Øyvind Edvardsen

A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors

A database system and computer programs for storage and retrieval of information about guanine nucleotide‐binding protein (G protein) ‐coupled receptor mutants and associated biological effects have been developed. Mutation data on the receptors were collected from the literature and a database of mutants and effects of mutations was developed. The G protein‐coupled receptor, family A, point mutation database (GRAP) provides detailed information on ligand‐binding and signal transduction properties of more than 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned, and from this alignment mutation data may be retrieved. Alternatively, a search form allowing detailed specification of which mutants to retrieve may be used, for example, to search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://www‐grap.fagmed.uit.no/GRAP/homepage.html) using the World‐Wide Web system.

TinyGRAP database: a bioinformatics tool to mine G-protein-coupled receptor mutant data.

We gratefully acknowledge the European Union for sponsoring the GPCRDB (project PL950224), and the Norwegian Research Council and the Forskerakademiet, Denmark for their support of O. Edvardsen and K. Kristiansen, respectively.

A putative model of the dopamine transporter

A three-dimensional model of the human dopamine transporter was constructed by molecular modeling techniques from its amino acid sequence, based on sequence analysis of this and 9 other transporter proteins. The model has 12 membrane spanning alpha-helices arranged in two 7-helical bundles, loops between helices and amino- and carboxy termini. The molecular electrostatic potentials were mainly negative at the synaptic side and positive in the cytoplasmic domains of the transporter model, strongly positive in some of the transmembrane domains, and strongly negative in other transmembrane domains. The model suggests specific binding sites for dopamine and cocaine, a functional role for chloride ions, and accounts for known structure-activity relationships of cocaine analogs at the dopamine transporter.

Molecular dynamics of serotonin and ritanserin interacting with the 5-HT2 receptor

A three-dimensional model of the serotonin (5-hydroxytrytamine; 5-HT) 5-HT2 receptor was constructed from the amino acid sequence by molecular graphics techniques, molecular mechanics energy calculations and molecular dynamics simulations. The receptor model has 7 alpha helical segments which form a membrane-spanning duct with a putative ligand binding site. Most of the synaptic domains and the ligand binding site were surrounded by negative electrostatic potentials, suggesting that positively charged ligands are attracted to the receptor by electrostatic forces. The cytoplasmic domains, except the C-terminal tail, had mainly positive electrostatic potentials. The molecular dynamics of the receptor-ligand complex was examined in 100 ps simulations with 5-HT or ritanserin at a postulated binding site. During the simulations the helices moved from an initial circular arrangement into a more oval arrangement, and became slightly tilted relative to each other. The protonated ligands neutralized the negative electrostatic potentials around Asp 120 and Asp 155 in the central core of the receptor. 5-HT had only weak interactions with Asp 155 but strong interactions with Asp 120 during the simulations, with the amino group of 5-HT tightly bound to the carboxylic side chain of Asp 120. Ritanserin showed similarly strong interactions with Asp 120 and Asp 155 during the simulations.

A putative three-dimensional arrangement of the human serotonin transporter transmembrane helices: a tool to aid experimental studies.

The human serotonin transporter is the molecular target for selective serotonin reuptake inhibitor drugs which are being used for treatment of depression. A three-dimensional model of the membrane spanning parts of the transporter was constructed. The transporter was assumed to consist of 12 transmembrane alpha-helices. The model was based on published experimental data of cocaine binding to mutant transporters, amino acid sequence analysis, and interactive molecular graphics. The model suggests that a high affinity cocaine binding site is situated in a region of the model where Asp98 acts like an anchor, while a putative low affinity site is situated in another region with Glu508 as the anchoring amino acid. A series of docking experiments with various reuptake inhibitors were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. Experiments involving molecular mapping of ligand binding areas may benefit from using the current model in experimental design. From the current model, several amino acids were proposed as prime candidates for mutagenesis and subsequent ligand binding studies. Also for evaluation of results from site directed mutagenesis experiments with SERT and similar transporters we assume the model will be helpful.

tGRAP, the G-protein coupled receptors mutant database.

The searchable mutant database tGRAP (previously called tinyGRAP) at the University of Tromsø contains data on mutated G-protein coupled receptors (GPCRs). All data have been extracted from scientific papers and entered manually into the database. The current version of the tGRAP mutant database (tGRAP.uit.no, release 10, April 2001) contains around 10 500 mutants extracted from almost 1400 research papers containing mutant data on five families of GPCRs, i.e. Family A, rhodopsin-like; Family B, secretin-like; Family C, metabotropic glutamate-like; Family D, pheromone; Family E, cAMP receptors. A query form provides rapid and simple access to relevant mutant information. In addition to this query form, a tool that enables the user to access mutation data via sequence alignments has been introduced. The ability to access mutant data from such alignments increases the usefulness of the mutant database and facilitates comparison of mutagenesis data between receptors. Moreover, this tool allows the construction of tailor-made sequence alignment views from any combination of receptors belonging to the same class. The database is available at http://tGRAP.uit.no/.

The Ligand-binding Site of Buspirone Analogues at the 5-HT1A Receptor

A three‐dimensional model of the 5‐HT1A receptor in man was constructed by molecular‐modelling techniques and used to study the molecular interactions of a series of buspirone analogues with the 5‐HT1A receptor by molecular‐mechanical‐energy minimization and molecular‐dynamics simulations.

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.

Molecular dynamics of 5-HT1A and 5-HT2A serotonin receptors with methylated buspirone analogues.

In the present study experimentally determined ligand selectivity of three methylated buspirone analogues (denoted as MM2, MM5 and P55) towards 5-HT1A and 5-HT2A serotonin receptors was theoretically investigated on a molecular level. The relationships between the ligand structure and 5-HT1A and 5-HT2A receptor affinities were studied and the results were found to be in agreement with the available site-directed mutagenesis and binding affinity data. Molecular dynamics (MD) simulations of ligand-receptor complexes were performed for each investigated analogue, docked twice into the central cavity of 5-HT1A/5-HT2A, each time in a different orientation. Present results were compared with our previous theoretical results, obtained for buspirone and its non-methylated analogues. It was found that due to the presence of the methyl group in the piperazine ring the ligand position alters and the structure of the ligand-receptor complex is modified. Further, the positions of derivatives with pyrimidinyl aromatic moiety and quinolinyl moiety are significantly different at the 5-HT2A receptor. Thus, methylation of such derivatives alters the 3D structures of ligand-receptor complexes in different ways. The ligand-induced changes of the receptor structures were also analysed. The obtained results suggest, that helical domains of both receptors have different dynamical behaviour. Moreover, both location and topography of putative binding sites for buspirone analogues are different at 5-HT1A and 5-HT2A receptors.

A world-wide web service for calculating approximate AMBER CC and CN bond stretching and torsional parameters

Abstract A computer program implementing linear interpolation for the purpose of calculating approximate CC and CN bond stretching and twofold torsional parameters for the AMBER force fields has been constructed. The computer program is based on published data from the Kollman group, and the method has been extended to enable the use of π-bond orders for parameter calculation. The computer program is available for use through a user-friendly World-Wide Web page.