Functional genomics of HMGN3a and SMARCAL1 in early mammalian embryogenesis.

BACKGROUND: Embryonic genome activation (EGA) is a critical event for the preimplantation embryo, which is manifested by changes in chromatin structure, transcriptional machinery, expression of embryonic genes, and degradation of maternal transcripts. The objectives of this study were to determine transcript abundance of HMGN3a and SMARCAL1 in mature bovine oocytes and early bovine embryos, to perform comparative functional genomics analysis of these genes across mammals. RESULTS: New annotations of both HMGN3a and SMARCAL1 were submitted to the Bovine Genome Annotation Submission Database at BovineGenome.org. Careful analysis of the bovine SMARCAL1 consensus gene set for this protein (GLEAN_20241) showed that the NCBI protein contains sequencing errors, and that the actual bovine protein has a high degree of homology to the human protein. Our results showed that there was a high degree of structural conservation of HMGN3a and SMARCAL1 in the mammalian species studied. HMGN3a transcripts were present at similar levels in bovine matured oocytes and 2-4-cell embryos but at higher levels in 8-16-cell embryos, morulae and blastocysts. On the other hand, transcript levels of SMARCAL1 decreased throughout preimplantation development. CONCLUSION: The high levels of structural conservation of these proteins highlight the importance of chromatin remodeling in the regulation of gene expression, particularly during early mammalian embryonic development. The greater similarities of human and bovine HMGN3a and SMARCAL1 proteins may suggest the cow as a valuable model to study chromatin remodeling at the onset of mammalian development. Understanding the roles of chromatin remodeling proteins during embryonic development emphasizes the importance of epigenetics and could shed light on the underlying mechanisms of early mammalian development.

An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.

TOPOFIT-DB, a database of protein structural alignments based on the TOPOFIT method.

TOPOFIT-DB (T-DB) is a public web-based database of protein structural alignments based on the TOPOFIT method, providing a comprehensive resource for comparative analysis of protein structure families. The TOPOFIT method is based on the discovery of a saturation point on the alignment curve (topomax point) which presents an ability to objectively identify a border between common and variable parts in a protein structural family, providing additional insight into protein comparison and functional annotation. TOPOFIT also effectively detects non-sequential relations between protein structures. T-DB provides users with the convenient ability to retrieve and analyze structural neighbors for a protein; do one-to-all calculation of a user provided structure against the entire current PDB release with T-Server, and pair-wise comparison using the TOPOFIT method through the T-Pair web page. All outputs are reported in various web-based tables and graphics, with automated viewing of the structure-sequence alignments in the Friend software package for complete, detailed analysis. T-DB presents researchers with the opportunity for comprehensive studies of the variability in proteins and is publicly available at http://mozart.bio.neu.edu/topofit/index.php.

A comprehensive analysis of non-sequential alignments between all protein structures.

BACKGROUND: The majority of relations between proteins can be represented as a conventional sequential alignment. Nevertheless, unusual non-sequential alignments with different connectivity of the aligned fragments in compared proteins have been reported by many researchers. It is interesting to understand those non-sequential alignments; are they unique, sporadic cases or they occur frequently; do they belong to a few specific folds or spread among many different folds, as a common feature of protein structure. We present here a comprehensive large-scale study of non-sequential alignments between available protein structures in Protein Data Bank. RESULTS: The study has been conducted on a non-redundant set of 8,865 protein structures aligned with the aid of the TOPOFIT method. It has been estimated that between 17.4% and 35.2% of all alignments are non-sequential depending on variations in the parameters. Analysis of the data revealed that non-sequential relations between proteins do occur systematically and in large quantities. Various sizes and numbers of non-sequential fragments have been observed with all possible complexities of fragment rearrangements found for alignments consisting of up to 12 fragments. It has been found that non-sequential alignments are not limited to proteins of any particular fold and are present in more than two hundred of them. Moreover, many of them are found between proteins with different fold assignments. It has been shown that protein structure symmetry does not explain non-sequential alignments. Therefore, compelling evidences have been provided that non-sequential alignments between proteins are systematic and widespread across the protein universe. CONCLUSION: The phenomenon of the widespread occurrence of non-sequential alignments between proteins might represent a missing rule of protein structure organization. More detailed study of this phenomenon will enhance our understanding of protein stability, folding, and evolution.

MODBASE, a database of annotated comparative protein structure models.

MODBASE (http://guitar.rockefeller.edu/modbase) is a relational database of annotated comparative protein structure models for all available protein sequences matched to at least one known protein structure. The models are calculated by MODPIPE, an automated modeling pipeline that relies on PSI-BLAST, IMPALA and MODELLER. MODBASE uses the MySQL relational database management system for flexible and efficient querying, and the MODVIEW Netscape plugin for viewing and manipulating multiple sequences and structures. It is updated regularly to reflect the growth of the protein sequence and structure databases, as well as improvements in the software for calculating the models. For ease of access, MODBASE is organized into different datasets. The largest dataset contains models for domains in 304 517 out of 539 171 unique protein sequences in the complete TrEMBL database (23 March 2001); only models based on significant alignments (PSI-BLAST E-value < 10(-4)) and models assessed to have the correct fold are included. Other datasets include models for target selection and structure-based annotation by the New York Structural Genomics Research Consortium, models for prediction of genes in the Drosophila melanogaster genome, models for structure determination of several ribosomal particles and models calculated by the MODWEB comparative modeling web server.

Tools for comparative protein structure modeling and analysis.

The following resources for comparative protein structure modeling and analysis are described (http://salilab.org): MODELLER, a program for comparative modeling by satisfaction of spatial restraints; MODWEB, a web server for automated comparative modeling that relies on PSI-BLAST, IMPALA and MODELLER; MODLOOP, a web server for automated loop modeling that relies on MODELLER; MOULDER, a CPU intensive protocol of MODWEB for building comparative models based on distant known structures; MODBASE, a comprehensive database of annotated comparative models for all sequences detectably related to a known structure; MODVIEW, a Netscape plugin for Linux that integrates viewing of multiple sequences and structures; and SNPWEB, a web server for structure-based prediction of the functional impact of a single amino acid substitution.

Structural alignment of proteins by a novel TOPOFIT method, as a superimposition of common volumes at a topomax point.

Similarity of protein structures has been analyzed using three-dimensional Delaunay triangulation patterns derived from the backbone representation. It has been found that structurally related proteins have a common spatial invariant part, a set of tetrahedrons, mathematically described as a common spatial subgraph volume of the three-dimensional contact graph derived from Delaunay tessellation (DT). Based on this property of protein structures, we present a novel common volume superimposition (TOPOFIT) method to produce structural alignments. Structural alignments usually evaluated by a number of equivalent (aligned) positions (N(e)) with corresponding root mean square deviation (RMSD). The superimposition of the DT patterns allows one to uniquely identify a maximal common number of equivalent residues in the structural alignment. In other words, TOPOFIT identifies a feature point on the RMSD N(e) curve, a topomax point, until which the topologies of two structures correspond to each other, including backbone and interresidue contacts, whereas the growing number of mismatches between the DT patterns occurs at larger RMSD (N(e)) after the topomax point. It has been found that the topomax point is present in all alignments from different protein structural classes; therefore, the TOPOFIT method identifies common, invariant structural parts between proteins. The alignments produced by the TOPOFIT method have a good correlation with alignments produced by other current methods. This novel method opens new opportunities for the comparative analysis of protein structures and for more detailed studies on understanding the molecular principles of tertiary structure organization and functionality. The TOPOFIT method also helps to detect conformational changes, topological differences in variable parts, which are particularly important for studies of variations in active/ binding sites and protein classification.

Different roles of two gamma-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature.

Gamma-tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of gamma-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, gamma-tubulin is encoded by two genes, gamma-T1 and gamma-T2, that produce distinct isotypes. Comparison of the gamma-T1 and gamma-T2 primary sequences to a Euplotesgamma-tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in gamma-T2. Structural models of gamma-T1 and gamma-T2, obtained using the 3D structure of human gamma-tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal 'plus end' contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to gamma-T1, the gamma-T2 gene is amplified by approximately 18-fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the gamma-tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non-motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only gamma-T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the gamma-T1 isotype associates only with basal bodies. Our results suggest that gamma-T1 and gamma-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that gamma-T1 and gamma-T2 have evolved sequence-based structural alterations that facilitate template nucleation of microtubules by the gamma-tubulin ring complex at cold temperatures.

Friend, an integrated analytical front-end application for bioinformatics.

UNLABELLED: Friend is a bioinformatics application designed for simultaneous analysis and visualization of multiple structures and sequences of proteins and/or DNA/RNA. The application provides basic functionalities, such as structure visualization, with different rendering and coloring, sequence alignment and simple phylogeny analysis, along with a number of extended features to perform more complex analyses of sequence structure relationships, including structural alignment of proteins, investigation of specific interaction motifs, studies of protein-protein and protein-DNA interactions and protein super-families. It is also useful for functional annotation of proteins, protein modeling and protein folding studies. Friend provides three levels of usage: (1) an extensive GUI for a scientist with no programming experience, (2) a command line interface for scripting for a scientist with some programming experience and (3) the ability to extend Friend with user written libraries for an experienced programmer. The application is linked and communicates with local and remote sequence and structure databases. AVAILABILITY: http://mozart.bio.neu.edu/friend.

Structural exon database, SEDB, mapping exon boundaries on multiple protein structures.

UNLABELLED: Comparative analysis of exon/intron organization of genes and their resulting protein structures is important for understanding evolutionary relationships between species, rules of protein organization and protein functionality. We present Structural Exon Database (SEDB), with a Web interface, an application that allows users to retrieve the exon/intron organization of genes and map the location of the exon boundaries and the intron phase onto a multiple structural alignment. SEDB is linked with Friend, an integrated analytical multiple sequence/structure viewer, which allows simultaneous visualization of exon boundaries on structure and sequence alignments. With SEDB researchers can study the correlations of gene structure with the properties of the encoded three-dimensional protein structures across eukaryotic organisms. AVAILABILITY: SEDB is publicly available at http://glinka.bio.neu.edu/SEDB/SEDB.html SUPPLEMENTARY INFORMATION: On the SEDB Web site.

LigBase: a database of families of aligned ligand binding sites in known protein sequences and structures.

A database comprising all ligand-binding sites of known structure aligned with all related protein sequences and structures is described. Currently, the database contains approximately 50000 ligand-binding sites for small molecules found in the Protein Data Bank (PDB). The structure-structure alignments are obtained by the Combinatorial Extension (CE) program (Shindyalov and Bourne, Protein Eng., 11, 739-747, 1998) and sequence-structure alignments are extracted from the ModBase database of comparative protein structure models for all known protein sequences (Sanchez et al., Nucleic Acids Res., 28, 250-253, 2000). It is possible to search for binding sites in LigBase by a variety of criteria. LigBase reports summarize ligand data including relevant structural information from the PDB file, such as ligand type and size, and contain links to all related protein sequences in the TrEMBL database. Residues in the binding sites are graphically depicted for comparison with other structurally defined family members. LigBase provides a resource for the analysis of families of related binding sites.

ModView, visualization of multiple protein sequences and structures.

SUMMARY: We describe ModView, a web application for visualization of multiple protein sequences and structures. ModView integrates a multiple structure viewer, a multiple sequence alignment editor, and a database querying engine. It is possible to interactively manipulate hundreds of proteins, to visualize conservative and variable residues, active and binding sites, fragments, and domains in protein families, as well as to display large macromolecular complexes such as ribosomes or viruses. As a Netscape plug-in, ModView can be included in HTML pages along with text and figures, which makes it useful for teaching and presentations. ModView is also suitable as a graphical interface to various databases because it can be controlled through JavaScript commands and called from CGI scripts. AVAILABILITY: ModView is available at http://guitar.rockefeller.edu/modview.