein far away from the active site. These results have implications for high-throughput docking, which makes use of a rigid structure of the protein target.

  2. Extra-helical binding site of a glucagon receptor antagonist.

    PubMed

    Jazayeri, Ali; Doré, Andrew S; Lamb, Daniel; Krishnamurthy, Harini; Southall, Stacey M; Baig, Asma H; Bortolato, Andrea; Koglin, Markus; Robertson, Nathan J; Errey, James C; Andrews, Stephen P; Teobald, Iryna; Brown, Alastair J H; Cooke, Robert M; Weir, Malcolm; Marshall, Fiona H

    2016-05-12

    Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors. PMID:27111510

  3. Distortion of Flavin Geometry Is Linked to Ligand Binding in Cholesterol Oxidase

    SciTech Connect

    Lyubimov, A.Y.; Heard, K.; Tang, H.; Sampson, N.S.; Vrielink, A.

    2009-06-03

    Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.

  4. Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi

    SciTech Connect

    Xu, Xueqing; Chang, Bianca W.; Ribeiro, Jose M. C.; Andersen, John F.

    2013-01-01

    Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein. Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity.

  5. The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins.

    PubMed

    Kozakov, Dima; Grove, Laurie E; Hall, David R; Bohnuud, Tanggis; Mottarella, Scott E; Luo, Lingqi; Xia, Bing; Beglov, Dmitri; Vajda, Sandor

    2015-05-01

    FTMap is a computational mapping server that identifies binding hot spots of macromolecules-i.e., regions of the surface with major contributions to the ligand-binding free energy. To use FTMap, users submit a protein, DNA or RNA structure in PDB (Protein Data Bank) format. FTMap samples billions of positions of small organic molecules used as probes, and it scores the probe poses using a detailed energy expression. Regions that bind clusters of multiple probe types identify the binding hot spots in good agreement with experimental data. FTMap serves as the basis for other servers, namely FTSite, which is used to predict ligand-binding sites, FTFlex, which is used to account for side chain flexibility, FTMap/param, used to parameterize additional probes and FTDyn, for mapping ensembles of protein structures. Applications include determining the druggability of proteins, identifying ligand moieties that are most important for binding, finding the most bound-like conformation in ensembles of unliganded protein structures and providing input for fragment-based drug design. FTMap is more accurate than classical mapping methods such as GRID and MCSS, and it is much faster than the more-recent approaches to protein mapping based on mixed molecular dynamics. By using 16 probe molecules, the FTMap server finds the hot spots of an average-size protein in <1 h. As FTFlex performs mapping for all low-energy conformers of side chains in the binding site, its completion time is proportionately longer.

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

    SciTech Connect

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

    1986-03-01

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

  7. Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket.

    PubMed

    Uno, Tadayuki; Sakamoto, Rikiharu; Tomisugi, Yoshikazu; Ishikawa, Yoshinobu; Wilkinson, Anthony J

    2003-09-01

    A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.

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

    PubMed

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

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

  9. Enthalpy/entropy compensation effects from cavity desolvation underpin broad ligand binding selectivity for rat odorant binding protein 3.

    PubMed

    Portman, Katherine L; Long, Jed; Carr, Stephen; Briand, Loïc; Winzor, Donald J; Searle, Mark S; Scott, David J

    2014-04-15

    Evolution has produced proteins with exquisite ligand binding specificity, and manipulating this effect has been the basis for much of modern rational drug design. However, there are general classes of proteins with broader ligand selectivity linked to function, the origin of which is poorly understood. The odorant binding proteins (OBPs) sequester volatile molecules for transportation to the olfactory receptors. Rat OBP3, which we characterize by X-ray crystallography and NMR, binds a homologous series of aliphatic γ-lactones within its aromatic-rich hydrophobic pocket with remarkably little variation in affinity but extensive enthalpy/entropy compensation effects. We show that the binding energetics are modulated by two desolvation processes with quite different thermodynamic signatures. Ligand desolvation follows the classical hydrophobic effect; however, cavity desolvation is consistent with the liberation of "high energy" water molecules back into bulk solvent with a strong, but compensated, enthalpic contribution, which together underpin the origins of broad ligand binding selectivity.

  10. Tissue specificity of endothelin binding sites

    SciTech Connect

    Bolger, G.T.; Liard, F.; Krogsrud, R.; Thibeault, D.; Jaramillo, J. )

    1990-09-01

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

  11. smFRET studies of the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding

    PubMed Central

    Kinz-Thompson, Colin D.; Gonzalez, Ruben L.

    2016-01-01

    The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution. PMID:25066296

  12. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  13. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    SciTech Connect

    Williams,R.; Holyoak, T.; McDonald, G.; Gui, C.; Fenton, A.

    2006-01-01

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

  14. Targeting a homogeneously glycosylated antibody Fc to bind cancer cells using a synthetic receptor ligand.

    PubMed

    Xiao, Junpeng; Chen, Rui; Pawlicki, Mark A; Tolbert, Thomas J

    2009-09-30

    The targeting of a glycosylated antibody Fc fragment to bind to cancer cells by site-selective incorporation of a synthetic ligand is described. Homogeneously glycosylated immunoglobulin G subclass 1 fragment crystallizable (IgG1 Fc) was produced by expression in a glycosylation-deficient yeast strain and subsequent treatment with mannosidase IA. A N-terminal cysteine was generated on the expressed IgG1 Fc by utilizing proteolytic processing enzymes in the yeast secretory pathway. A cyclic RGD peptide thioester 2 was synthesized and then site-selectively attached to the N-terminus of the IgG1 Fc glycoprotein using native chemical ligation. The resulting chemically modified antibody fragment, RGD-Man(5)-IgG1 Fc (5), retained biological activity similar to that of the free cyclic RGD peptide 1 when assayed for its ability to both promote and inhibit the adhesion of alpha(v)beta(3) integrin receptor-expressing WM-115 melanoma cells. In addition, fluorescent microscopy experiments were conducted using FITC-labeled 5 and confirmed binding of 5 to WM-115 melanoma cells. Site-selectively modified antibody fragments such as the one described here may be used to combine the beneficial properties of synthetic receptor ligands with antibody fragments to develop useful biochemical tools and improved therapeutics. The methods described here can also be used to produce glycoprotein fragments for the chemoenzymatic synthesis of homogeneous glycoproteins.

  15. Evaluation of water displacement energetics in protein binding sites with grid cell theory.

    PubMed

    Gerogiokas, G; Southey, M W Y; Mazanetz, M P; Heifetz, A; Hefeitz, A; Bodkin, M; Law, R J; Michel, J

    2015-04-01

    Excess free energies, enthalpies and entropies of water in protein binding sites were computed via classical simulations and Grid Cell Theory (GCT) analyses for three pairs of congeneric ligands in complex with the proteins scytalone dehydratase, p38α MAP kinase and EGFR kinase respectively. Comparative analysis is of interest since the binding modes for each ligand pair differ in the displacement of one binding site water molecule, but significant variations in relative binding affinities are observed. Protocols that vary in their use of restraints on protein and ligand atoms were compared to determine the influence of protein-ligand flexibility on computed water structure and energetics, and to assess protocols for routine analyses of protein-ligand complexes. The GCT-derived binding affinities correctly reproduce experimental trends, but the magnitude of the predicted changes in binding affinities is exaggerated with respect to results from a previous Monte Carlo Free Energy Perturbation study. Breakdown of the GCT water free energies into enthalpic and entropic components indicates that enthalpy changes dominate the observed variations in energetics. In EGFR kinase GCT analyses revealed that replacement of a pyrimidine by a cyanopyridine perturbs water energetics up three hydration shells away from the ligand.

  16. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    PubMed

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  17. Copper binding to the prion protein: Structural implications of four identical cooperative binding sites

    PubMed Central

    Viles, John H.; Cohen, Fred E.; Prusiner, Stanley B.; Goodin, David B.; Wright, Peter E.; Dyson, H. Jane

    1999-01-01

    Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP. PMID:10051591

  18. Crystal structures and ligand binding of PurM proteins from Thermus thermophilus and Geobacillus kaustophilus.

    PubMed

    Kanagawa, Mayumi; Baba, Seiki; Watanabe, Yuzo; Nakagawa, Noriko; Ebihara, Akio; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sampei, Gen-Ichi; Kawai, Gota

    2016-03-01

    Crystal structures of 5-aminoimidazole ribonucleotide (AIR) synthetase, also known as PurM, from Thermus thermophilus (Tt) and Geobacillus kaustophilus (Gk) were determined. For TtPurM, the maximum resolution was 2.2 Å and the space group was P21212 with four dimers in an asymmetric unit. For GkPurM, the maximum resolution was 2.2 Å and the space group was P21212 with one monomer in asymmetric unit. The biological unit is dimer for both TtPurM and GkPurM and the dimer structures were similar to previously determined structures of PurM in general. For TtPurM, ∼50 residues at the amino terminal were disordered in the crystal structure whereas, for GkPurM, the corresponding region covered the ATP-binding site forming an α helix in part, suggesting that the N-terminal region of PurM changes its conformation upon binding of ligands. FGAM binding site was predicted by the docking simulation followed by the MD simulation based on the SO4 (2-) binding site found in the crystal structure of TtPurM.

  19. How to deal with multiple binding poses in alchemical relative protein-ligand binding free energy calculations.

    PubMed

    Kaus, Joseph W; Harder, Edward; Lin, Teng; Abel, Robert; McCammon, J Andrew; Wang, Lingle

    2015-06-01

    Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the

  20. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    PubMed

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  1. 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.

  2. 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.

  3. 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.

  4. A Unitary Anesthetic Binding Site at High Resolution*

    PubMed Central

    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.

    2009-01-01

    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 GABAA 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 GABAA 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. PMID:19605349

  5. 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

  6. A tandem regression-outlier analysis of a ligand cellular system for key structural modifications around ligand binding

    PubMed Central

    2013-01-01

    Background A tandem technique of hard equipment is often used for the chemical analysis of a single cell to first isolate and then detect the wanted identities. The first part is the separation of wanted chemicals from the bulk of a cell; the second part is the actual detection of the important identities. To identify the key structural modifications around ligand binding, the present study aims to develop a counterpart of tandem technique for cheminformatics. A statistical regression and its outliers act as a computational technique for separation. Results A PPARγ (peroxisome proliferator-activated receptor gamma) agonist cellular system was subjected to such an investigation. Results show that this tandem regression-outlier analysis, or the prioritization of the context equations tagged with features of the outliers, is an effective regression technique of cheminformatics to detect key structural modifications, as well as their tendency of impact to ligand binding. Conclusions The key structural modifications around ligand binding are effectively extracted or characterized out of cellular reactions. This is because molecular binding is the paramount factor in such ligand cellular system and key structural modifications around ligand binding are expected to create outliers. Therefore, such outliers can be captured by this tandem regression-outlier analysis. PMID:23627990

  7. 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.

  8. Molecular dynamics simulations of the ligand-binding domain of the ionotropic glutamate receptor GluR2.

    PubMed Central

    Arinaminpathy, Yalini; Sansom, Mark S P; Biggin, Philip C

    2002-01-01

    Ionotropic glutamate receptors are essential for fast synaptic nerve transmission. Recent x-ray structures for the ligand-binding (S1S2) region of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive receptor have suggested how differences in protein/ligand interactions may determine whether a ligand will behave as a full agonist. We have used multiple molecular dynamics simulations of 2-5 ns duration to explore the structural dynamics of GluR2 S1S2 in the presence and absence of glutamate and in a complex with kainate. Our studies indicate that not only is the degree of domain closure dependent upon interactions with the ligand, but also that protein/ligand interactions influence the motion of the S2 domain with respect to S1. Differences in domain mobility between the three states (apo-S1S2, glutamate-bound, and kainate-bound) are surprisingly clear-cut. We discuss how these changes in dynamics may provide an explanation relating the mechanism of transmission of the agonist-binding event to channel opening. We also show here how the glutamate may adopt an alternative mode of binding not seen in the x-ray structure, which involves a key threonine (T480) side chain flipping into a new conformation. This new conformation results in an altered pattern of hydrogen bonding at the agonist-binding site. PMID:11806910

  9. Mixed ligand ruthenium(III) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones with triphenylphosphine/triphenylarsine co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and cytotoxic activity

    NASA Astrophysics Data System (ADS)

    Sampath, K.; Sathiyaraj, S.; Raja, G.; Jayabalakrishnan, C.

    2013-08-01

    The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide (HL2), were prepared and characterized by various physico-chemical and spectroscopic methods. The title compounds act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the ligands and complexes were investigated by absorption spectroscopy and IR spectroscopy. It reveals that the compounds bind to nitrogenous bases of DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  10. Effects of aggressive encounters on plasma corticosteroid-binding globulin and its ligands in white-crowned sparrows.

    PubMed

    Charlier, Thierry D; Underhill, Caroline; Hammond, Geoffrey L; Soma, Kiran K

    2009-09-01

    In birds, corticosteroid-binding globulin (CBG) binds corticosterone, progesterone and testosterone. The concentration of each ligand can alter the binding of the other ligands through competitive interactions. Thus, an increase in corticosterone or progesterone may displace testosterone bound to CBG, leading to an increase in bioactive free testosterone levels without affecting total testosterone levels in the circulation. Aggressive interactions increase plasma total testosterone levels in some birds but not in others. Here, we tested the hypothesis that aggressive encounters in the late breeding season would not increase total testosterone levels in plasma, but would alter CBG, total corticosterone or total progesterone levels in such a way as to modify the number of available binding sites and therefore occupancy by testosterone. A marked decrease in CBG occupancy by testosterone would indirectly suggest an increase in free testosterone levels in plasma. Wild male white-crowned sparrows were exposed to a simulated territorial intrusion (STI) or control for 30 min. Subjects were then caught and bled. We measured CBG using a ligand-binding assay and corticosterone, progesterone and testosterone using highly sensitive radioimmunoassays. STI significantly increased aggressive behaviors but did not affect plasma total testosterone levels. STI significantly increased plasma CBG and total corticosterone levels and decreased plasma total progesterone levels. We predict that CBG occupancy by corticosterone will increase slightly following an aggressive encounter. However, this small change is unlikely to increase free testosterone levels, because of the large number of seemingly unoccupied CBG binding sites in these subjects.

  11. Reduced steric hindrance and optimized spatial arrangement of carbohydrate ligands in imprinted monolayers for enhanced protein binding.

    PubMed

    Zheng, Haifu; Du, Xuezhong

    2013-02-01

    Imprinted monolayers provide several advantages over bulk imprinting methods. This is especially important for large templates such as proteins. Concanavalin A (Con A)-imprinted binary monolayers consisting of glycolipids with oligo(ethylene glycol) (OEG) spacers and zwitterionic phospholipids (DPPC) were constructed and investigated. The shorter phosphorylcholine (PC) headgroups with an almost flat-on orientation in the binary monolayers gave rise to reduced steric hindrance favorable to the accommodation of Con A with greater ease and facilitated the access of the OEG-linked mannose moieties for enhanced protein binding. Further enhanced binding resulted from optimized spatial rearrangement of the glycolipids at the air-water interface directed by Con A in the subphase to create bivalent binding sites and to minimize steric crowding of neighboring mannose ligands. The combination of the exposed carbohydrate ligands from biologically inert surfaces and the optimized ligand arrangement is the most reasonable solution to enhancement of protein affinity. The bivalent carbohydrate binding sites protruding from the imprinted monolayers were created to be complementary to the Con A binding pockets. This strategy generates tailor-made surfaces with enhanced protein binding and opens the possibility of controlled assembly of intellectual biomaterials and preparation of biosensors.

  12. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form.

    PubMed

    Baum, Bernhard; Lecker, Laura S M; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N; Brenk, Ruth

    2015-08-01

    Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data. PMID:27092480

  17. Effects of ligand binding on the mechanical stability of protein GB1 studied by steered molecular dynamics simulation.

    PubMed

    Su, Ji-Guo; Zhao, Shu-Xin; Wang, Xiao-Feng; Li, Chun-Hua; Li, Jing-Yuan

    2016-08-01

    Regulation of the mechanical properties of proteins plays an important role in many biological processes, and sheds light on the design of biomaterials comprised of protein. At present, strategies to regulate protein mechanical stability focus mainly on direct modulation of the force-bearing region of the protein. Interestingly, the mechanical stability of GB1 can be significantly enhanced by the binding of Fc fragments of human IgG antibody, where the binding site is distant from the force-bearing region of the protein. The mechanism of this long-range allosteric control of protein mechanics is still elusive. In this work, the impact of ligand binding on the mechanical stability of GB1 was investigated using steered molecular dynamics simulation, and a mechanism underlying the enhanced protein mechanical stability is proposed. We found that the external force causes deformation of both force-bearing region and ligand binding site. In other words, there is a long-range coupling between these two regions. The binding of ligand restricts the distortion of the binding site and reduces the deformation of the force-bearing region through a long-range allosteric communication, which thus improves the overall mechanical stability of the protein. The simulation results are very consistent with previous experimental observations. Our studies thus provide atomic-level insights into the mechanical unfolding process of GB1, and explain the impact of ligand binding on the mechanical properties of the protein through long-range allosteric regulation, which should facilitate effective modulation of protein mechanical properties.

  18. Effects of ligand binding on the mechanical stability of protein GB1 studied by steered molecular dynamics simulation.

    PubMed

    Su, Ji-Guo; Zhao, Shu-Xin; Wang, Xiao-Feng; Li, Chun-Hua; Li, Jing-Yuan

    2016-08-01

    Regulation of the mechanical properties of proteins plays an important role in many biological processes, and sheds light on the design of biomaterials comprised of protein. At present, strategies to regulate protein mechanical stability focus mainly on direct modulation of the force-bearing region of the protein. Interestingly, the mechanical stability of GB1 can be significantly enhanced by the binding of Fc fragments of human IgG antibody, where the binding site is distant from the force-bearing region of the protein. The mechanism of this long-range allosteric control of protein mechanics is still elusive. In this work, the impact of ligand binding on the mechanical stability of GB1 was investigated using steered molecular dynamics simulation, and a mechanism underlying the enhanced protein mechanical stability is proposed. We found that the external force causes deformation of both force-bearing region and ligand binding site. In other words, there is a long-range coupling between these two regions. The binding of ligand restricts the distortion of the binding site and reduces the deformation of the force-bearing region through a long-range allosteric communication, which thus improves the overall mechanical stability of the protein. The simulation results are very consistent with previous experimental observations. Our studies thus provide atomic-level insights into the mechanical unfolding process of GB1, and explain the impact of ligand binding on the mechanical properties of the protein through long-range allosteric regulation, which should facilitate effective modulation of protein mechanical properties. PMID:27444879

  19. Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling.

    PubMed

    Pawar, Parag; Jadhav, Sameer; Eggleton, Charles D; Konstantopoulos, Konstantinos

    2008-10-01

    Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.

  20. trans-Parinaric acid as a versatile spectroscopic label to study ligand binding properties of bovine β-lactoglobulin

    NASA Astrophysics Data System (ADS)

    Zsila, Ferenc; Bikádi, Zsolt

    2005-11-01

    Advantageous spectroscopic properties of the plant derived polyunsaturated trans-parinaric acid ( tPnA) was demonstrated in obtaining valuable data on the ligand binding characteristics of the lipocalin member bovine β-lactoglobulin A (BLG-A). Titration of the protein with tPnA resulted in the appearance of an intense negative induced circular dichroism (CD) band and bathochromic shift of the ultraviolet (UV) peak of the ligand. The extrinsic optical activity was interpreted by the chiral contribution of the allylic axial C sbnd H bonds of tPnA to the π-π * transition of the planar tetraene chromophore. Analysis of the series of induced CD curves obtained by CD titration experiment indicated the complexation of a single ligand molecule to a uniform protein binding site. Additionally, the dramatic increase of fluorescence intensity of the lactoglobulin bound ligand suggested the hydrophobic nature of the binding site. CD and fluorescence titration data were utilized to calculate the binding constant ( Ka) of which high value (≈10 6 M -1) refers to strong protein association of tPnA. pH dependent reversible dis- and reappearance of the induced CD signal unambigously proved the inclusion of tPnA into the central hydrophobic cavity of the lactoglobulin governed by the protonation induced conformational movement of the EF loop at the opening of the calyx. This conclusion was supported and complemented by molecular docking calculations.

  1. Identification of a Ligand Binding Pocket in LdtR from Liberibacter asiaticus.

    PubMed

    Pagliai, Fernando A; Gonzalez, Claudio F; Lorca, Graciela L

    2015-01-01

    LdtR is a transcriptional activator involved in the regulation of a putative L,D transpeptidase in Liberibacter asiaticus, an unculturable pathogen and one of the causative agents of Huanglongbing disease. Using small molecule screens we identified benzbromarone as an inhibitor of LdtR activity, which was confirmed using in vivo and in vitro assays. Based on these previous results, the objective of this work was to identify the LdtR ligand binding pocket and characterize its interactions with benzbromarone. A structural model of LdtR was constructed and the molecular interactions with the ligand were predicted using the SwissDock interface. Using site-directed mutagenesis, these residues were changed to alanine. Electrophoretic mobility shift assays, thermal denaturation, isothermal titration calorimetry experiments, and in vivo assays were used to identify residues T43, L61, and F64 in the Benz1 pocket of LdtR as the amino acids most likely involved in the binding to benzbromarone. These results provide new information on the binding mechanism of LdtR to a modulatory molecule and provide a blue print for the design of therapeutics for other members of the MarR family of transcriptional regulators involved in pathogenicity. PMID:26635775

  2. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  3. Appropriate calibration curve fitting in ligand binding assays.

    PubMed

    Findlay, John W A; Dillard, Robert F

    2007-06-29

    Calibration curves for ligand binding assays are generally characterized by a nonlinear relationship between the mean response and the analyte concentration. Typically, the response exhibits a sigmoidal relationship with concentration. The currently accepted reference model for these calibration curves is the 4-parameter logistic (4-PL) model, which optimizes accuracy and precision over the maximum usable calibration range. Incorporation of weighting into the model requires additional effort but generally results in improved calibration curve performance. For calibration curves with some asymmetry, introduction of a fifth parameter (5-PL) may further improve the goodness of fit of the experimental data to the algorithm. Alternative models should be used with caution and with knowledge of the accuracy and precision performance of the model across the entire calibration range, but particularly at upper and lower analyte concentration areas, where the 4- and 5-PL algorithms generally outperform alternative models. Several assay design parameters, such as placement of calibrator concentrations across the selected range and assay layout on multiwell plates, should be considered, to enable optimal application of the 4- or 5-PL model. The fit of the experimental data to the model should be evaluated by assessment of agreement of nominal and model-predicted data for calibrators.

  4. Quantitative Analysis of Multivalent Ligand Presentation on Gold Glyconanoparticles and Their Effects on Protein Binding

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Ramström, Olof; Yan, Mingdi

    2010-03-01

    Bio-functionalized nanomaterials, which combine functions of biological ligands and unique properties of nano-sized building blocks, have exhibited increased potential applications in biosensing, therapeutics, and diagnostics. Glyconanoparitcles carrying a monolayer of carbohydrate ligands on nanoparticles provide an excellent platform for sensitive protein recognitions. Using Au nanoparticles as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, quantitative analysis especially the binding affinity of the resulting glyconanoparticles is challenging to determine. Here we present a new characterization technique, based on fluorescent competition binding assays, for measuring dissociation constants for glyconanoparticles-protein interactions. Au nanoparticles coupled with a series of un-derivatized carbohydrates were prepared by a photocoupling chemistry. Dramatic binding affinity enhancement was observed due to the high ligand density on nanoparticles, which was highly relevant to ligand display, controlled by the linker type, chain length, ligand size and density.

  5. DNA-Based Nanostructures: Changes of Mechanical Properties of DNA upon Ligand Binding

    NASA Astrophysics Data System (ADS)

    Nechipurenko, Yury; Grokhovsky, Sergey; Gursky, Georgy; Nechipurenko, Dmitry; Polozov, Robert

    The formation of DNA-based nanostructures involves the binding of different kinds of ligands to DNA as well as the interaction of DNA molecules with each other. Complex formation between ligand and DNA can alter physicochemical properties of the DNA molecule. In the present work, the accessibility of DNA-ligand complexes to cleavage by DNase I are considered, and the exact algorithms for analysis of diagrams of DNase I footprinting for ligand-DNA complexes are obtained. Changes of mechanical properties of the DNA upon ligand binding are also demonstrated by the cleavage patterns generated upon ultrasound irradiation of cis-platin-DNA complexes. Propagation of the mechanical perturbations along DNA in the presence of bound ligands is considered in terms of a string model with a heterogeneity corresponding to the position of a bound ligand on DNA. This model can reproduce qualitatively the cleavage patterns obtained upon ultrasound irradiation of cis-platin-DNA complexes.

  6. Ligand binding cooperativity: Bioisosteric replacement of CO with SO2 among thrombin inhibitors.

    PubMed

    Said, Ahmed M; Hangauer, David G

    2016-08-15

    Ligand-protein binding is a complex process that involves the formation of number of non-covalent interactions, e.g. H-bonds and hydrophobic interactions, between the ligand and the protein host. Upon binding, ligand functional groups can act synergistically (positive cooperativity) to improve the overall ligand binding affinity beyond what would be expected from their individual contributions. In this study, using thrombin as a protein model system, we evaluated the effect of the bioisosteric replacement of a carbonyl functionality with a sulphonyl functionality on positive cooperativity between their H-bonds with thrombin and hydrophobic binding in the adjacent S3 pocket. The positive cooperativity observed was greatly reduced when replacing the carbonyl group with a sulphonyl group. Evaluating how bioisosteric replacements affect cooperativity is important for making better informed ligand optimization SAR decisions. PMID:27445170

  7. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    NASA Technical Reports Server (NTRS)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  8. Specific binding of a ligand of sigma-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    SciTech Connect

    Samovilova, N.N.; Yarygin, K.N.; Vinogradov, V.A.

    1986-08-01

    A ligand of the sigma-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the sigma-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.).

  9. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    SciTech Connect

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.

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

    PubMed

    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.

  11. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities

    PubMed Central

    Genheden, Samuel; Ryde, Ulf

    2015-01-01

    Introduction: The molecular mechanics energies combined with the Poisson–Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA and MM/GBSA) methods are popular approaches to estimate the free energy of the binding of small ligands to biological macromolecules. They are typically based on molecular dynamics simulations of the receptor–ligand complex and are therefore intermediate in both accuracy and computational effort between empirical scoring and strict alchemical perturbation methods. They have been applied to a large number of systems with varying success. Areas covered: The authors review the use of MM/PBSA and MM/GBSA methods to calculate ligand-binding affinities, with an emphasis on calibration, testing and validation, as well as attempts to improve the methods, rather than on specific applications. Expert opinion: MM/PBSA and MM/GBSA are attractive approaches owing to their modular nature and that they do not require calculations on a training set. They have been used successfully to reproduce and rationalize experimental findings and to improve the results of virtual screening and docking. However, they contain several crude and questionable approximations, for example, the lack of conformational entropy and information about the number and free energy of water molecules in the binding site. Moreover, there are many variants of the method and their performance varies strongly with the tested system. Likewise, most attempts to ameliorate the methods with more accurate approaches, for example, quantum-mechanical calculations, polarizable force fields or improved solvation have deteriorated the results. PMID:25835573

  12. Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Peränen, Johan; Olkkonen, Vesa M

    2015-05-01

    Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.

  13. Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Peränen, Johan; Olkkonen, Vesa M

    2015-05-01

    Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling. PMID:25420878

  14. 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.

  15. Influence of length and flexibility of spacers on the binding affinity of divalent ligands.

    PubMed

    Liese, Susanne; Netz, Roland R

    2015-01-01

    We present a quantitative model for the binding of divalent ligand-receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.

  16. Dynamics of Transcription Factor Binding Site Evolution

    PubMed Central

    Tuğrul, Murat; Paixão, Tiago; Barton, Nicholas H.; Tkačik, Gašper

    2015-01-01

    Evolution of gene regulation is crucial for our understanding of the phenotypic differences between species, populations and individuals. Sequence-specific binding of transcription factors to the regulatory regions on the DNA is a key regulatory mechanism that determines gene expression and hence heritable phenotypic variation. We use a biophysical model for directional selection on gene expression to estimate the rates of gain and loss of transcription factor binding sites (TFBS) in finite populations under both point and insertion/deletion mutations. Our results show that these rates are typically slow for a single TFBS in an isolated DNA region, unless the selection is extremely strong. These rates decrease drastically with increasing TFBS length or increasingly specific protein-DNA interactions, making the evolution of sites longer than ∼ 10 bp unlikely on typical eukaryotic speciation timescales. Similarly, evolution converges to the stationary distribution of binding sequences very slowly, making the equilibrium assumption questionable. The availability of longer regulatory sequences in which multiple binding sites can evolve simultaneously, the presence of “pre-sites” or partially decayed old sites in the initial sequence, and biophysical cooperativity between transcription factors, can all facilitate gain of TFBS and reconcile theoretical calculations with timescales inferred from comparative genomics. PMID:26545200

  17. Dewetting-Controlled Binding of Ligands to Hydrophobic Pockets

    PubMed Central

    Setny, P.; Wang, Z.; Cheng, L.-T.; Li, B.; McCammon, J. A.; Dzubiella, J.

    2010-01-01

    We report on a combined atomistic molecular dynamics simulation and implicit solvent analysis of a generic hydrophobic pocket-ligand (host-guest) system. The approaching ligand induces complex wetting-dewetting transitions in the weakly solvated pocket. The transitions lead to bimodal solvent fluctuations which govern magnitude and range of the pocket-ligand attraction. A recently developed implicit water model, based on the minimization of a geometric functional, captures the sensitive aqueous interface response to the concave-convex pocket-ligand configuration semiquantitatively. PMID:19905832

  18. Role of carbohydrates in thyrotropin binding sites.

    PubMed

    Pekonen, F

    1980-07-01

    The role of carbohydrates in thyrotropin binding was studied by glycosidase treatment of human thyroid membranes. Removal of over 75% of membrane sialic acid resulted in a 50% increase of TSH binding, measured in 10 mM Tris-HCl, 50 mM NaCl, 0.1% bSA, pH 7.4, 37 degrees C (buffer A). This augmentation was due to an increase in binding to high affinity sites (Ka 1 X 10(10)M-1). The binding was highly specific and was not significantly inhibited by gangliosides. In contrast, low affinity binding of TSH was unchanged either in buffer A or in 10 mM Tris-acetate, 0.1% bSA pH 6.0, 4 degrees C (buffer B) and was inhibited by gangliosides. Treatment of membranes with beta-galactosidase, beta-N-acetylglucosaminidase and alpha-L-fucosidase had little effect on TSH binding. The data suggests that membrane-associated sialic acid inhibits TSH binding to high affinity receptors and that gangliosides are not involved in tthis TSH-receptor interaction.

  19. 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

  20. 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.

  1. Structural studies of neuropilin-2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket.

    PubMed

    Tsai, Yi-Chun Isabella; Fotinou, Constantina; Rana, Rohini; Yelland, Tamas; Frankel, Paul; Zachary, Ian; Djordjevic, Snezana

    2016-05-01

    Neuropilin-2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin-2 and its homologous protein neuropilin-1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin-2. The neuropilin-2-specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X-ray crystal structure of the neuropilin-2 b1 domain in a complex with the C-terminal sub-domain of VEGF-A. Zn(2+) binding to neuropilin-2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin-2 ligand binding and signalling activity. PMID:26991001

  2. Site Identification by Ligand Competitive Saturation (SILCS) simulations for fragment-based drug design.

    PubMed

    Faller, Christina E; Raman, E Prabhu; MacKerell, Alexander D; Guvench, Olgun

    2015-01-01

    Fragment-based drug design (FBDD) involves screening low molecular weight molecules ("fragments") that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind nonoverlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy.The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is "soaked" in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called "FragMaps" can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine "Grid Free Energies (GFEs)," which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

  3. Site Identification by Ligand Competitive Saturation (SILCS) Simulations for Fragment-Based Drug Design

    PubMed Central

    Faller, Christina E.; Raman, E. Prabhu; MacKerell, Alexander D.; Guvench, Olgun

    2015-01-01

    Fragment-based drug design (FBDD) involves screening low molecular weight molecules (“fragments”) that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind non-overlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy. The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is “soaked” in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called “FragMaps” can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine “Grid Free Energies (GFEs),” which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

  4. A computational study of ligand binding affinities in iron(III) porphine and protoporphyrin IX complexes.

    PubMed

    Durrant, Marcus C

    2014-07-01

    The search for novel anti-malarial drugs that can disrupt biomineralization of ferriprotoporphyrin IX to haemozoin requires an understanding of the fundamental chemistry of the porphyrin's iron(iii) centre at the water-lipid interface. Towards this end, the binding affinities for a diverse set of 31 small ligands with iron(iii) porphine have been calculated using density functional theory, in the gas phase and also with implicit solvent corrections for both water and n-octanol. In addition, the binding of hydroxide, chloride, acetate, methylamine and water to ferriprotoporphyrin IX has been studied, and very similar trends are observed for the smaller and larger models. Anionic ligands generally give stronger binding than neutral ones; the strongest binding is observed for RO(-) and OH(-) ligands, whilst acetate binds relatively weakly among the anions studied. Electron-rich nitrogen donors tend to bind more strongly than electron-deficient ones, and the weakest binding is found for neutral O and S donors such as oxazole and thiophene. In all cases, ligand binding is stronger in n-octanol than in water, and the differences in binding energies for the two solvents are greater for ionic ligands than for neutrals. Finally, dimerization of ferriprotoporphyrin IX by means of iron(iii)-carboxylate bond formation has been modelled. The results are discussed in terms of haemozoin crystal growth and its disruption by known anti-malarial drugs.

  5. Binding of flexible and constrained ligands to the Grb2 SH2 domain: structural effects of ligand preorganization

    SciTech Connect

    Clements, John H.; DeLorbe, John E.; Benfield, Aaron P.; Martin, Stephen F.

    2010-10-01

    Structures of the Grb2 SH2 domain complexed with a series of flexible and constrained replacements of the phosphotyrosine residue in tripeptides derived from Ac-pYXN (where X = V, I, E and Q) were compared to determine what, if any, structural differences arise as a result of ligand preorganization. Structures of the Grb2 SH2 domain complexed with a series of pseudopeptides containing flexible (benzyl succinate) and constrained (aryl cyclopropanedicarboxylate) replacements of the phosphotyrosine (pY) residue in tripeptides derived from Ac-pYXN-NH{sub 2} (where X = V, I, E and Q) were elucidated by X-ray crystallography. Complexes of flexible/constrained pairs having the same pY + 1 amino acid were analyzed in order to ascertain what structural differences might be attributed to constraining the phosphotyrosine replacement. In this context, a given structural dissimilarity between complexes was considered to be significant if it was greater than the corresponding difference in complexes coexisting within the same asymmetric unit. The backbone atoms of the domain generally adopt a similar conformation and orientation relative to the ligands in the complexes of each flexible/constrained pair, although there are some significant differences in the relative orientations of several loop regions, most notably in the BC loop that forms part of the binding pocket for the phosphate group in the tyrosine replacements. These variations are greater in the set of complexes of constrained ligands than in the set of complexes of flexible ligands. The constrained ligands make more direct polar contacts to the domain than their flexible counterparts, whereas the more flexible ligand of each pair makes more single-water-mediated contacts to the domain; there was no correlation between the total number of protein–ligand contacts and whether the phosphotyrosine replacement of the ligand was preorganized. The observed differences in hydrophobic interactions between the complexes of

  6. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-12-31

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  7. Molecular design of substrate binding sites

    SciTech Connect

    Shelnutt, J.A.; Hobbs, J.D.

    1991-01-01

    Computer-aided molecular design methods were used to tailor binding sites for small substrate molecules, including CO{sub 2} and methane. The goal is to design a cavity, adjacent to a catalytic metal center, into which the substrate will selectively bind through only non-bonding interactions with the groups lining the binding pocket. Porphyrins are used as a basic molecular structure, with various substituents added to construct the binding pocket. The conformations of these highly-substituted porphyrins are predicted using molecular mechanics calculations with a force field that gives accurate predictions for metalloporhyrins. Dynamics and energy-minimization calculations of substrate molecules bound to the cavity indicate high substrate binding affinity. The size, shape and charge-distribution of groups surrounding the cavity provide molecular selectivity. Specifically, calculated binding energies of methane, benzene, dichloromethane, CO{sub 2} and chloroform vary by about 10 kcal/mol for metal octaethyl-tetraphenylporphyrins (OETPPs) with chloroform, dichloromethane, and CO{sub 2} having the lowest. Significantly, a solvent molecule is found in the cavity in the X-ray structures of Co- and CuOETPP crystals obtained from dichloromethane. 5 refs., 3 figs., 3 tabs.

  8. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    SciTech Connect

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  9. Salt effects on polyelectrolyte-ligand binding: comparison of Poisson-Boltzmann, and limiting law/counterion binding models.

    PubMed

    Sharp, K A; Friedman, R A; Misra, V; Hecht, J; Honig, B

    1995-08-01

    The theory for salt dependence of the free energy, entropy, and enthalpy of a polyelectrolyte in the PB (PB) model is extended to treat the nonspecific salt dependence of polyelectrolyte-ligand binding reactions. The salt dependence of the binding constant (K) is given by the difference in osmotic pressure terms between the reactants and products. For simple 1-1 salts it is shown that this treatment is equivalent to the general preferential interaction model for the salt dependence of binding [C. Anderson and M. Record (1993) Journal of Physical Chemistry, Vol. 97, pp. 7116-7126]. The salt dependence, entropy, and enthalpy are compared for the PB model and one specific form of the preferential interaction coefficient model that uses counterion condensation/limiting law (LL) behavior. The PB and LL models are applied to three ligand-polyelectrolyte systems with the same net ligand charge: a model sphere-cylinder binding reaction, a drug-DNA binding reaction, and a protein-DNA binding reaction. For the small ligands both the PB and limiting law models give (In K vs. In[salt]) slopes close in magnitude to the net ligand charge. However, the enthalpy/entropy breakdown of the salt dependence is quite different. In the PB model there are considerable contributions from electrostatic enthalpy and dielectric (water reorientation) entropy, compared to the predominant ion cratic (release) entropy in the limiting law model. The relative contributions of these three terms in the PB model depends on the ligand: For the protein, ion release entropy is the smallest contribution to the salt dependence of binding. The effect of three approximations made in the LL model is examined: These approximations are (1) the ligand behaves ideally, (2) the preferential interaction coefficient of the polyelectrolyte is unchanged upon ligand binding, and (3) the polyelectrolyte preferential interaction coefficient is given by the limiting law/counterion-condensation value. Analysis of the PB

  10. Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins.

    PubMed

    Venkitakrishnan, Rani Parvathy; Benard, Outhiriaradjou; Max, Marianna; Markley, John L; Assadi-Porter, Fariba M

    2012-01-01

    Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.

  11. Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

    PubMed

    Kuznetsova, Irina M; Sulatskaya, Anna I; Povarova, Olga I; Turoverov, Konstantin K

    2012-01-01

    In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.

  12. Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites

    NASA Astrophysics Data System (ADS)

    Vukovic, Sinisa; Brennan, Paul E.; Huggins, David J.

    2016-09-01

    The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process.

  13. Exploring the role of water in molecular recognition: predicting protein ligandability using a combinatorial search of surface hydration sites.

    PubMed

    Vukovic, Sinisa; Brennan, Paul E; Huggins, David J

    2016-09-01

    The interaction between any two biological molecules must compete with their interaction with water molecules. This makes water the most important molecule in medicine, as it controls the interactions of every therapeutic with its target. A small molecule binding to a protein is able to recognize a unique binding site on a protein by displacing bound water molecules from specific hydration sites. Quantifying the interactions of these water molecules allows us to estimate the potential of the protein to bind a small molecule. This is referred to as ligandability. In the study, we describe a method to predict ligandability by performing a search of all possible combinations of hydration sites on protein surfaces. We predict ligandability as the summed binding free energy for each of the constituent hydration sites, computed using inhomogeneous fluid solvation theory. We compared the predicted ligandability with the maximum observed binding affinity for 20 proteins in the human bromodomain family. Based on this comparison, it was determined that effective inhibitors have been developed for the majority of bromodomains, in the range from 10 to 100 nM. However, we predict that more potent inhibitors can be developed for the bromodomains BPTF and BRD7 with relative ease, but that further efforts to develop inhibitors for ATAD2 will be extremely challenging. We have also made predictions for the 14 bromodomains with no reported small molecule K d values by isothermal titration calorimetry. The calculations predict that PBRM1(1) will be a challenging target, while others such as TAF1L(2), PBRM1(4) and TAF1(2), should be highly ligandable. As an outcome of this work, we assembled a database of experimental maximal K d that can serve as a community resource assisting medicinal chemistry efforts focused on BRDs. Effective prediction of ligandability would be a very useful tool in the drug discovery process.

  14. On the interaction of luminol with human serum albumin: Nature and thermodynamics of ligand binding

    NASA Astrophysics Data System (ADS)

    Moyon, N. Shaemningwar; Mitra, Sivaprasad

    2010-09-01

    The mechanism and thermodynamic parameters for the binding of luminol (LH 2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH 2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH 2 binds into the sub-domain IIA of the protein.

  15. Influence of length and flexibility of spacers on the binding affinity of divalent ligands

    PubMed Central

    Liese, Susanne

    2015-01-01

    Summary We present a quantitative model for the binding of divalent ligand–receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket. PMID:26124882

  16. Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein

    SciTech Connect

    Calvo, Eric; Mans, Ben J.; Ribeiro, José M.C.; Andersen, John F.

    2009-04-07

    The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describe the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.

  17. Cationic Gold Clusters Ligated with Differently Substituted Phosphines: Effect of Substitution on Ligand Reactivity and Binding

    SciTech Connect

    Johnson, Grant E.; Olivares, Astrid M.; Hill, David E.; Laskin, Julia

    2015-01-01

    We present a systematic study of the effect of the number of methyl (Me) and cyclohexyl (Cy) functional groups in monodentate phosphine ligands on the solution-phase synthesis of ligated sub-nanometer gold clusters and their gas-phase fragmentation pathways. Small mixed ligand cationic gold clusters were synthesized using ligand exchange reactions between pre-formed triphenylphosphine ligated (PPh3) gold clusters and monodentate Me- and Cy-substituted ligands in solution and characterized using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation (CID) experiments. Under the same experimental conditions, larger gold-PPh3 clusters undergo efficient exchange of unsubstituted PPh3 ligands for singly Me- and Cy-substituted PPh2Me and PPh2Cy ligands. The efficiency of ligand exchange decreases with an increasing number of Me or Cy groups in the substituted phosphine ligands. CID experiments performed for a series of ligand-exchanged gold clusters indicate that loss of a neutral Me-substituted ligand is preferred over loss of a neutral PPh¬3 ligand while the opposite trend is observed for Cy-substituted ligands. The branching ratio of the competing ligand loss channels is strongly correlated with the electron donating ability of the phosphorous lone pair as determined by the relative proton affinity of the ligand. The results indicate that the relative ligand binding energies increase in the order PMe3 < PPhMe2 < PPh2Me < PPh3< PPh2Cy < PPhCy2< PCy3. Furthermore, the difference in relative ligand binding energies increases with the number of substituted PPh3-mMem or PPh3-mCym ligands (L) exchanged onto each cluster. This study provides the first experimental determination of the relative binding energies of ligated gold clusters containing differently substituted monophosphine ligands, which are important to controlling their synthesis and reactivity in solution. The results also indicate that ligand substitution is an important

  18. Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy.

    PubMed

    Le Sueur, Amanda L; Schaugaard, Richard N; Baik, Mu-Hyun; Thielges, Megan C

    2016-06-01

    The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes. PMID:27164303

  19. The Free Energy Landscape of GABA Binding to a Pentameric Ligand-Gated Ion Channel and Its Disruption by Mutations.

    PubMed

    Comitani, Federico; Limongelli, Vittorio; Molteni, Carla

    2016-07-12

    Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily are important neuroreceptors that mediate fast synaptic transmission. They are activated by the binding of a neurotransmitter, but the details of this process are still not fully understood. As a prototypical pLGIC, here we choose the insect resistance to dieldrin (RDL) receptor involved in resistance to insecticides and investigate the binding of the neurotransmitter GABA to its extracellular domain at the atomistic level. We achieve this by means of μ-sec funnel-metadynamics simulations, which efficiently enhance the sampling of bound and unbound states by using a funnel-shaped restraining potential to limit the exploration in the solvent. We reveal the sequence of events in the binding process from the capture of GABA from the solvent to its pinning between the charged residues Arg111 and Glu204 in the binding pocket. We characterize the associated free energy landscapes in the wild-type RDL receptor and in two mutant forms, where the key residues Arg111 and Glu204 are mutated to Ala. Experimentally these mutations produce nonfunctional channels, which is reflected in the reduced ligand binding affinities due to the loss of essential interactions. We also analyze the dynamical behavior of the crucial loop C, whose opening allows the access of GABA to the binding site and closure locks the ligand into the protein. The RDL receptor shares structural and functional features with other pLGICs; hence, our work outlines a valuable protocol to study the binding of ligands to pLGICs beyond conventional docking and molecular dynamics techniques. PMID:27228114

  20. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

    PubMed Central

    2012-01-01

    Background The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM). In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists) to their targets (ecdysone receptors) leads to an adaptive response (resistance), is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella. PMID:23078528

  1. PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma: A case report

    SciTech Connect

    Pappata, S.; Cornu, P.; Samson, Y.; Prenant, C.; Benavides, J.; Scatton, B.; Crouzel, C.; Hauw, J.J.; Syrota, A. )

    1991-08-01

    The utility of the peripheral type benzodiazepine site ligand 11C-PK 11195, for imaging human glioma in conjunction with Positron Emission Tomography, relies on a high specific binding of the tracer to tumoral peripheral type benzodiazepines sites. In a patient with glioblastoma, the authors found that 11C-PK 11195 binding was two-fold higher in the tumor than in normal gray matter and that 30% of tumoral binding could be displaced by a large excess of unlabeled drug. These findings suggest that tumoral retention of the ligand is due, in part, to specific binding.

  2. Binding of flexible and constrained ligands to the Grb2 SH2 domain: structural effects of ligand preorganization

    PubMed Central

    Clements, John H.; DeLorbe, John E.; Benfield, Aaron P.; Martin, Stephen F.

    2010-01-01

    Structures of the Grb2 SH2 domain complexed with a series of pseudopeptides containing flexible (benzyl succinate) and constrained (aryl cyclopropanedicarboxylate) replacements of the phosphotyrosine (pY) residue in tripeptides derived from Ac-pYXN-NH2 (where X = V, I, E and Q) were elucidated by X-ray crystallography. Complexes of flexible/constrained pairs having the same pY + 1 amino acid were analyzed in order to ascertain what structural differences might be attributed to constraining the phosphotyrosine replacement. In this context, a given structural dissimilarity between complexes was considered to be significant if it was greater than the corresponding difference in complexes coexisting within the same asymmetric unit. The backbone atoms of the domain generally adopt a similar conformation and orientation relative to the ligands in the complexes of each flexible/constrained pair, although there are some significant differences in the relative orientations of several loop regions, most notably in the BC loop that forms part of the binding pocket for the phosphate group in the tyrosine replacements. These variations are greater in the set of complexes of constrained ligands than in the set of complexes of flexible ligands. The constrained ligands make more direct polar contacts to the domain than their flexible counterparts, whereas the more flexible ligand of each pair makes more single-water-mediated contacts to the domain; there was no correlation between the total number of protein–ligand contacts and whether the phosphotyrosine replacement of the ligand was preorganized. The observed differences in hydrophobic interactions between the complexes of each flexible/constrained ligand pair were generally similar to those observed upon comparing such contacts in coexisting complexes. The average adjusted B factors of the backbone atoms of the domain and loop regions are significantly greater in the complexes of constrained ligands than in the complexes of

  3. Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

    PubMed Central

    Michaloski, Jussara S.; Redondo, Alexandre R.; Magalhães, Leila S.; Cambui, Caio C.; Giordano, Ricardo J.

    2016-01-01

    Receptor tyrosine kinases (RTKs) are key molecules in numerous cellular processes, the inhibitors of which play an important role in the clinic. Among them are the vascular endothelial growth factor (VEGF) family members and their receptors (VEGFR), which are essential in the formation of new blood vessels by angiogenesis. Anti-VEGF therapy has already shown promising results in oncology and ophthalmology, but one of the challenges in the field is the design of specific small-molecule inhibitors for these receptors. We show the identification and characterization of small 6-mer peptides that target the extracellular ligand-binding domain of all three VEGF receptors. These peptides specifically prevent the binding of VEGF family members to all three receptors and downstream signaling but do not affect other angiogenic RTKs and their ligands. One of the selected peptides was also very effective at preventing pathological angiogenesis in a mouse model of retinopathy, normalizing the vasculature to levels similar to those of a normal developing retina. Collectively, our results suggest that these peptides are pan-VEGF inhibitors directed at a common binding pocket shared by all three VEGFRs. These peptides and the druggable binding site they target might be important for the development of novel and selective small-molecule, extracellular ligand-binding inhibitors of RTKs (eTKIs) for angiogenic-dependent diseases.

  4. 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.

  5. Principles of Ligand Binding within a Completely Buried Cavity in HIF2[alpha] PAS-B

    SciTech Connect

    Key, Jason; Scheuermann, Thomas H.; Anderson, Peter C.; Daggett, Valerie; Gardner, Kevin H.

    2010-04-19

    Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment. The C-terminal Per-ARNT-Sim (PAS) domain of HIF2{alpha} (HIF2{alpha} PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures and equilibrium and transition-state thermodynamics of HIF2{alpha} PAS-B with several artificial ligands using isothermal titration calorimetry, NMR exchange spectroscopy, and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in the NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.

  6. Structural transitions in ion coordination driven by changes in competition for ligand binding

    PubMed Central

    Varma, Sameer; Rempe, Susan B.

    2009-01-01

    Transferring Na+ and K+ ions from their preferred coordination states in water to states having different coordination numbers incurs a free energy cost. In several examples in nature, however, these ions readily partition from aqueous-phase coordination states into spatial regions having much higher coordination numbers. Here we utilize statistical theory of solutions, quantum chemical simulations, classical mechanics simulations and structural informatics to understand this aspect of ion partitioning. Our studies lead to the identification of a specific role of the solvation environment in driving transitions in ion coordination structures. Although ion solvation in liquid media is an exergonic reaction overall, we find it is also associated with considerable free energy penalties for extracting ligands from their solvation environments to form coordinated ion complexes. Reducing these penalties increases the stabilities of higher-order coordinations and brings down the energetic cost to partition ions from water into over-coordinated binding sites in biomolecules. These penalties can be lowered via a reduction in direct favorable interactions of the coordinating ligands with all atoms other than the ions themselves. A significant reduction in these penalties can, in fact, also drive up ion coordination preferences. Similarly, an increase in these penalties can lower ion coordination preferences, akin to a Hofmeister effect. Since such structural transitions are effected by the properties of the solvation phase, we anticipate that they will also occur for other ions. The influence of other factors, including ligand density, ligand chemistry and temperature, on the stabilities of ion coordination structures are also explored. PMID:18954053

  7. DBSI: DNA-binding site identifier

    PubMed Central

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

    2013-01-01

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

  8. Computational Identification of Uncharacterized Cruzain Binding Sites

    PubMed Central

    Wilson, Benjamin A.; McCammon, J. Andrew

    2010-01-01

    Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention. PMID:20485483

  9. Protein conformational plasticity and complex ligand-binding kinetics explored by atomistic simulations and Markov models

    PubMed Central

    Plattner, Nuria; Noé, Frank

    2015-01-01

    Understanding the structural mechanisms of protein–ligand binding and their dependence on protein sequence and conformation is of fundamental importance for biomedical research. Here we investigate the interplay of conformational change and ligand-binding kinetics for the serine protease Trypsin and its competitive inhibitor Benzamidine with an extensive set of 150 μs molecular dynamics simulation data, analysed using a Markov state model. Seven metastable conformations with different binding pocket structures are found that interconvert at timescales of tens of microseconds. These conformations differ in their substrate-binding affinities and binding/dissociation rates. For each metastable state, corresponding solved structures of Trypsin mutants or similar serine proteases are contained in the protein data bank. Thus, our wild-type simulations explore a space of conformations that can be individually stabilized by adding ligands or making suitable changes in protein sequence. These findings provide direct evidence of conformational plasticity in receptors. PMID:26134632

  10. Inclusion of multiple fragment types in the Site Identification by Ligand Competitive Saturation (SILCS) approach

    PubMed Central

    Raman, E. Prabhu; Yu, Wenbo; Lakkaraju, Sirish K.; MacKerell, Alexander D.

    2014-01-01

    The Site Identification by Ligand Competitive Saturation (SILCS) method identifies the location and approximate affinities of small molecular fragments on a target macromolecular surface by performing Molecular Dynamics (MD) simulations of the target in an aqueous solution of small molecules representative of different chemical functional groups. In this study, we introduce a set of small molecules to map potential interactions made by neutral hydrogen bond donors and acceptors, and charged donor and acceptor fragments in addition to nonpolar fragments. The affinity pattern is obtained in the form of discretized probability or, equivalently, free energy maps, called FragMaps, which can be visualized with the target surface. We performed SILCS simulations for four proteins for which structural and thermodynamic data is available for multiple, diverse ligands. Good overlap is shown between high affinity regions identified by the FragMaps and the crystallographic positions of ligand functional groups with similar chemical functionality, thus demonstrating the validity of the qualitative information obtained from the simulations. To test the ability of FragMaps in providing quantitative predictions, we calculate the previously introduced Ligand Grid Free Energy (LGFE) metric and observe its correspondence with experimentally measured binding affinity. LGFE is computed for different conformational ensembles and improvement in prediction is shown with increasing ligand conformational sampling. Ensemble generation includes a Monte Carlo sampling approach that uses the GFE FragMaps directly as the energy function. The results show some, but not all experimental trends are predicted, and warrant improvements in the scoring methodology. In addition, the potential utility of atom-based free energy contributions to the LGFE scores and the use of multiple ligands in SILCS to identify displaceable water molecules during ligand design are discussed. PMID:24245913

  11. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory

    NASA Astrophysics Data System (ADS)

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R.

    2015-12-01

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  12. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory.

    PubMed

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  13. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  14. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

  15. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    SciTech Connect

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E. )

    1991-06-04

    Tritium-labeled {alpha}- and {beta}-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10{degrees}C, MBP bound {alpha}-maltose with 2.7 {plus minus} 0.5-fold higher affinity than {beta}-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound {alpha}-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound {beta}-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the {beta}-maltodextrin is bound by its reducing end, and, in the other complex, the {beta}-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

  16. Thermodynamics of binding of di- and tetrasubstituted naphthalene diimide ligands to DNA G-quadruplex.

    PubMed

    Prato, Gary; Silvent, Samantha; Saka, Sammy; Lamberto, Massimiliano; Kosenkov, Dmytro

    2015-02-26

    Naphthalene diimide ligands have the potential to stabilize human telomeric G-quadruplex DNA via noncovalent interactions. Stabilization of G-quadruplex high order structures has become an important strategy to develop novel anticancer therapeutics. In this study four naphthalene diimide based ligands were analyzed in order to elucidate the principal factors determining contributions to G-quadruplex-ligand binding. Three possible modes of binding and their respective Gibbs free energies for two naphthalene diimide based di-N-alkylpyridinium substituted ligands have been determined using a molecular docking technique and compared to experimental results. The structures obtained from the molecular docking calculations, were analyzed using the ab initio based fragment molecular orbital (FMO) method in order to determine the major enthalpic contributions to the binding and types of interactions between the ligand and specific residues of the G-quadruplex. A computational methodology for the efficient and inexpensive ligand optimization as compared to fully ab initio methods based on the estimation of binding affinities of the naphthalene diimide derived ligands to G-quadruplex is proposed.

  17. Thermodynamics of binding of di- and tetrasubstituted naphthalene diimide ligands to DNA G-quadruplex.

    PubMed

    Prato, Gary; Silvent, Samantha; Saka, Sammy; Lamberto, Massimiliano; Kosenkov, Dmytro

    2015-02-26

    Naphthalene diimide ligands have the potential to stabilize human telomeric G-quadruplex DNA via noncovalent interactions. Stabilization of G-quadruplex high order structures has become an important strategy to develop novel anticancer therapeutics. In this study four naphthalene diimide based ligands were analyzed in order to elucidate the principal factors determining contributions to G-quadruplex-ligand binding. Three possible modes of binding and their respective Gibbs free energies for two naphthalene diimide based di-N-alkylpyridinium substituted ligands have been determined using a molecular docking technique and compared to experimental results. The structures obtained from the molecular docking calculations, were analyzed using the ab initio based fragment molecular orbital (FMO) method in order to determine the major enthalpic contributions to the binding and types of interactions between the ligand and specific residues of the G-quadruplex. A computational methodology for the efficient and inexpensive ligand optimization as compared to fully ab initio methods based on the estimation of binding affinities of the naphthalene diimide derived ligands to G-quadruplex is proposed. PMID:25635929

  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. Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA*

    PubMed Central

    Bui, Soi H.; McLean, Kirsty J.; Cheesman, Myles R.; Bradley, Justin M.; Rigby, Stephen E. J.; Levy, Colin W.; Leys, David; Munro, Andrew W.

    2012-01-01

    The Rhodococcus rhodochrous strain 11Y XplA enzyme is an unusual cytochrome P450-flavodoxin fusion enzyme that catalyzes reductive denitration of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX). We show by light scattering that XplA is a monomeric enzyme. XplA has high affinity for imidazole (Kd = 1.6 μm), explaining previous reports of a red-shifted XplA Soret band in pure enzyme. The true Soret maximum of XplA is at 417 nm. Similarly, unusually weak XplA flavodoxin FMN binding (Kd = 1.09 μm) necessitates its purification in the presence of the cofactor to produce hallmark flavin contributions absent in previously reported spectra. Structural and ligand-binding data reveal a constricted active site