Krzysztof Ginalski

Practical lessons from protein structure prediction

Despite recent efforts to develop automated protein structure determination protocols, structural genomics projects are slow in generating fold assignments for complete proteomes, and spatial structures remain unknown for many protein families. Alternative cheap and fast methods to assign folds using prediction algorithms continue to provide valuable structural information for many proteins. The development of high-quality prediction methods has been boosted in the last years by objective community-wide assessment experiments. This paper gives an overview of the currently available practical approaches to protein structure prediction capable of generating accurate fold assignment. Recent advances in assessment of the prediction quality are also discussed.

A comprehensive update of the sequence and structure classification of kinases

BackgroundA comprehensive update of the classification of all available kinases was carried out. This survey presents a complete global picture of this large functional class of proteins and confirms the soundness of our initial kinase classification scheme.ResultsThe new survey found the total number of kinase sequences in the protein database has increased more than three-fold (from 17,310 to 59,402), and the number of determined kinase structures increased two-fold (from 359 to 702) in the past three years. However, the framework of the original two-tier classification scheme (in families and fold groups) remains sufficient to describe all available kinases. Overall, the kinase sequences were classified into 25 families of homologous proteins, wherein 22 families (~98.8% of all sequences) for which three-dimensional structures are known fall into 10 fold groups. These fold groups not only include some of the most widely spread proteins folds, such as the Rossmann-like fold, ferredoxin-like fold, TIM-barrel fold, and antiparallel β-barrel fold, but also all major classes (all α, all β, α+β, α/β) of protein structures. Fold predictions are made for remaining kinase families without a close homolog with solved structure. We also highlight two novel kinase structural folds, riboflavin kinase and dihydroxyacetone kinase, which have recently been characterized. Two protein families previously annotated as kinases are removed from the classification based on new experimental data.ConclusionStructural annotations of all kinase families are now revealed, including fold descriptions for all globular kinases, making this the first large functional class of proteins with a comprehensive structural annotation. Potential uses for this classification include deduction of protein function, structural fold, or enzymatic mechanism of poorly studied or newly discovered kinases based on proteins in the same family.

Detecting distant homology with Meta-BASIC

Meta-BASIC (http://basic.bioinfo.pl) is a novel sensitive approach for recognition of distant similarity between proteins based on consensus alignments of meta profiles. Specifically, Meta-BASIC compares sequence profiles combined with predicted secondary structure by utilizing several scoring systems and alignment algorithms. In our benchmarking tests, Meta-BASIC outperforms many individual servers, including fold recognition servers, and it can compete with meta predictors that base their strength on the structural comparison of models. In addition, Meta-BASIC, which enables detection of very distant relationships even if the tertiary structure for the reference protein is not known, has a high-throughput capability. This new method is applied to 860 PfamA protein families with unknown function (DUF) and provides many novel structure-functional assignments available on-line at http://basic.bioinfo.pl/duf.pl. Detailed discussion is provided for two of the most interesting assignments. DUF271 and DUF431 are predicted to be a nucleotide-diphospho-sugar transferase and an alpha/beta-knot SAM-dependent RNA methyltransferase, respectively.

Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade.

Site‐2 proteases (S2Ps) form a large family of membrane‐embedded metalloproteases that participate in cellular signaling pathways through sequential cleavage of membrane‐tethered substrates. Using sequence similarity searches, we extend the S2P family to include remote homologs that help define a conserved structural core consisting of three predicted transmembrane helices with traditional metalloprotease functional motifs and a previously unrecognized motif (GxxxN/S/G). S2P relatives were identified in genomes from Bacteria, Archaea, and Eukaryota including protists, plants, fungi, and animals. The diverse S2P homologs divide into several groups that differ in various inserted domains and transmembrane helices. Mammalian S2P proteases belong to the major ubiquitous group and contain a PDZ domain. Sequence and structural analysis of the PDZ domain support its mediating the sequential cleavage of membrane‐tethered substrates. Finally, conserved genomic neighborhoods of S2P homologs allow functional predictions for PDZ‐containing transmembrane proteases in extra‐cytoplasmic stress response and lipid metabolism.

Structure-based sequence alignment for the β-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A–G). The β‐trefoil subdomain of the C‐terminal part of the heavy chain (HC), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re‐examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the β‐trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure‐based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re‐examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.

BOF: a novel family of bacterial OB-fold proteins.

Using top‐of‐the‐line fold recognition methods, we assigned an oligonucleotide/oligosaccharide‐binding (OB)‐fold structure to a family of previously uncharacterized hypothetical proteins from several bacterial genomes. This novel family of bacterial OB‐fold (BOF) proteins present in a number of pathogenic strains encompasses sequences of unknown function from DUF388 (in Pfam database) and COG3111. The BOF proteins can be linked evolutionarily to other members of the OB‐fold nucleic acid‐binding superfamily (anticodon‐binding and single strand DNA‐binding domains), although they probably lack nucleic acid‐binding properties as implied by the analysis of the potential binding site. The presence of conserved N‐terminal predicted signal peptide indicates that BOF family members localize in the periplasm where they may function to bind proteins, small molecules, or other typical OB‐fold ligands. As hypothesized for the distantly related OB‐fold containing bacterial enterotoxins, the loss of nucleotide‐binding function and the rapid evolution of the BOF ligand‐binding site may be associated with the presence of BOF proteins in mobile genetic elements and their potential role in bacterial pathogenicity.

Addressing the issue of sequence‐to‐structure alignments in comparative modeling of CASP3 target proteins

During a blind protein structure prediction experiment (the third round of the Critical Assessment of Techniques for Protein Structure Prediction; URL http://PredictionCenter.llnl.gov/casp3/), four target proteins, T0047, T0048, T0055, and T0070, were modeled by comparison. These proteins display 62%, 29%, 24%, and 19% sequence identity, respectively, to the structurally homologous proteins most similar in sequence. The issue of sequence‐to‐structure alignment in cases of low sequence homology was the main emphasis. Selection of alignments was made by constructing and evaluating three‐dimensional models based on series of samples produced mainly by automatic multiple sequence alignments. Sequence‐to‐structure alignments were correct in all but two regions, in which significant changes in target structures compared with related proteins were the source of errors. Template choice is an important determinant of model quality, and a correct selection was made of a lower homology template for modeling of T0070; however, in the case of T0055, a template with 8% greater sequence homology proved deceptive. Loops and some ungapped template regions were assigned conformations taken from other proteins. Using fragments from homologous structures led to improvement over template backbone more often than cases in which nonhomologous structures were the source. The results also indicate that side‐chain prediction accuracy depends not only on sequence similarity but also on accuracy of the backbone. Proteins Suppl 1999;3:73–80. Published 1999 Wiley‐Liss, Inc.

Sequence-structure mapping errors in the PDB: OB-fold domains

The Protein Data Bank (PDB) is the single most important repository of structural data for proteins and other biologically relevant molecules. Therefore, it is critically important to keep the PDB data, as much as possible, error‐free. In this study, we have analyzed PDB crystal structures possessing oligonucleotide/oligosaccharide binding (OB)‐fold, one of the highly populated folds, for the presence of sequence‐structure mapping errors. Using energy‐based structure quality assessment coupled with sequence analyses, we have found that there are at least five OB‐structures in the PDB that have regions where sequences have been incorrectly mapped onto the structure. We have demonstrated that the combination of these computation techniques is effective not only in detecting sequence‐structure mapping errors, but also in providing guidance to correct them. Namely, we have used results of computational analysis to direct a revision of X‐ray data for one of the PDB entries containing a fairly inconspicuous sequence‐structure mapping error. The revised structure has been deposited with the PDB. We suggest use of computational energy assessment and sequence analysis techniques to facilitate structure determination when homologs having known structure are available to use as a reference. Such computational analysis may be useful in either guiding the sequence‐structure assignment process or verifying the sequence mapping within poorly defined regions.

Protein Domain of Unknown Function DUF1023 is an α/β hydrolase

Pfam family DUF1023 consists entirely of uncharacterized proteins generated by sequencing the genomes of Actinobacteria (Bateman A., et al., Nucleic Acids Res. 2004;32 Database issue:D138–141.) Utilizing sequence similarity detection methods, we infer homology between DUF1023 and α/β hydrolases. DUF1023 proteins conserve the core secondary structures in α/β hydrolase fold, and share similar catalytic machinery as that of α/β hydrolases. We predict DUF1023 spatial structure and deduce that they function as hydrolases utilizing catalytic Ser‐His‐Asp triad with the serine as a nucleophile. Proteins 2005.