V. S. Gowri

Integration of related sequences with protein three-dimensional structural families in an updated version of PALI database

The database of Phylogeny and ALIgnment of homologous protein structures (PALI) contains three-dimensional (3-D) structure-dependent sequence alignments as well as structure-based phylogenetic trees of protein domains in various families. The latest updated version (Release 2.1) comprises of 844 families of homologous proteins involving 3863 protein domain structures with each of these families having at least two members. Each member in a family has been structurally aligned with every other member in the same family using two proteins at a time. In addition, an alignment of multiple structures has also been performed using all the members in a family. Every family with at least three members is associated with two dendrograms, one based on a structural dissimilarity metric and the other based on similarity of topologically equivalenced residues for every pairwise alignment. Apart from these multi-member families, there are 817 single member families in the updated version of PALI. A new feature in the current release of PALI is the integration, with 3-D structural families, of sequences of homologues from the sequence databases. Alignments between homologous proteins of known 3-D structure and those without an experimentally derived structure are also provided for every family in the enhanced version of PALI. The database with several web interfaced utilities can be accessed at: http://pauling.mbu.iisc.ernet.in/~pali.

SUPFAM: A database of sequence superfamilies of protein domains

BackgroundSUPFAM database is a compilation of superfamily relationships between protein domain families of either known or unknown 3-D structure. In SUPFAM, sequence families from Pfam and structural families from SCOP are associated, using profile matching, to result in sequence superfamilies of known structure. Subsequently all-against-all family profile matches are made to deduce a list of new potential superfamilies of yet unknown structure.DescriptionThe current version of SUPFAM (release 1.4) corresponds to significant enhancements and major developments compared to the earlier and basic version. In the present version we have used RPS-BLAST, which is robust and sensitive, for profile matching. The reliability of connections between protein families is ensured better than before by use of benchmarked criteria involving strict e-value cut-off and a minimal alignment length condition. An e-value based indication of reliability of connections is now presented in the database. Web access to a RPS-BLAST-based tool to associate a query sequence to one of the family profiles in SUPFAM is available with the current release. In terms of the scientific content the present release of SUPFAM is entirely reorganized with the use of 6190 Pfam families and 2317 structural families derived from SCOP. Due to a steep increase in the number of sequence and structural families used in SUPFAM the details of scientific content in the present release are almost entirely complementary to previous basic version. Of the 2286 families, we could relate 245 Pfam families with apparently no structural information to families of known 3-D structures, thus resulting in the identification of new families in the existing superfamilies. Using the profiles of 3904 Pfam families of yet unknown structure, an all-against-all comparison involving sequence-profile match resulted in clustering of 96 Pfam families into 39 new potential superfamilies.ConclusionSUPFAM presents many non-trivial superfamily relationships of sequence families involved in a variety of functions and hence the information content is of interest to a wide scientific community. The grouping of related proteins without a known structure in SUPFAM is useful in identifying priority targets for structural genomics initiatives and in the assignment of putative functions. Database URL: http://pauling.mbu.iisc.ernet.in/~supfam.

MulPSSM: a database of multiple position-specific scoring matrices of protein domain families

Representation of multiple sequence alignments of protein families in terms of position-specific scoring matrices (PSSMs) is commonly used in the detection of remote homologues. A PSSM is generated with respect to one of the sequences involved in the multiple sequence alignment as a reference. We have shown recently that the use of multiple PSSMs corresponding to an alignment, with several sequences in the family used as reference, improves the sensitivity of the remote homology detection dramatically. MulPSSM contains PSSMs for a large number of sequence and structural families of protein domains with multiple PSSMs for every family. The approach involves use of a clustering algorithm to identify most distinct sequences corresponding to a family. With each one of the distinct sequences as reference, multiple PSSMs have been generated. The current release of MulPSSM contains ∼33 000 and ∼38 000 PSSMs corresponding to 7868 sequence and 2625 structural families. A RPS_BLAST interface allows sequence search against PSSMs of sequence or structural families or both. An analysis interface allows display and convenient navigation of alignments and domain hits. MulPSSM can be accessed at .

Use of multiple profiles corresponding to a sequence alignment enables effective detection of remote homologues

MOTIVATION Position specific scoring matrices (PSSMs) corresponding to aligned sequences of homologous proteins are commonly used in homology detection. A PSSM is generated on the basis of one of the homologues as a reference sequence, which is the query in the case of PSI-BLAST searches. The reference sequence is chosen arbitrarily while generating PSSMs for reverse BLAST searches. In this work we demonstrate that the use of multiple PSSMs corresponding to a given alignment and variable reference sequences is more effective than using traditional single PSSMs and hidden Markov models. RESULTS Searches for proteins with known 3-D structures have been made against three databases of protein family profiles corresponding to known structures: (1) One PSSM per family; (2) multiple PSSMs corresponding to an alignment and variable reference sequences for every family; and (3) hidden Markov models. A comparison of the performances of these three approaches suggests that the use of multiple PSSMs is most effective. CONTACT ns@mbu.iisc.ernet.in.

A glutathione-specific aldose reductase of Leishmania donovani and its potential implications for methylglyoxal detoxification pathway

Methylglyoxal is mainly catabolized by two major enzymatic pathways. The first is the ubiquitous detoxification pathway, the glyoxalase pathway. In addition to the glyoxalase pathway, aldose reductase pathway also plays a crucial role in lowering the levels of methylglyoxal. The gene encoding aldose reductase (ALR) has been cloned from Leishmania donovani, a protozoan parasite causing visceral leishmaniasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 855 bp encoding a putative protein of 284 amino acids with a calculated molecular mass of 31.7 kDa and a predicted isoelectric point of 5.85. The sequence identity between L. donovani ALR (LdALR) and mammals and plants is only 36-44%. The ORF is a single copy gene. A protein with a molecular mass that matched the estimated approximately 74 kDa according to the amino acid composition of LdALR with a maltose binding tag present at its N-terminal end was induced by heterologous expression of LdALR in Escherichia coli. In the presence of glutathione, recombinant LdALR reduced methylglyoxal with a K(m) of approximately 112 microM. Comparative structural analysis of the human ALR structure with LdALR model suggests that the active site anchoring the N-terminal end of the glutathione is highly conserved. However, the C-terminal end of the glutathione backbone is expected to be exposed in LdALR, as the residues anchoring the C-terminal end of the glutathione backbone come from the three loop regions in human, which are apparently shortened in the LdALR structure. Thus, the computational analysis provides clues about the expected mode of glutathione binding and its interactions with the protein. This is the first report of the role of an ALR in the metabolic disposal of methylglyoxal in L. donovani and of thiol binding to a kinetoplastid aldose reductase.

Strategies for the effective identification of remotely related sequences in multiple PSSM search approach

Searches using position specific scoring matrices (PSSMs) have been commonly used in remote homology detection procedures such as PSI‐BLAST and RPS‐BLAST. A PSSM is generated typically using one of the sequences of a family as the reference sequence. In the case of PSI‐BLAST searches the reference sequence is same as the query. Recently we have shown that searches against the database of multiple family‐profiles, with each one of the members of the family used as a reference sequence, are more effective than searches against the classical database of single family‐profiles. Despite relatively a better overall performance when compared with common sequence‐profile matching procedures, searches against the multiple family‐profiles database result in a few false positives and false negatives. Here we show that profile length and divergence of sequences used in the construction of a PSSM have major influence on the performance of multiple profile based search approach. We also identify that a simple parameter defined by the number of PSSMs corresponding to a family that is hit, for a query, divided by the total number of PSSMs in the family can distinguish effectively the true positives from the false positives in the multiple profiles search approach. Proteins 2007.

Evolutionary divergence of Plasmodium falciparum: sequences, protein-protein interactions, pathways and processes.

In this article we review the organism-wide biological data available for Plasmodium falciparum (P. falciparum), a malarial parasite, in relation to the data available for other organisms. We provide comparisons at different levels such as amino acid sequences of proteins encoded in the genomes, protein-protein interaction features, metabolic and signaling pathways and processes. Our comparative analyses highlights that P. falciparum is highly diverged compared to most other eukaryotes at all these levels. Despite the extensive variation some of the physical associations between proteins, such as RNA polymerase complex and CDK-cyclin complex are expected to be conserved given their fundamental importance and ubiquitous nature. We also discuss examples of protein-protein interactions across human and P. falciparum potentially happening during pathogenesis.

Molecular and structural basis of drift in the functions of closely-related homologous enzyme domains: implications for function annotation based on homology searches and structural genomics.

Using a large database of protein domain families of known 3-D structure we present an analysis on the relationships among sequences, structures and functions of closely-related enzymes performed at the level of catalytic domains. Only in 38% of the pairs of homologous catalytic domains characterized by over about 60% of sequence identity the functions are almost completely identical. Nearly 43% of the pairs differ in their substrate specificity. Hence the most common variation of enzyme function among the closely-related homologues is the differences in the substrate specificity. For homologous pairs characterized by a sequence identity of 30-60%, if the structural difference metric is less than about 30, the functions are highly conserved. For clearly homologous protein domain pairs, usually sharing less than 40% sequence identity, we observe that often the chemical groups involved in the functions, and the cofactors differ. We also report of extremely unusual cases of closely-related homologues belonging to entirely different classes of enzymes. Such drastic shifts in the gross functions of homologues seem to be achieved by retooling of catalytic residues or by altering the stability of the intermediates in the biochemical reactions. Our work provides guidelines on the functional annotation based on homology searches and in structural genomics initiatives.

Analysis on sliding helices and strands in protein structural comparisons: A case study with protein kinases

Protein structural alignments are generally considered as ‘golden standard’ for the alignment at the level of amino acid residues. In this study we have compared the quality of pairwise and multiple structural alignments of about 5900 homologous proteins from 718 families of known 3-D structures. We observe shifts in the alignment of regular secondary structural elements (helices and strands) between pairwise and multiple structural alignments. The differences between pairwise and multiple structural alignments within helical and β-strand regions often correspond to 4 and 2 residue positions respectively. Such shifts correspond approximately to “one turn” of these regular secondary structures. We have performed manual analysis explicitly on the family of protein kinases. We note shifts of one or two turns in helix-helix alignments obtained using pairwise and multiple structural alignments. Investigations on the quality of the equivalent helix-helix, strand-strand pairs in terms of their residue side-chain accessibilities have been made. Our results indicate that the quality of the pairwise alignments is comparable to that of the multiple structural alignments and, in fact, is often better. We propose that pairwise alignment of protein structures should also be used in formulation of methods for structure prediction and evolutionary analysis.