Homology Modeling of Transporter Proteins.

Membrane transporter proteins are divided into channels/pores and carriers and constitute protein families of physiological and pharmacological importance. Several presently used therapeutic compounds elucidate their effects by targeting membrane transporter proteins, including anti-arrhythmic, anesthetic, antidepressant, anxiolytic and diuretic drugs. The lack of three-dimensional structures of human transporters hampers experimental studies and drug discovery. In this chapter, the use of homology modeling for generating structural models of membrane transporter proteins is reviewed. The increasing number of atomic resolution structures available as templates, together with improvements in methods and algorithms for sequence alignments, secondary structure predictions, and model generation, in addition to the increase in computational power have increased the applicability of homology modeling for generating structural models of transporter proteins. Different pitfalls and hints for template selection, multiple-sequence alignments, generation and optimization, validation of the models, and the use of transporter homology models for structure-based virtual ligand screening are discussed.

In Silico Site-Directed Mutagenesis Informs Species-Specific Predictions of Chemical Susceptibility Derived From the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) Tool.

Chemical hazard assessment requires extrapolation of information from model organisms to all species of concern. The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was developed as a rapid, cost-effective method to aid cross-species extrapolation of susceptibility to chemicals acting on specific protein targets through evaluation of protein structural similarities and differences. The greatest resolution for extrapolation of chemical susceptibility across species involves comparisons of individual amino acid residues at key positions involved in protein-chemical interactions. However, a lack of understanding of whether specific amino acid substitutions among species at key positions in proteins affect interaction with chemicals made manual interpretation of alignments time consuming and potentially inconsistent. Therefore, this study used in silico site-directed mutagenesis coupled with docking simulations of computational models for acetylcholinesterase (AChE) and ecdysone receptor (EcR) to investigate how specific amino acid substitutions impact protein-chemical interaction. This study found that computationally derived substitutions in identities of key amino acids caused no change in protein-chemical interaction if residues share the same side chain functional properties and have comparable molecular dimensions, while differences in these characteristics can change protein-chemical interaction. These findings were considered in the development of capabilities for automatically generated species-specific predictions of chemical susceptibility in SeqAPASS. These predictions for AChE and EcR were shown to agree with SeqAPASS predictions comparing the primary sequence and functional domain sequence of proteins for more than 90% of the investigated species, but also identified dramatic species-specific differences in chemical susceptibility that align with results from standard toxicity tests. These results provide a compelling line of evidence for use of SeqAPASS in deriving screening level, species-specific, susceptibility predictions across broad taxonomic groups for application to human and ecological hazard assessment.

From Homology Models to a Set of Predictive Binding Pockets-a 5-HT1A Receptor Case Study.

Despite its remarkable importance in the arena of drug design, serotonin 1A receptor (5-HT1A) has been elusive to the X-ray crystallography community. This lack of direct structural information not only hampers our knowledge regarding the binding modes of many popular ligands (including the endogenous neurotransmitter-serotonin), but also limits the search for more potent compounds. In this paper we shed new light on the 3D pharmacological properties of the 5-HT1A receptor by using a ligand-guided approach (ALiBERO) grounded in the Internal Coordinate Mechanics (ICM) docking platform. Starting from a homology template and set of known actives, the method introduces receptor flexibility via Normal Mode Analysis and Monte Carlo sampling, to generate a subset of pockets that display enriched discrimination of actives from inactives in retrospective docking. Here, we thoroughly investigated the repercussions of using different protein templates and the effect of compound selection on screening performance. Finally, the best resulting protein models were applied prospectively in a large virtual screening campaign, in which two new active compounds were identified that were chemically distinct from those described in the literature.

Environmental Chemicals Modulate Polar Bear (Ursus maritimus) Peroxisome Proliferator-Activated Receptor Gamma (PPARG) and Adipogenesis in Vitro.

We studied interactions between polar bear peroxisome proliferator-activated receptor gamma (pbPPARG) and selected compounds using a luciferase reporter assay and predictions through molecular docking. Furthermore, we studied adipogenesis by liver and adipose tissue extracts from a polar bear and three synthetic mixtures of contaminants in murine 3T3-L1 preadipocytes and polar bear adipose tissue-derived stem cells (pbASCs). PCB153 and p,p'-DDE antagonized pbPPARG, although their predicted receptor-ligand affinity was weak. PBDEs, tetrabromobisphenol A, and PCB170 had a weak agonistic effect on pbPPARG, while hexabromocyclododecane, bisphenol A, oxychlordane, and endosulfan were weak antagonists. pbPPARG-mediated luciferase activity was suppressed by synthetic contaminant mixtures reflecting levels measured in polar bear adipose tissue, as were transcript levels of PPARG and the PPARG target gene fatty acid binding protein 4 (FABP4) in pbASCs. Contaminant extracts from polar bear tissues enhanced triglyceride accumulation in murine 3T3-L1 cells and pbASCs, whereas triglyceride accumulation was not affected by the synthetic mixtures. Chemical characterization of extracts using nontarget methods revealed presence of exogenous compounds that have previously been reported to induce adipogenesis. These compounds included phthalates, tonalide, and nonylphenol. In conclusion, major legacy contaminants in polar bear adipose tissue exert antagonistic effects on PPARG, but adipogenesis by a mixture containing emerging compounds may be enhanced through PPARG or other pathways.

Identification of novel serotonin transporter compounds by virtual screening.

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) plays an essential role in the termination of serotonergic neurotransmission by removing 5-HT from the synaptic cleft into the presynaptic neuron. It is also of pharmacological importance being targeted by antidepressants and psychostimulant drugs. Here, five commercial databases containing approximately 3.24 million drug-like compounds have been screened using a combination of two-dimensional (2D) fingerprint-based and three-dimensional (3D) pharmacophore-based screening and flexible docking into multiple conformations of the binding pocket detected in an outward-open SERT homology model. Following virtual screening (VS), selected compounds were evaluated using in vitro screening and full binding assays and an in silico hit-to-lead (H2L) screening was performed to obtain analogues of the identified compounds. Using this multistep VS/H2L approach, 74 active compounds, 46 of which had K(i) values of ≤1000 nM, belonging to 16 structural classes, have been identified, and multiple compounds share no structural resemblance with known SERT binders.

A linear combination of pharmacophore hypotheses as a new tool in search of new active compounds--an application for 5-HT1A receptor ligands.

This study explores a new approach to pharmacophore screening involving the use of an optimized linear combination of models instead of a single hypothesis. The implementation and evaluation of the developed methodology are performed for a complete known chemical space of 5-HT1AR ligands (3616 active compounds with K i < 100 nM) acquired from the ChEMBL database. Clusters generated from three different methods were the basis for the individual pharmacophore hypotheses, which were assembled into optimal combinations to maximize the different coefficients, namely, MCC, accuracy and recall, to measure the screening performance. Various factors that influence filtering efficiency, including clustering methods, the composition of test sets (random, the most diverse and cluster population-dependent) and hit mode (the compound must fit at least one or two models from a final combination) were investigated. This method outmatched both single hypothesis and random linear combination approaches.

Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin.

BACKGROUND: Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2). RESULTS: During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2. CONCLUSION: These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

Substrate binding and translocation of the serotonin transporter studied by docking and molecular dynamics simulations.

The serotonin (5-HT) transporter (SERT) plays an important role in the termination of 5-HT-mediated neurotransmission by transporting 5-HT away from the synaptic cleft and into the presynaptic neuron. In addition, SERT is the main target for antidepressant drugs, including the selective serotonin reuptake inhibitors (SSRIs). The three-dimensional (3D) structure of SERT has not yet been determined, and little is known about the molecular mechanisms of substrate binding and transport, though such information is very important for the development of new antidepressant drugs. In this study, a homology model of SERT was constructed based on the 3D structure of a prokaryotic homologous leucine transporter (LeuT) (PDB id: 2A65). Eleven tryptamine derivates (including 5-HT) and the SSRI (S)-citalopram were docked into the putative substrate binding site, and two possible binding modes of the ligands were found. To study the conformational effect that ligand binding may have on SERT, two SERT-5-HT and two SERT-(S)-citalopram complexes, as well as the SERT apo structure, were embedded in POPC lipid bilayers and comparative molecular dynamics (MD) simulations were performed. Our results show that 5-HT in the SERT-5-HT(B) complex induced larger conformational changes in the cytoplasmic parts of the transmembrane helices of SERT than any of the other ligands. Based on these results, we suggest that the formation and breakage of ionic interactions with amino acids in transmembrane helices 6 and 8 and intracellular loop 1 may be of importance for substrate translocation.

Protein binding site analysis by means of structural interaction fingerprint patterns.

We introduce a new approach to the known concept of interaction profiles, based on Structural Interaction Fingerprints (SIFt), for precise and rapid binding site description. A set of scripts for batch generation and analysis of SIFt were prepared, and the implementation is computationally efficient and supports parallelization. It is based on a 9-digit binary interaction pattern that describes physical ligand-protein interactions in structures and models of ligand-protein complexes. The tool performs analysis and identifies binding site residues (crucial and auxiliary) and classifies interactions according to type (hydrophobic, aromatic, charge, polar, side chain, and backbone). It is convenient and easy to use, and gives manageable output data for both, interpretation and further processing. In the presented Letter, SIFts are applied to analyze binding sites in models of antagonist-5-HT7 receptor complexes and structures of cyclin dependent kinase 2-ligand complexes.

The molecular interactions of buspirone analogues with the serotonin transporter.

A major problem with the selective serotonin reuptake inhibitors (SSRIs) is the delayed onset of action. A reason for that may be that the initial SSRI-induced increase in serotonin levels activates somatodendritic 5-HT(1A) autoreceptors, causing a decrease in serotonin release in major forebrain areas. It has been suggested that compounds combining inhibition of the serotonin transport protein with antagonistic effects on the 5-HT(1A) receptor will shorten the onset time. The anxiolytic drug buspirone is known as 5-HT(1A) partial agonist. In the present work, we are studying the inhibition of the serotonin transporter protein by a series of buspirone analogues by molecular modelling and by experimental affinity measurements. Models of the transporter protein were constructed using the crystal structure of the Escherichia coli major facilitator family transporter-LacY and the X-ray structure of the neurotransmitter symporter family (NSS) transporter-LeuT(Aa) as templates. The buspirone analogues were docked into both SERT models and the interactions with amino acids within the protein were analyzed. Two putative binding sites were identified on the LeuT(Aa) based model, one suggested to be a high-affinity site, and the other suggested to be a low-affinity binding site. Molecular dynamic simulations of the LacY based model in complex with ligands did not induce a helical architecture of the LacY based model into an arrangement more similar to that of the LeuT(Aa) based model.

Putative drug binding conformations of monoamine transporters.

Structural information about monoamine transporters and their interactions with psychotropic drugs is important for understanding their molecular mechanisms of action and for drug development. The crystal structure of a Major Facilitator Superfamily (MFS) transporter, the lactose permease symporter (lac permease), has provided insight into the three-dimensional structure and mechanisms of secondary transporters. Based on the hypothesis that the 12 transmembrane alpha-helix (TMH) secondary transporters belong to a common folding class, the lac permease structure was used for molecular modeling of the serotonin transporter (SERT), the dopamine transporter (DAT), and the noradrenaline transporter (NET). The molecular modeling methods used included amino acid sequence alignment, homology modeling, and molecular mechanical energy calculations. The lac permease crystal structure has an inward-facing conformation, and construction of outward-facing SERT, DAT, and NET conformations allowing ligand binding was the most challenging step of the modeling procedure. The psychomotor stimulants cocaine and S-amphetamine, and the selective serotonin reuptake inhibitor (SSRI) S-citalopram, were docked into putative binding sites on the transporters to examine their molecular binding mechanisms. In the inward-facing conformation of SERT the translocation pore was closed towards the extracellular side by hydrophobic interactions between the conserved amino acids Phe105, Pro106, Phe117, and Ala372. An unconserved amino acid, Asp499 in TMH10 in NET, may contribute to the low affinity of S-citalopram to NET.

The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions.

The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6DeltaPD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein-protein interactions, but not DNA binding of Pax6DeltaPD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein-protein interactions.

Subtype-specific sorting of the ETA endothelin receptor by a novel endocytic recycling signal for G protein-coupled receptors.

We have previously reported that endocytic sorting of ET(A) endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxyl-terminal recycling motif of the ET(A) receptor. Assay of truncation mutants of the ET(A) receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 to 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel beta-strand structures (beta-finger). One of these segments was the beta-finger motif of neuronal nitric-oxide synthase reported to function as an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domain-binding ligand. Based on these findings, the three-dimensional structure of the recycling motif of ET(A) receptor was predicted to attain a beta-finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative beta-finger conformation or PDZ ligand function prevented recycling of the ET(A) receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxylterminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ET(A) receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.

Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function.

The superfamily of G-protein-coupled receptors (GPCRs) could be subclassified into 7 families (A, B, large N-terminal family B-7 transmembrane helix, C, Frizzled/Smoothened, taste 2, and vomeronasal 1 receptors) among mammalian species. Cloning and functional studies of GPCRs have revealed that the superfamily of GPCRs comprises receptors for chemically diverse native ligands including (1) endogenous compounds like amines, peptides, and Wnt proteins (i.e., secreted proteins activating Frizzled receptors); (2) endogenous cell surface adhesion molecules; and (3) photons and exogenous compounds like odorants. The combined use of site-directed mutagenesis and molecular modeling approaches have provided detailed insight into molecular mechanisms of ligand binding, receptor folding, receptor activation, G-protein coupling, and regulation of GPCRs. The vast majority of family A, B, C, vomeronasal 1, and taste 2 receptors are able to transduce signals into cells through G-protein coupling. However, G-protein-independent signaling mechanisms have also been reported for many GPCRs. Specific interaction motifs in the intracellular parts of these receptors allow them to interact with scaffold proteins. Protein engineering techniques have provided information on molecular mechanisms of GPCR-accessory protein, GPCR-GPCR, and GPCR-scaffold protein interactions. Site-directed mutagenesis and molecular dynamics simulations have revealed that the inactive state conformations are stabilized by specific interhelical and intrahelical salt bridge interactions and hydrophobic-type interactions. Constitutively activating mutations or agonist binding disrupts such constraining interactions leading to receptor conformations that associates with and activate G-proteins.

Interaction codes within the family of mammalian Phox and Bem1p domain-containing proteins.

The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, lambda/iota- and zeta PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling. Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity. By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62. A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction. Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain. We report several novel interactions within this family. An interaction between the cell polarity scaffold protein Par6 and MEK5 was found. Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs. Evidence for involvement of p62 in MEK5-ERK5 signaling is presented.

Gene conversion as a source of nucleotide diversity in Plasmodium falciparum.

Examination of polymorphisms in the Plasmodium falciparum gene for falcipain 2 revealed that this gene is one of two paralogs separated by 10.8 kb in chromosome 11. We designate the annotated gene denoted chr11.gen_424 as encoding falcipain 2A and the annotated gene denoted chr11.gen_427 as encoding falcipain 2B. The paralogs are 96% identical at the nucleotide level and 93% identical at the amino acid level. The consensus sequences differ in 31/309 synonymous sites and 45/1140 nonsynonymous sites, including three amino acid replacements (V393I, A400P, and Q414E) that are near the catalytic site and that may affect substrate affinity or specificity. In six reference isolates, among 36 synonymous sites and 46 nonsynonymous sites that are polymorphic in the gene for falcipain 2A, falcipain 2B, or both, significant spatial clustering is observed. All but one of the polymorphisms appear to result from gene conversion between the paralogs. The estimated rate of gene conversion between the paralogs may be as many as 1,400 to 1,700 times greater than the rate of mutation. Owing to gene conversion, one of the falcipain 2A alleles is more similar to the falcipain 2B alleles than it is to other falcipain 2A alleles. Divergence among the synonymous sites suggests that the paralogous genes last shared a common ancestor 15.2 MYA, with a range of 8.8 to 20.6 MYA. During this period, the paralogs have acquired 0.10 synonymous substitutions per synonymous site in the coding region. The 5' and 3' flanking regions differ in 47.7% and 39.8% of the nucleotide sites, respectively. Hence synonymous sites and flanking regions are not conserved in sequence in spite of their high AT content and T skew.

Bioinformatics: from genome to drug targets.

The complete sequence determination of the human genome marks the start of a new era in biological science, with focus shifting from sequencing to functional mechanisms of gene products. In addition to effects on gene expression, most of the currently used therapeutic drugs either have enzymes or membrane proteins as their molecular targets of action. These membrane proteins include ion channels and transporters of small molecules, and receptors that convey signals from one side of a membrane to the other. Membrane proteins are thus involved in a variety of cellular processes and have a large potential as targets for new drug discovery. However, detailed structural information is still lacking for the majority of membrane proteins since their association with membrane constituents make NMR (nuclear magnetic resonance) spectroscopic and X-ray diffraction determinations difficult. Molecular modelling by biocomputing is a methodological alternative for structural studies of membrane proteins, but has to be based on experimental structural information in addition to computational techniques. A combination of bioinformatics and experimental techniques was used to model membrane proteins from two different classes, secondary transporters of the sodium:neurotransmitter symporter family (SNF transporters), and G-protein coupled receptors (GPCRs). The protein models were used to examine ligand-protein interactions and signalling/transport mechanisms, and to design experimental site-directed mutagenesis studies. Such studies have provided new insight into the detailed molecular mechanisms of two important classes of membrane proteins, which may be of value in the discovery and development of new pharmaceuticals.

Molecular biology of serotonin receptors structure and function at the molecular level.

5-hydroxytryptamine (5-HT; serotonin) is a neurotransmitter essential for a large number of physiological processes including the regulation of vascular and non-vascular smooth muscle contraction, modulation of platelet aggregation, and the regulation of appetite, mood, anxiety, wakefulness and perception. To mediate this astonishing array of functions, no fewer than 15 separate receptors have evolved, of which all but two (5-HT(3A) and 5-HT(3B)) are G-protein coupled receptors. This review will summarize our current understanding of the structure and function of the G-protein coupled 5-HT receptors. In particular, a systematic review of the available mutagenesis studies of 5-HT receptors will be presented. This information will be synthesized to provide a working model of agonist and antagonist actions at a prototypic 5-HT receptor the 5-HT(2A) receptor. Finally, examples will be given to demonstrate that a detailed knowledge of the predicted structure of one receptor can be useful for structure-based drug design.

Evidence for a model of agonist-induced activation of 5-hydroxytryptamine 2A serotonin receptors that involves the disruption of a strong ionic interaction between helices 3 and 6.

5-Hydroxytryptamine 2A (5-HT2A) receptors are essential for the actions of serotonin (5-hydroxytryptamine (5-HT)) on physiological processes as diverse as vascular smooth muscle contraction, platelet aggregation, perception, and emotion. In this study, we investigated the molecular mechanism(s) by which 5-HT activates 5-HT2A receptors using a combination of approaches including site-directed mutagenesis, molecular modeling, and pharmacological analysis using the sensitive, cell-based functional assay R-SAT. Alanine-scanning mutagenesis of residues close to the intracellular end of H6 of the 5-HT2A receptor implicated glutamate Glu-318(6.30) in receptor activation, as also predicted by a newly constructed molecular model of the 5-HT2A receptor, which was based on the x-ray structure of bovine rhodopsin. Close examination of the molecular model suggested that Glu-318(6.30) could form a strong ionic interaction with Arg-173(3.50) of the highly conserved "(D/E)RY motif" located at the interface between the third transmembrane segment and the second intracellular loop (i2). A direct prediction of this hypothesis, that disrupting this ionic interaction by an E318(6.30)R mutation would lead to a highly constitutively active receptor with enhanced affinity for agonist, was confirmed using R-SAT. Taken together, these results predict that the disruption of a strong ionic interaction between transmembrane helices 3 and 6 of 5-HT2A receptors is essential for agonist-induced receptor activation and, as recently predicted by ourselves (B. L. Roth and D. A. Shapiro (2001) Expert Opin. Ther. Targets 5, 685-695) and others, that this may represent a general mechanism of activation for many, but not all, G-protein-coupled receptors.

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/.