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The ionotropic N-methyl- d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares approximately 40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations. [figure: see text]. The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-A radius of the ligand (glutamate).

Background: Multiple sequence alignment algorithms are very important tools in molecular biology today. Accurate alignment of proteins is central to several areas such as homology modelling, docking studies, understanding evolutionary trends and study of structure-function relationships. In recent times, improvement of existing progressing programs and implementation of new iterative algorithms have made a significant change in this field., Results: We report an alignment algorithm that combines progressive dynamic algorithm, local substructure alignment and iterative refinement to achieve an improved, user-interactive tool. Large-scale benchmarking studies show that this FMALIGN server produces alignments that, aside from preservation of functional and structural conservation, have accuracy comparable to other popular multiple alignment programs., Conclusions: The FMALIGN server allows the user to fix conserved regions in equivalent position in the alignment thereby reducing the chance of global misalignment to a great extent. FMALIGN is available at http://caps.ncbs.res.in/FMALIGN/Home.html.

Functional selection and three-dimensional structural constraints of proteins relate to the retention of significant sequence similarity between proteins of similar fold and function despite poor overall sequence identity and evolutionary pressures. We report the availability of 'iMOT' (interacting MOTif) server, an interactive package for the automatic identification of spatially interacting motifs among distantly related proteins sharing similar folds and possessing common ancestral lineage. Spatial interactions between conserved stretches of a protein are evaluated by calculations of pseudo-potentials that describe the strength of interactions. Such an evaluation permits the automatic identification of highly interacting conserved regions of a protein. Interacting motifs have been shown to be useful in searching for distant homologues and establishing remote homologies among the largely unassigned sequences in genome databases. Information on such motifs should also be of value in protein folding, modelling and engineering experiments. The iMOT server can be accessed from http://www.ncbs.res.in/~faculty/mini/imot/iMOTserver.html. Supplementary Material can be accessed from: http://www.ncbs.res.in/~faculty/mini/imot/supplementary.html.

Background: The functional selection and three-dimensional structural constraints of proteins in nature often relates to the retention of significant sequence similarity between proteins of similar fold and function despite poor sequence identity. Organization of structure-based sequence alignments for distantly related proteins, provides a map of the conserved and critical regions of the protein universe that is useful for the analysis of folding principles, for the evolutionary unification of protein families and for maximizing the information return from experimental structure determination. The Protein Alignment organised as Structural Superfamily (PASS2) database represents continuously updated, structural alignments for evolutionary related, sequentially distant proteins., Description: An automated and updated version of PASS2 is, in direct correspondence with SCOP 1.63, consisting of sequences having identity below 40% among themselves. Protein domains have been grouped into 628 multi-member superfamilies and 566 single member superfamilies. Structure-based sequence alignments for the superfamilies have been obtained using COMPARER, while initial equivalencies have been derived from a preliminary superposition using LSQMAN or STAMP 4.0. The final sequence alignments have been annotated for structural features using JOY4.0. The database is supplemented with sequence relatives belonging to different genomes, conserved spatially interacting and structural motifs, probabilistic hidden markov models of superfamilies based on the alignments and useful links to other databases. Probabilistic models and sensitive position specific profiles obtained from reliable superfamily alignments aid annotation of remote homologues and are useful tools in structural and functional genomics. PASS2 presents the phylogeny of its members both based on sequence and structural dissimilarities. Clustering of members allows us to understand diversification of the family members. The search engine has been improved for simpler browsing of the database., Conclusions: The database resolves alignments among the structural domains consisting of evolutionarily diverged set of sequences. Availability of reliable sequence alignments of distantly related proteins despite poor sequence identity and single-member superfamilies permit better sampling of structures in libraries for fold recognition of new sequences and for the understanding of protein structure-function relationships of individual superfamilies. PASS2 is accessible at http://www.ncbs.res.in/~faculty/mini/campass/pass2.html

Protein comparative modeling has useful applications in large-scale structural initiatives and in rational design of drug targets in medicinal chemistry. The reliability of a homology model is dependent on the sequence identity between the query and the structural homologue used as a template for modeling. Here, we present a method for the utilization and conservation of important structural features of template structures by providing additional spatial restraints in comparative modeling programs like MODELLER. We show that root mean square deviation at C(alpha) positions between the model and the corresponding experimental structure and the quality of the models can be significantly improved for distantly related systems by utilizing additional spatial restraints of the template structures. We demonstrate the influence of such approaches to homology modeling during distant relationships in understanding functional properties of protein such as ligand binding using cytochrome P450 as an example.

DSDBASE is a database of disulphide bonds in proteins, which provides information on native disulphides and those that are stereochemically possible between pairs of residues for all known protein structural entries. The modelling of disulphides has been performed, using MODIP, by the identification of residue pairs that can strainlessly accommodate a covalent cross-link. We also assess the stereochemical quality of the covalent cross-link and grade them appropriately. One of the potential uses of the database is to design site-directed mutants in order to enhance the thermal stability of a protein. The proposed sites of mutations can be viewed specifically with respect to active sites of enzymes and across physiological dimers. The occurrence of native and modelled disulphides increases the dimensions of the database enormously. This database can also be employed for proposing three-dimensional models of disulphide-rich short polypeptides. The database can be accessed from http://www.ncbs.res.in/ approximately faculty/mini/dsdbase/dsdbase.html. Supplementary information can be accessed from http://www.ncbs.res.in/ approximately faculty/mini/dsdbase/nar/suppl.htm.

Tyrosine phosphatases play an important role in cellular signalling and networking that is antagonistic to the kinases. Near completion of the human genome- sequencing project permits us to review the distribution of this family and study its involvement in different pathways. Ninety-six homologues of the classical and dual- specific tyrosine phosphatases (DuSPs) were identified in the human genome using sensitive sequence search techniques. Uncommon domain architectures were encountered, including an example where a kinase and a phosphatase domain are found to co-exist in a single polypeptide. The evolutionary rate is higher for the DuSP compared with the classical tyrosine phosphatases. Orthologues of the 96 putative human tyrosine phosphatases were identified in four model organisms to study the conservation of the family members. Three nuclear localized tyrosine phosphatases retain an orthologous relationship with all model systems considered but still differ in their domain architectures. The diversity in the multi-domain members of the superfamily occurs mainly through domain recruitment, especially in receptor tyrosine phosphatases. The curation of human tyrosine phosphatases provides a convenient framework for characterizing and analysing the functional and structural properties of this diverse family of proteins.

The Structural Motifs of Superfamilies (SMoS) database provides information about the structural motifs of aligned protein domain superfamilies. Such motifs among structurally aligned multiple members of protein superfamilies are recognized by the conservation of amino acid preference and solvent inaccessibility and are examined for the conservation of other features like secondary structural content, hydrogen bonding, non-polar interaction and residue packing. These motifs, along with their sequence and spatial orientation, represent the conserved core structure of each superfamily and also provide the minimal requirement of sequence and structural information to retain each superfamily fold.

Motivation: Although many methods are available for the identification of structural domains from protein three-dimensional structures, accurate definition of protein domains and the curation of such data for a large number of proteins are often possible only after manual intervention. The availability of domain definitions for protein structural entries is useful for the sequence analysis of aligned domains, structure comparison, fold recognition procedures and understanding protein folding, domain stability and flexibility., Results: We have improved our method of domain identification starting from the concept of clustering secondary structural elements, but with an intention of reducing the number of discontinuous segments in identified domains. The results of our modified and automatic approach have been compared with the domain definitions from other databases. On a test data set of 55 proteins, this method acquires high agreement (88%) in the number of domains with the crystallographers' definition and resources such as SCOP, CATH, DALI, 3Dee and PDP databases. This method also obtains 98% overlap score with the other resources in the definition of domain boundaries of the 55 proteins. We have examined the domain arrangements of 4592 non-redundant protein chains using the improved method to include 5409 domains leading to an update of the structural domain database., Availability: The latest version of the domain database and online domain identification methods are available from http://www.ncbs.res.in/~faculty/mini/ddbase/ddbase.html, Supplementary Information: http://www.ncbs.res.in/~faculty/mini/ddbase/supplementary/supplementary.html

Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL synthase superfamily members adopt the fold of the N-acetyltransferase superfamily. We suggest, by the identification of intermediate sequences, that the two superfamilies are evolutionarily related. Evolutionary trace analyses of aligned sequences and docking studies have been used to discuss functionally important residues of EsaI homologues.

Hot water epilepsy (HWE) is a benign and rare form of reflex epilepsy that occurs most commonly in humans. Bdm1 is one of the proteins whose mRNA transcript is overexpressed during HWE in a rat model. We show, by sequence analysis and fold recognition methods, that Bdm1 has strong structural similarities to alpha/beta hydrolases like the thioesterases. A three-dimensional model derived by comparative modeling methods allowed the search for catalytic residues using a flexible functional template characteristic of these enzymes. We predict that Bdm1 might be regulated by homocysteine levels by means of direct participation in degradation pathways.

Members of a superfamily of proteins could result from divergent evolution of homologues with insignificant similarity in the amino acid sequences. A superfamily relationship is detected commonly after the three-dimensional structures of the proteins are determined using X-ray analysis or NMR. The SUPFAM database described here relates two homologous protein families in a multiple sequence alignment database of either known or unknown structure. The present release (1.1), which is the first version of the SUPFAM database, has been derived by analysing Pfam, which is one of the commonly used databases of multiple sequence alignments of homologous proteins. The first step in establishing SUPFAM is to relate Pfam families with the families in PALI, which is an alignment database of homologous proteins of known structure that is derived largely from SCOP. The second step involves relating Pfam families which could not be associated reliably with a protein superfamily of known structure. The profile matching procedure, IMPALA, has been used in these steps. The first step resulted in identification of 1280 Pfam families (out of 2697, i.e. 47%) which are related, either by close homologous connection to a SCOP family or by distant relationship to a SCOP family, potentially forming new superfamily connections. Using the profiles of 1417 Pfam families with apparently no structural information, an all-against-all comparison involving a sequence-profile match using IMPALA resulted in clustering of 67 homologous protein families of Pfam into 28 potential new superfamilies. Expansion of groups of related proteins of yet unknown structural information, as proposed in SUPFAM, should help in identifying 'priority proteins' for structure determination in structural genomics initiatives to expand the coverage of structural information in the protein sequence space. For example, we could assign 858 distinct Pfam domains in 2203 of the gene products in the genome of Mycobacterium tubercolosis. Fifty-one of these Pfam families of unknown structure could be clustered into 17 potentially new superfamilies forming good targets for structural genomics. SUPFAM database can be accessed at http://pauling.mbu.iisc.ernet.in/~supfam.

PASS2 is a nearly automated version of CAMPASS and contains sequence alignments of proteins grouped at the level of superfamilies. This database has been created to fall in correspondence with SCOP database (1.53 release) and currently consists of 110 multi-member superfamilies and 613 superfamilies corresponding to single members. In multi-member superfamilies, protein chains with no more than 25% sequence identity have been considered for the alignment and hence the database aims to address sequence alignments which represent 26 219 protein domains under the SCOP 1.53 release. Structure-based sequence alignments have been obtained by COMPARER and the initial equivalences are provided automatically from a MALIGN alignment and subsequently augmented using STAMP4.0. The final sequence alignments have been annotated for the structural features using JOY4.0. Several interesting links are provided to other related databases and genome sequence relatives. Availability of reliable sequence alignments of distantly related proteins, despite poor sequence identity and single-member superfamilies, permit better sampling of structures in libraries for fold recognition of new sequences and for the understanding of protein structure-function relationships of individual superfamilies. The database can be queried by keywords and also by sequence search, interfaced by PSI-BLAST methods. Structure-annotated sequence alignments and several structural accessory files can be retrieved for all the superfamilies including the user-input sequence. The database can be accessed from http://www.ncbs.res.in/%7Efaculty/mini/campass/pass.html.

Inositol 1,4,5-triphosphate receptors (Insp(3)Rs) and ryanodine receptors (ryRs) act as cationic channels transporting calcium ions from the endoplasmic reticulum to cytosol by forming tetramers and are proteins localized to the endoplasmic reticulum (ER). Despite the absence of classical calcium-binding motifs, calcium channeling occurs at the transmembrane domain. We have investigated putative calcium binding motifs in these sequences. Prediction methods indicate the presence of six transmembrane helices in the C-terminal domain, one of the three domains conserved between Insp(3)R and ryR receptors. The recently identified crystal structure of the K(+) channel, which also forms tetramers, revealed that two transmembrane helices, an additional pore helix and a selectivity filter are responsible for selective K(+) ion channeling. The last three TM helices of Insp(3)R and ryR are particularly well conserved and we found analogous pore helix and selectivity filter motif in these sequences. We obtained a three-dimensional structural model for the transmembrane tetramer by extrapolating the distant structural similarity to the K(+) channels.

With a growing number of structures available in the Brookhaven Protein Data Bank, automatic methods for domain identification are required for the construction of databases. Domains are considered to be clusters of secondary structure elements. Thus, helices and strands are first clustered using intersecondary structural distances between C alpha positions, and dendrograms based on this distance measure are used to identify domains. Individual domains are recognized by a disjoint factor, which enables the automatic identification and classification into disjoint, interacting, and conjoint domains. Application to a database of 83 protein families and 18 unique structures shows that the approach provides an effective delineation of boundaries and identifies those proteins that can be considered as a single domain. A quantitative estimate of the interaction between domains has been proposed. The database of protein domains is a useful tool for understanding protein folding, for recognizing protein folds, and for understanding structure-activity relationships.

The explosion of DNA sequence data from genome projects presents many challenges. For instance, we must extend our current knowledge of protein structure and function so that it can be applied to these new sequences. The derivation of rules for the relationships between sequence and structure allow us to recognize a common fold by the use of tertiary templates. New techniques enable us to begin to meet the challenge of rule-based modelling of distantly related proteins. This paper describes an integrated and knowledge-based approach to the prediction of protein structure and function which can maximize the value of sequence information.

Knowledge, both from the three-dimensional structures of homologous proteins and from the general analysis of protein structure, is of value in modeling a protein of known sequence but unknown structure. While many models are still constructed at least in part by manual methods on graphics devices, automated procedures have come into greater use. These procedures include those that assemble fragments of structure from other known structures and those that derive coordinates for the model from the satisfaction of restraints placed on atomic positions.

A general procedure for arriving at 3-D models of disulphide-rich polypeptide systems based on the covalent cross-link constraints has been developed. The procedure, which has been coded as a computer program, RANMOD, assigns a large number of random, permitted backbone conformations to the polypeptide and identifies stereochemically acceptable structures as plausible models based on strainless disulphide bridge modelling. Disulphide bond modelling is performed using the procedure MODIP developed earlier, in connection with the choice of suitable sites where disulphide bonds could be engineered in proteins (Sowdhamini, R., Srinivasan, N., Shoichet, B., Santi, D.V., Ramakrishnan, C. and Balaram, P. (1989) Protein Engng, 3, 95-103). The method RANMOD has been tested on small disulphide loops and the structures compared against preferred backbone conformations derived from an analysis of putative disulphide subdatabase and model calculations. RANMOD has been applied to disulphide-rich peptides and found to give rise to several stereochemically acceptable structures. The results obtained on the modelling of two test cases, alpha-conotoxin GI and endothelin I, are presented. Available NMR data suggest that such small systems exhibit conformational heterogeneity in solution. Hence, this approach for obtaining several distinct models is particularly attractive for the study of conformational excursions.

A super-secondary structural motif comprising two orthogonally oriented beta-strands connected by short linking segments of less than or equal to 5 residues has been identified from a data set of 65 independent protein crystal structures. Of the 42 examples from 14 proteins, a vast majority have only a single residue as the linking element. Analysis of the conformational angles at the junction reveals that the recently described type VIII beta-turn occurs frequently at the connecting hinge, while the type II beta-turn is also fairly common.

A computer modeling procedure for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cystine disulfide crosslinks has been developed. Residue pairs with C alpha-C alpha distances of less than or equal to 6.5 A and C beta-C beta distances of less than or equal to 4.5 A are chosen for geometrical fixation of S atoms using the program MODIP. The stereochemistry of the modeled disulfides is evaluated using limits for the structural parameters of the various torsion angles and S-S bond length in the disulfide bridge. The ability of the procedure to correctly model disulfides has been checked with examples of cystine peptides of known crystal structures and 103 disulfide bridges from 25 available protein crystal structures determined at less than or equal to 2 A resolution. An analysis of results on three proteins with engineered disulfides, T4 lysozyme, dihydrofolate reductase and subtilisin, is presented. Two positions for the introduction of 'stereochemically optimal' disulfides are identified in subtilisin.