Search

Abstract Background Detection of common evolutionary origin (homology) is a primary means of inferring protein structure and function. At present, comparison of protein families represented as sequence profiles is arguably the most effective homology detection strategy. However, finding the best way to represent evolutionary information of a protein sequence family in the profile, to compare profiles and to estimate the biological significance of such comparisons, remains an active area of research. Results Here, we present a new homology detection method based on sequence profile-profile comparison. The method has a number of new features including position-dependent gap penalties and a global score system. Position-dependent gap penalties provide a more biologically relevant way to represent and align protein families as sequence profiles. The global score system enables an analytical solution of the statistical parameters needed to estimate the statistical significance of profile-profile similarities. The new method, together with other state-of-the-art profile-based methods (HHsearch, COMPASS and PSI-BLAST), is benchmarked in all-against-all comparison of a challenging set of SCOP domains that share at most 20% sequence identity. For benchmarking, we use a reference ("gold standard") free model-based evaluation framework. Evaluation results show that at the level of protein domains our method compares favorably to all other tested methods. We also provide examples of the new method outperforming structure-based similarity detection and alignment. The implementation of the new method both as a standalone software package and as a web server is available at http://www.ibt.lt/bioinformatics/coma. Conclusion Due to a number of developments, the new profile-profile comparison method shows an improved ability to match distantly related protein domains. Therefore, the method should be useful for annotation and homology modeling of uncharacterized proteins.

Abstract Background Sequence searches are routinely employed to detect and annotate related proteins. However, a rapid growth of databases necessitates a frequent repetition of sequence searches and subsequent analysis of obtained results. Although there are several automatic systems available for executing periodical sequence searches and reporting results, they all suffer either from a lack of sensitivity, restrictive database choice or limited flexibility in setting up search strategies. Here, a new sequence search and reporting software package designed to address these shortcomings is described. Results Re-searcher is an open-source highly configurable system for recurrent detection and reporting of new homologs for the sequence of interest in specified protein sequence databases. Searches are performed using PSI-BLAST at desired time intervals either within NCBI or local databases. In addition to searches against individual databases, the system can perform "PDB-BLAST"-like combined searches, when PSI-BLAST profile generated during search against the first database is used to search the second database. The system supports multiple users enabling each to separately keep track of multiple queries and query-specific results. Conclusions Re-searcher features a large number of options enabling automatic periodic detection of both close and distant homologs. At the same time it has a simple and intuitive interface, making the analysis of results even for a large number of queries a straightforward task.

Abstract Background Protein sequence alignments have become indispensable for virtually any evolutionary, structural or functional study involving proteins. Modern sequence search and comparison methods combined with rapidly increasing sequence data often can reliably match even distantly related proteins that share little sequence similarity. However, even highly significant matches generally may have incorrectly aligned regions. Therefore when exact residue correspondence is used to transfer biological information from one aligned sequence to another, it is critical to know which alignment regions are reliable and which may contain alignment errors. Results PSI-BLAST-ISS is a standalone Unix-based tool designed to delineate reliable regions of sequence alignments as well as to suggest potential variants in unreliable regions. The region-specific reliability is assessed by producing multiple sequence alignments in different sequence contexts followed by the analysis of the consistency of alignment variants. The PSI-BLAST-ISS output enables the user to simultaneously analyze alignment reliability between query and multiple homologous sequences. In addition, PSI-BLAST-ISS can be used to detect distantly related homologous proteins. The software is freely available at: http://www.ibt.lt/bioinformatics/iss. Conclusion PSI-BLAST-ISS is an effective reliability assessment tool that can be useful in applications such as comparative modelling or analysis of individual sequence regions. It favorably compares with the existing similar software both in the performance and functional features.

We present the results for CAPRI Round 54, the 5th joint CASP-CAPRI protein assembly prediction challenge. The Round offered 37 targets, including 14 homodimers, 3 homo-trimers, 13 heterodimers including 3 antibody-antigen complexes, and 7 large assemblies. On average ~70 CASP and CAPRI predictor groups, including more than 20 automatics servers, submitted models for each target. A total of 21 941 models submitted by these groups and by 15 CAPRI scorer groups were evaluated using the CAPRI model quality measures and the DockQ score consolidating these measures. The prediction performance was quantified by a weighted score based on the number of models of acceptable quality or higher submitted by each group among their five best models. Results show substantial progress achieved across a significant fraction of the 60+ participating groups. High-quality models were produced for about 40% of the targets compared to 8% two years earlier. This remarkable improvement is due to the wide use of the AlphaFold2 and AlphaFold2-Multimer software and the confidence metrics they provide. Notably, expanded sampling of candidate solutions by manipulating these deep learning inference engines, enriching multiple sequence alignments, or integration of advanced modeling tools, enabled top performing groups to exceed the performance of a standard AlphaFold2-Multimer version used as a yard stick. This notwithstanding, performance remained poor for complexes with antibodies and nanobodies, where evolutionary relationships between the binding partners are lacking, and for complexes featuring conformational flexibility, clearly indicating that the prediction of protein complexes remains a challenging problem., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163-aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex.

The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1. Mutations in ELG1 lead to DNA damage sensitivity and genome instability. To characterize the role of Elg1 in maintaining genomic integrity, we used homology modeling to generate a number of site-specific mutations in ELG1 that exhibit different PCNA unloading capabilities. We show that the sensitivity to DNA damaging agents and hyper-recombination of these alleles correlate with their ability to unload PCNA from the chromatin. Our results indicate that retention of modified and unmodified PCNA on the chromatin causes genomic instability. We also show, using purified proteins, that the Elg1 complex inhibits DNA synthesis by unloading SUMOylated PCNA from the DNA. Additionally, we find that mutations in ELG1 suppress the sensitivity of rad5Δ mutants to DNA damage by allowing trans-lesion synthesis to take place. Taken together, the data indicate that the Elg1-RLC complex plays an important role in the maintenance of genomic stability by unloading PCNA from the chromatin., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Summary: The PPI3D web server is focused on searching and analyzing the structural data on protein-protein interactions. Reducing the data redundancy by clustering and analyzing the properties of interaction interfaces using Voronoi tessellation makes this software a highly effective tool for addressing different questions related to protein interactions., Availability and Implementation: The server is freely accessible at http://bioinformatics.lt/software/ppi3d/ ., Contact: ceslovas.venclovas@bti.vu.lt., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Motivation: Herpesviruses are large DNA viruses causing a variety of diseases in humans and animals. To develop effective treatment, it is important to understand the mechanisms of their replication. One of the components of the herpesviral DNA replication system is a helicase-primase complex, consisting of UL5 (helicase), UL52 (primase) and UL8. UL8 is an essential herpesviral protein involved in multiple protein-protein interactions. Intriguingly, so far no UL8 homologs outside of herpesviruses could be identified. Moreover, nothing is known about its structure or domain organization., Results: Here, combining sensitive homology detection methods and homology modeling, we found that the UL8 protein family is related to B-family polymerases. In the course of evolution, UL8 has lost the active site and has undergone a reduction of DNA-binding motifs. The loss of active site residues explains the failure to detect any catalytic activity of UL8. A structural model of human herpes virus 1 UL8 constructed as part of the study is consistent with the mutation data targeting its interaction with primase UL52. It also provides a platform for studying multiple interactions that UL8 is involved in. The two other components of helicase-primase complex show evolutionary links with a newly characterized human primase that also has DNA polymerase activity (PrimPol) and the Pif1 helicase, respectively. The role of these enzymes in recovering stalled replication forks suggests mechanistic and functional similarities with herpesviral proteins., Contact: venclovas@ibt.lt, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Contact Area Difference score (CAD-score) web server provides a universal framework to compute and analyze discrepancies between different 3D structures of the same biological macromolecule or complex. The server accepts both single-subunit and multi-subunit structures and can handle all the major types of macromolecules (proteins, RNA, DNA and their complexes). It can perform numerical comparison of both structures and interfaces. In addition to entire structures and interfaces, the server can assess user-defined subsets. The CAD-score server performs both global and local numerical evaluations of structural differences between structures or interfaces. The results can be explored interactively using sortable tables of global scores, profiles of local errors, superimposed contact maps and 3D structure visualization. The web server could be used for tasks such as comparison of models with the native (reference) structure, comparison of X-ray structures of the same macromolecule obtained in different states (e.g. with and without a bound ligand), analysis of nuclear magnetic resonance (NMR) structural ensemble or structures obtained in the course of molecular dynamics simulation. The web server is freely accessible at: http://www.ibt.lt/bioinformatics/cad-score., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Growing interest in computational prediction of ribonucleic acid (RNA) three-dimensional structure has highlighted the need for reliable and meaningful methods to compare models and experimental structures. We present a structure superposition-free method to quantify both the local and global accuracy of RNA structural models with respect to the reference structure. The method, initially developed for proteins and here extended to RNA, closely reflects physical interactions, has a simple definition, a fixed range of values and no arbitrary parameters. It is based on the correspondence of respective contact areas between nucleotides or their components (base or backbone). The better is the agreement between respective contact areas in a model and the reference structure, the more accurate the model is considered to be. Since RNA bases account for the largest contact areas, we further distinguish stacking and non-stacking contacts. We have extensively tested the contact area-based evaluation method and found it effective in both revealing local discrepancies and ranking models by their overall quality. Compared to other reference-based RNA model evaluation methods, the new method shows a stronger emphasis on stereochemical quality of models. In addition, it takes into account model completeness, enabling a meaningful evaluation of full models and those missing some residues., (© The Author(s) 2014. Published by Oxford University Press.)

The Voronoi diagram of balls, corresponding to atoms of van der Waals radii, is particularly well-suited for the analysis of three-dimensional structures of biological macromolecules. However, due to the shortage of practical algorithms and the corresponding software, simpler approaches are often used instead. Here, we present a simple and robust algorithm for computing the vertices of the Voronoi diagram of balls. The vertices of Voronoi cells correspond to the centers of the empty tangent spheres defined by quadruples of balls. The algorithm is implemented as an open-source software tool, Voronota. Large-scale tests show that Voronota is a fast and reliable tool for processing both experimentally determined and computationally modeled macromolecular structures. Voronota can be easily deployed and may be used for the development of various other structure analysis tools that utilize the Voronoi diagram of balls. Voronota is available at: http://www.ibt.lt/bioinformatics/voronota., (Copyright © 2014 Wiley Periodicals, Inc.)

Vitamin B₁₂-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B₁₂ in vitro, it is uncertain whether the organism is able to scavenge B₁₂ during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B₁₂ transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B₁₂. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B₁₂ assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B₁₂ acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B₁₂ and related corrinoids in vitro. Our results establish an alternative to the canonical BtuCD-type system for B₁₂ uptake in M. tuberculosis, and elucidate a role in B₁₂ metabolism for an ABC protein implicated in chronic mycobacterial infection.

Evaluation of protein models against the native structure is essential for the development and benchmarking of protein structure prediction methods. Although a number of evaluation scores have been proposed to date, many aspects of model assessment still lack desired robustness. In this study we present CAD-score, a new evaluation function quantifying differences between physical contacts in a model and the reference structure. The new score uses the concept of residue-residue contact area difference (CAD) introduced by Abagyan and Totrov (J Mol Biol 1997; 268:678-685). Contact areas, the underlying basis of the score, are derived using the Voronoi tessellation of protein structure. The newly introduced CAD-score is a continuous function, confined within fixed limits, free of any arbitrary thresholds or parameters. The built-in logic for treatment of missing residues allows consistent ranking of models of any degree of completeness. We tested CAD-score on a large set of diverse models and compared it to GDT-TS, a widely accepted measure of model accuracy. Similarly to GDT-TS, CAD-score showed a robust performance on single-domain proteins, but displayed a stronger preference for physically more realistic models. Unlike GDT-TS, the new score revealed a balanced assessment of domain rearrangement, removing the necessity for different treatment of single-domain, multi-domain, and multi-subunit structures. Moreover, CAD-score makes it possible to assess the accuracy of inter-domain or inter-subunit interfaces directly. In addition, the approach offers an alternative to the superposition-based model clustering. The CAD-score implementation is available both as a web server and a standalone software package at http://www.ibt.lt/bioinformatics/cad-score/., (Copyright © 2012 Wiley Periodicals, Inc.)

Motivation: Eukaryote-infecting nucleo-cytoplasmic large DNA viruses (NCLDVs) feature some of the largest genomes in the viral world. These viruses typically do not strongly depend on the host DNA replication systems. In line with this observation, a number of essential DNA replication proteins, such as DNA polymerases, primases, helicases and ligases, have been identified in the NCLDVs. One other ubiquitous component of DNA replisomes is the single-stranded DNA-binding (SSB) protein. Intriguingly, no NCLDV homologs of canonical OB-fold-containing SSB proteins had previously been detected. Only in poxviruses, one of seven NCLDV families, I3 was identified as the SSB protein. However, whether I3 is related to any known protein structure has not yet been established., Results: Here, we addressed the case of 'missing' canonical SSB proteins in the NCLDVs and also probed evolutionary origins of the I3 family. Using advanced computational methods, in four NCLDV families, we detected homologs of the bacteriophage T7 SSB protein (gp2.5). We found the properties of these homologs to be consistent with the SSB function. Moreover, we implicated specific residues in single-stranded DNA binding. At the same time, we found no evolutionary link between the T7 gp2.5-like NCLDV SSB homologs and the poxviral SSB protein (I3). Instead, we identified a distant relationship between I3 and small protein B (SmpB), a bacterial RNA-binding protein. Thus, apparently, the NCLDVs have the two major distinct sets of SSB proteins having bacteriophage and bacterial origins, respectively.

Homology modeling is based on the observation that related protein sequences adopt similar three-dimensional structures. Hence, a homology model of a protein can be derived using related protein structure(s) as modeling template(s). A key step in this approach is the establishment of correspondence between residues of the protein to be modeled and those of modeling template(s). This step, often referred to as sequence-structure alignment, is one of the major determinants of the accuracy of a homology model. This chapter gives an overview of methods for deriving sequence-structure alignments and discusses recent methodological developments leading to improved performance. However, no method is perfect. How to find alignment regions that may have errors and how to make improvements? This is another focus of this chapter. Finally, the chapter provides a practical guidance of how to get the most of the available tools in maximizing the accuracy of sequence-structure alignments.

Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding.

Unlabelled: We present Voroprot, an interactive cross-platform software tool that provides a unique set of capabilities for exploring geometric features of protein structure. Voroprot allows the construction and visualization of the Apollonius diagram (also known as the additively weighted Voronoi diagram), the Apollonius graph, protein alpha shapes, interatomic contact surfaces, solvent accessible surfaces, pockets and cavities inside protein structure., Availability: Voroprot is available for Windows, Linux and Mac OS X operating systems and can be downloaded from http://www.ibt.lt/bioinformatics/voroprot/.

PD-(D/E)XK nucleases, initially represented by only Type II restriction enzymes, now comprise a large and extremely diverse superfamily of proteins. They participate in many different nucleic acids transactions including DNA degradation, recombination, repair and RNA processing. Different PD-(D/E)XK families, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. This makes the identification of new superfamily members using standard homology search techniques challenging. To tackle this problem, we developed a method for the detection of PD-(D/E)XK families based on the binary classification of profile-profile alignments using support vector machines (SVMs). Using a number of both superfamily-specific and general features, SVMs were trained to identify true positive alignments of PD-(D/E)XK representatives. With this method we identified several PFAM families of uncharacterized proteins as putative new members of the PD-(D/E)XK superfamily. In addition, we assigned several unclassified restriction enzymes to the PD-(D/E)XK type. Results show that the new method is able to make confident assignments even for alignments that have statistically insignificant scores. We also implemented the method as a freely accessible web server at http://www.ibt.lt/bioinformatics/software/pdexk/.

Radicicol is a natural antibiotic that specifically inhibits chaperone Hsp90 activity and binds to its active site with nanomolar affinity. Radicicol has been widely used as a lead compound to generate synthetic analogs with reduced toxicity and increased stability that could be employed clinically. Here we present a detailed thermodynamic description of radicicol binding to human Hsp90 and yeast Hsc82 studied by isothermal titration calorimetry and thermal shift assay. Titrations as a function of pH showed a linked protonation event upon radicicol binding. The intrinsic binding constant and the thermodynamic parameters (including the enthalpy, entropy, and heat capacity) were determined for yeast Hsc82, and human alpha and beta Hsp90. Recent experimental evidence in literature shows that yeast Hsc82 has significant differences from human Hsp90 isozymes. Here we support this by demonstrating differences in radicicol binding thermodynamics to these proteins. The intrinsic enthalpy of radicicol binding to Hsc82 was -46.7 kJ/mol, to Hsp90alpha -70.7 kJ/mol, and to Hsp90beta was -66.8 kJ/mol. The enthalpies of binding were significantly different, while the intrinsic dissociation constants were quite similar, equal to 0.25, 0.04, and 0.15 nM, respectively. The structural features responsible for such large difference in binding enthalpy but small difference in the intrinsic binding Gibbs free energy are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Summary: Detection of distant homology is a widely used computational approach for studying protein evolution, structure and function. Here, we report a homology search web server based on sequence profile-profile comparison. The user may perform searches in one of several regularly updated profile databases using either a single sequence or a multiple sequence alignment as an input. The same profile databases can also be downloaded for local use. The capabilities of the server are illustrated with the identification of new members of the highly diverse PD-(D/E)XK nuclease superfamily., Availability: http://www.ibt.lt/bioinformatics/coma/

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA') and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA' and DnaE2, as well as with the beta-clamp. Moreover, disruption of the ImuB-beta clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA', imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the alpha-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA'-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.

Background: Detection of common evolutionary origin (homology) is a primary means of inferring protein structure and function. At present, comparison of protein families represented as sequence profiles is arguably the most effective homology detection strategy. However, finding the best way to represent evolutionary information of a protein sequence family in the profile, to compare profiles and to estimate the biological significance of such comparisons, remains an active area of research., Results: Here, we present a new homology detection method based on sequence profile-profile comparison. The method has a number of new features including position-dependent gap penalties and a global score system. Position-dependent gap penalties provide a more biologically relevant way to represent and align protein families as sequence profiles. The global score system enables an analytical solution of the statistical parameters needed to estimate the statistical significance of profile-profile similarities. The new method, together with other state-of-the-art profile-based methods (HHsearch, COMPASS and PSI-BLAST), is benchmarked in all-against-all comparison of a challenging set of SCOP domains that share at most 20% sequence identity. For benchmarking, we use a reference ("gold standard") free model-based evaluation framework. Evaluation results show that at the level of protein domains our method compares favorably to all other tested methods. We also provide examples of the new method outperforming structure-based similarity detection and alignment. The implementation of the new method both as a standalone software package and as a web server is available at http://www.ibt.lt/bioinformatics/coma., Conclusion: Due to a number of developments, the new profile-profile comparison method shows an improved ability to match distantly related protein domains. Therefore, the method should be useful for annotation and homology modeling of uncharacterized proteins.

Here, we describe our template-based protein modeling approach and its performance during the eighth community-wide experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP8, http://predictioncenter.org/casp8). In CASP8, our modeling approach was supplemented by the newly developed distant homology detection method based on sequence profile-profile comparison. Detection of structural homologs that could be used as modeling templates was largely achieved by automated profile-based searches. However, the other two major steps in template-based modeling (TBM) (selection of the best template(s) and construction of the optimal sequence-structure alignment) to a large degree relied on the combination of automatic tools and manual input. The analysis of 64 domains categorized by CASP8 assessors as TBM domains revealed that we missed correct structural templates for only four of them. The use of multiple templates or their fragments enabled us to improve over the structure of the single best PDB template in about 1/3 of our models for TBM domains. Our results for sequence-structure alignments are mixed. Although many models have optimal or near optimal sequence mapping, a large fraction contains one or more misaligned regions. Strikingly, in spite of this, our TBM models have the best overall alignment accuracy scores. This clearly suggests that the correct mapping of protein sequence onto three-dimensional structure remains one of the big challenges in protein structure prediction., (Copyright 2009 Wiley-Liss, Inc.)

The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

Background: Sequence searches are routinely employed to detect and annotate related proteins. However, a rapid growth of databases necessitates a frequent repetition of sequence searches and subsequent analysis of obtained results. Although there are several automatic systems available for executing periodical sequence searches and reporting results, they all suffer either from a lack of sensitivity, restrictive database choice or limited flexibility in setting up search strategies. Here, a new sequence search and reporting software package designed to address these shortcomings is described., Results: Re-searcher is an open-source highly configurable system for recurrent detection and reporting of new homologs for the sequence of interest in specified protein sequence databases. Searches are performed using PSI-BLAST at desired time intervals either within NCBI or local databases. In addition to searches against individual databases, the system can perform "PDB-BLAST"-like combined searches, when PSI-BLAST profile generated during search against the first database is used to search the second database. The system supports multiple users enabling each to separately keep track of multiple queries and query-specific results., Conclusions: Re-searcher features a large number of options enabling automatic periodic detection of both close and distant homologs. At the same time it has a simple and intuitive interface, making the analysis of results even for a large number of queries a straightforward task.

Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5'-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3'-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.

Two recent papers illuminate a key step in DNA sliding clamp loading: one reveals the structure of the PCNA clamp wrapped around DNA--still open from being loaded--while the other finds that the clamp may assist this process by forming a right-handed helix upon opening.

Background: Protein sequence alignments have become indispensable for virtually any evolutionary, structural or functional study involving proteins. Modern sequence search and comparison methods combined with rapidly increasing sequence data often can reliably match even distantly related proteins that share little sequence similarity. However, even highly significant matches generally may have incorrectly aligned regions. Therefore when exact residue correspondence is used to transfer biological information from one aligned sequence to another, it is critical to know which alignment regions are reliable and which may contain alignment errors., Results: PSI-BLAST-ISS is a standalone Unix-based tool designed to delineate reliable regions of sequence alignments as well as to suggest potential variants in unreliable regions. The region-specific reliability is assessed by producing multiple sequence alignments in different sequence contexts followed by the analysis of the consistency of alignment variants. The PSI-BLAST-ISS output enables the user to simultaneously analyze alignment reliability between query and multiple homologous sequences. In addition, PSI-BLAST-ISS can be used to detect distantly related homologous proteins. The software is freely available at: http://www.ibt.lt/bioinformatics/iss., Conclusion: PSI-BLAST-ISS is an effective reliability assessment tool that can be useful in applications such as comparative modelling or analysis of individual sequence regions. It favorably compares with the existing similar software both in the performance and functional features.

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.

Access to a nucleotide by its rotation out of the DNA helix (base flipping) is used by numerous DNA modification and repair enzymes. Despite extensive studies of the paradigm HhaI methyltransferase, initial events leading to base flipping remained elusive. Here we demonstrate that the replacement of the target C:G pair with the 2-aminopurine:T pair in the DNA or shortening of the side chain of Gln237 in the protein severely perturb base flipping, but retain specific DNA binding. Kinetic analyses and molecular modeling suggest that a steric interaction between the protruding side chain of Gln237 and the target cytosine in B-DNA reduces the energy barrier for flipping by 3 kcal/mol. Subsequent stabilization of an open state by further 4 kcal/mol is achieved through specific hydrogen bonding of the side chain to the orphan guanine. Gln237 thus plays a key role in actively opening the target C:G pair by a "push-and-bind" mechanism.

Purpose: The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells., Experimental Design: A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA., Results: Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K(a)) range between 8 x 10(7) and 10(9) M(-1). Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23)., Conclusions: In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.

The quality of structure models produced in the CASP5 experiment has been compared with that in earlier CASPs. The most significant progress is in the fold recognition regime, where the development of meta-servers has allowed more accurate consensus models to be generated. In contrast to this, there is little evidence of progress in producing more accurate comparative models, particularly those based on sequence identities > 30%. For comparative models based on low-sequence identity and for fold recognition models, accuracy depends primarily on the fraction of the target structure that is similar to an available template, and the quality of the alignment. Overall, these results indicate that there are still no effective methods of improving model quality beyond that obtained by successfully copying a template structure. For models of proteins with previously unknown folds, there appears to be a pause in the previous consistent improvement. There is some evidence that more groups are producing top-quality models, however. Although specific progress between successive experiments is sometimes difficulty to identify, over the history of all the CASPs there has been steady, if sometimes slow, progress in all modeling regimes., (Copyright 2003 Wiley-Liss, Inc.)

Models for 20 comparative modeling targets were submitted for the fifth round of the "blind" test of protein structure prediction methods (CASP5; http://predictioncenter.llnl.gov/casp5). The modeling approach used in CASP5 was similar to that used 2 years ago in CASP4 (Venclovas, Proteins 2001; Suppl 5:47-54). The main features of this approach include use of multiple templates, initial assessment of alignment reliability in a region-specific manner, and structure-based selection of alignment variants in unreliable regions. The CASP5 modeling results presented here show significant improvement in comparison to CASP4, especially in the area of distant homology. The improvements include more effective use of multiple templates and better alignments. However, a number of structurally conserved regions in submitted distant homology models were misaligned. Analysis of these errors indicates that the absolute majority of them occurred in regions deemed unreliable in the course of model building. Most of these error-prone regions can be characterized by their peripheral location and a lack of conserved sequence patterns. For a few of the error-prone regions, all methods evaluated during CASP5 proved ineffective, pointing to the need for more sensitive energy-based methods. Despite these remaining issues, the applicability of comparative modeling continues to expand into more distant evolutionary relationships, providing a means to structurally characterize a significant number of currently available protein sequences., (Copyright 2003 Wiley-Liss, Inc.)

Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.

Comparative modeling aims at constructing molecular models for proteins of unknown structure, by using known structures of related proteins as templates. To test the comparative modeling approach reported here, predictions for 13 target proteins were submitted during the fourth round of "blind" protein structure prediction experiment (CASP4; http://PredictionCenter.llnl.gov/casp4). Sequence identity between these target proteins and the closest known structures ranged from 13 to 58%, indicating a broad spectrum of prediction difficulty. Although this broad difficulty range required addressing a variety of issues, the most important proved to be sequence-structure alignment for distant homology targets. The alignment step was based on structure-based evaluation of alignment variants produced mainly with PSI-BLAST intermediate sequence search procedure (PSI-BLAST-ISS). Although a fraction of correctly aligned residues in resulting models was markedly better than the average in all cases, for distant homology targets it was still considerably below the estimated achievable level. Results with CASP4 targets show that, along with the correctness of sequence-structure alignments, effective use of multiple template structures may significantly increase accuracy of the model structure. Improvement in this area should also result in more accurate loop modeling and side-chain prediction., (Copyright 2002 Wiley-Liss, Inc.)

As the number of completed CASP (Critical Assessment of Protein Structure Prediction) experiments grows, so does the need for stable, standard methods for comparing performance in successive experiments. It is critical to develop methods for determining the areas in which there is progress and in which areas are static. We have added an analysis of the CASP4 results to that previously published for CASPs 1, 2, and 3. We again use a unified difficulty scale to permit comparison of performance as a function of target difficulty in the different CASPs. The scale is used to compare performance in aligning target sequences to a structural template. There was a clear improvement in alignment quality between CASP1 (1994) and CASP2 (1996). No change is apparent between CASP2 and CASP3 (1998). There is a small barely detectable improvement between CASP3 and the latest experiment (CASP4, 2000). Alignment remains the major source of error in all models based on less than about 30% sequence identity. Comparison of performance in the new fold modeling regime is complicated by issues in devising an objective target difficulty scale. We have found limited numerical support for significant progress between CASP3 and CASP4 in this area. More subjectively, most observers are convinced that there has been substantial progress. Progress is dominated by a single group., (Copyright 2002 Wiley-Liss, Inc.)

We present a measure for the evaluation of secondary structure prediction methods that is based on secondary structure segments rather than individual residues. The algorithm is an extension of the segment overlap measure Sov, originally defined by Rost et al. (J Mol Biol 1994;235:13-26). The new definition of Sov corrects the normalization procedure and improves Sov's ability to discriminate between similar and dissimilar segment distributions. The method has been comprehensively tested during the second Critical Assessment of Techniques for Protein Structure Prediction (CASP2). Here, we describe the underlying concepts, modifications to the original definition, and their significance.