The RNase H-like superfamily: new members,comparative structural analysis and evolutionary classification

Ribonuclease H-like (RNHL) superfamily, also called the retroviral integrase superfamily, groups together numerous enzymes involved in nucleic acid metabolism and implicated in many biological processes, including replication, homologous recombination, DNA repair, transposition and RNA interference. The RNHL superfamily proteins show extensive divergence of sequences and structures. We conducted database searches to identify members of the RNHL superfamily (including those previously unknown), yielding >60 000 unique domain sequences. Our analysis led to the identification of new RNHL superfamily members, such as RRXRR (PF14239), DUF460 (PF04312, COG2433), DUF3010 (PF11215), DUF429 (PF04250 and COG2410, COG4328, COG4923), DUF1092 (PF06485), COG5558, OrfB_IS605 (PF01385, COG0675) and Peptidase_A17 (PF05380). Based on the clustering analysis we grouped all identified RNHL domain sequences into 152 families. Phylogenetic studies revealed relationships between these families, and suggested a possible history of the evolution of RNHL fold and its active site. Our results revealed clear division of the RNHL superfamily into exonucleases and endonucleases. Structural analyses of features characteristic for particular groups revealed a correlation between the orientation of the C-terminal helix with the exonuclease/endonuclease function and the architecture of the active site. Our analysis provides a comprehensive picture of sequence-structure-function relationships in the RNHL superfamily that may guide functional studies of the previously uncharacterized protein families.     

Retroviral integrase superfamily: the structural perspective

The retroviral integrase superfamily (RISF) comprises numerous important nucleic acid‐processing enzymes, including transposases, integrases and various nucleases. These enzymes are involved in a wide range of processes such as transposition, replication and repair of DNA, homologous recombination, and RNA‐mediated gene silencing. Two out of the four enzymes that are encoded by the human immunodeficiency virus—RNase H1 and integrase—are members of this superfamily. RISF enzymes act on various substrates, and yet show remarkable mechanistic and structural similarities. All share a common fold of the catalytic core and the active site, which is composed primarily of carboxylate residues. Here, I present RISF proteins from a structural perspective, describing the individual members and the common and divergent elements of their structures, as well as the mechanistic insights gained from the structures of RNase H1 enzyme complexes with RNA/DNA hybrids.     

A comparative structural analysis of the ADF/cofilin family

Actin-depolymerizing factor (ADF) and cofilin define a family of actin-binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 A crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate-specific inserts and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and "other eukaryotes." Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in alpha-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding.     

The cation/Ca(2+) exchanger superfamily: phylogenetic analysis and structural implications

Cation/Ca(2+) exchangers are an essential component of Ca(2+) signaling pathways and function to transport cytosolic Ca(2+) across membranes against its electrochemical gradient by utilizing the downhill gradients of other cation species such as H(+), Na(+), or K(+). The cation/Ca(2+) exchanger superfamily is composed of H(+)/Ca(2+) exchangers and Na(+)/Ca(2+) exchangers, which have been investigated extensively in both plant cells and animal cells. Recently, information from completely sequenced genomes of bacteria, archaea, and eukaryotes has revealed the presence of genes that encode homologues of cation/Ca(2+) exchangers in many organisms in which the role of these exchangers has not been clearly demonstrated. In this study, we report a comprehensive sequence alignment and the first phylogenetic analysis of the cation/Ca(2+) exchanger superfamily of 147 sequences. The results present a framework for structure-function relationships of cation/Ca(2+) exchangers, suggesting unique signature motifs of conserved residues that may underlie divergent functional properties. Construction of a phylogenetic tree with inclusion of cation/Ca(2+) exchangers with known functional properties defines five protein families and the evolutionary relationships between the members. Based on this analysis, the cation/Ca(2+) exchanger superfamily is classified into the YRBG, CAX, NCX, and NCKX families and a newly recognized family, designated CCX. These findings will provide guides for future studies concerning structures, functions, and evolutionary origins of the cation/Ca(2+) exchangers.     

Functional diversity of the phosphoglucomutase superfamily: structural implications.

Three-dimensional structural models of three members of the phosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase, were constructed by homology modeling based on the known crystal structure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase each have 50% or more identity with rabbit muscle phosphoglucomutase at the amino acid level and all are reported to exhibit no or minor phosphoglucomutase activity. There are four major insertions and two deletions in the parafusin sequence relative to PGM, all of which are located in surface-exposed loops connecting secondary structural elements. The remaining amino acid substitutions are distributed throughout the sequence and are not predicted to alter the polypeptide fold. Parafusin contains a putative protein kinase C site located on a surface loop in domain II that is not present in the homologs. Although the general domain structure and the active site of rabbit muscle phosphoglucomutase are preserved in the model of phosphoglucomutase-related protein, a major structural difference is likely to occur in domain 1 due to the absence of 55 amino acid residues in PGM-RP. This deletion predicts the loss of three alpha-helices and one beta-strand from an anti-parallel beta-sheet in this domain as compared with the rabbit muscle phosphoglucomutase.     

Protein-DNA interactions: A structural analysis

A detailed analysis of the DNA-binding sites of 26 proteins is presented using data from the Nucleic Acid Database (NDB) and the Protein Data Bank (PDB). Chemical and physical properties of the protein-DNA interface, such as polarity, size, shape, and packing, were analysed. The DNA-binding sites shared common features, comprising many discontinuous sequence segments forming hydrophilic surfaces capable of direct and water-mediated hydrogen bonds. These interface sites were compared to those of protein-protein binding sites, revealing them to be more polar, with many more intermolecular hydrogen bonds and buried water molecules than the protein-protein interface sites. By looking at the number and positioning of protein residue-DNA base interactions in a series of interaction footprints, three modes of DNA binding were identified (single-headed, double-headed and enveloping). Six of the eight enzymes in the data set bound in the enveloping mode, with the protein presenting a large interface area effectively wrapped around the DNA.A comparison of structural parameters of the DNA revealed that some values for the bound DNA (including twist, slide and roll) were intermediate of those observed for the unbound B-DNA and A-DNA. The distortion of bound DNA was evaluated by calculating a root-mean-square deviation on fitting to a canonical B-DNA structure. Major distortions were commonly caused by specific kinks in the DNA sequence, some resulting in the overall bending of the helix. The helix bending affected the dimensions of the grooves in the DNA, allowing the binding of protein elements that would otherwise be unable to make contact. From this structural analysis a preliminary set of rules that govern the bending of the DNA in protein-DNA complexes, are proposed.     

A comparative structural analysis of Fab-fragments of monoclonal rheumatoid and non-rheumatoid immunoglobulins M

The spin-labeling method was used to study the Fab- and Fab-RF-fragments of IgM and IgM-RF, respectively. The spin-label 2,2,6,6-tetramethyl-4-dichloro-sym-triazinyl-aminopiperidine-1-oxyl was introduced into the peptide moiety of the proteins. The rotational correlation time t of the spin-label carrier was determined based on the temperature-viscosity dependence of the EPR spectra parameters of the spin-labeled proteins. The tau values for Fab- and Fab-RF-fragments were 21 +/- 2 and 12 +/- 1 ns, respectively. The data strongly suggest that the significantly lower tau value for the Fab-RF-fragment may be due to the local structural flexibility of the fragment, which in turn may explain the peculiarities of IgM-RF as an autoantibody.     

The maize ALDH protein superfamily: linking structural features to functional specificities

Background  

The completion of maize genome sequencing has resulted in the identification of a large number of uncharacterized genes. Gene annotation and functional characterization of gene products are important to uncover novel protein functionality.     

A systematic comparative and structural analysis of protein phosphorylation sites based on the mtcPTM database

mtcPTM is an online repository of human and mouse phosphosites in which data are hierarchically organized to preserve biologically relevant experimental information, thus allowing straightforward comparisons of phosphorylation patterns found under different conditions. The database also contains the largest available collection of atomic models of phosphorylatable proteins. Detailed analysis of this structural dataset reveals that phosphorylation sites are found in a heterogeneous range of structural and sequence contexts. mtcPTM is available on the web .     

antibacTR: dynamic antibacterial-drug-target ranking integrating comparative genomics,structural analysis and experimental annotation

Background

Development of novel antibacterial drugs is both an urgent healthcare necessity and a partially neglected field. The last decades have seen a substantial decrease in the discovery of novel antibiotics, which combined with the recent thrive of multi-drug-resistant pathogens have generated a scenario of general concern. The procedures involved in the discovery and development of novel antibiotics are economically challenging, time consuming and lack any warranty of success. Furthermore, the return-on-investment for an antibacterial drug is usually marginal when compared to other therapeutics, which in part explains the decrease of private investment.

Results

In this work we present antibacTR, a computational pipeline designed to aid researchers in the selection of potential drug targets, one of the initial steps in antibacterial-drug discovery. The approach was designed and implemented as part of two publicly funded initiatives aimed at discovering novel antibacterial targets, mechanisms and drugs for a priority list of Gram-negative pathogens: Acinetobacter baumannii, Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. However, at present this list has been extended to cover a total of 74 fully sequenced Gram-negative pathogens. antibacTR is based on sequence comparisons and queries to multiple databases (e.g. gene essentiality, virulence factors) to rank proteins according to their potential as antibacterial targets. The dynamic ranking of potential drug targets can easily be executed, customized and accessed by the user through a web interface which also integrates computational analyses performed in-house and visualizable on-site. These include three-dimensional modeling of protein structures and prediction of active sites among other functionally relevant ligand-binding sites.

Conclusions

Given its versatility and ease-of-use at integrating both experimental annotation and computational analyses, antibacTR may effectively assist microbiologists, medicinal-chemists and other researchers working in the field of antibacterial drug-discovery. The public web-interface for antibacTR is available at ‘http://bioinf.uab.cat/antibactr’.     

Leishmania: overexpression and comparative structural analysis of the stage-regulated meta 1 gene.

The meta 1 gene of Leishmania major is upregulated in metacyclic promastigotes and encodes an 11.5-kDa protein with no significant similarities to other proteins in the existing databases. In this paper, we characterize the homologous meta 1 genes in L. amazonensis and L. donovani. Proteins encoded by this gene in all three species present a high degree of identity. The meta 1 gene cannot be replaced by gene targeting in L. major, suggesting an essential role for the protein, at least in promastigotes. Overexpression of the meta 1 protein in L. amazonensis generates parasites that are more virulent than wild-type organisms in vivo.     

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight 'twist' rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.     

The cadherin superfamily in Anopheles gambiae: a comparative study with Drosophila melanogaster

The cadherin superfamily is a diverse and multifunctional group of proteins with extensive representation across genomes of phylogenetically distant species that is involved in cell-cell communication and adhesion. The mosquito Anopheles gambiae is an emerging model organism for the study of innate immunity and host-pathogen interactions, where the malaria parasite induces a profound rearrangement of the actin cytoskeleton at critical stages of infection. We have used bioinformatics tools to retrieve present sequence knowledge about the complete repertoire of cadherins in A. gambiae and compared it to that of the fruit fly, Drosophila melanogaster. In A. gambiae, we have identified 43 genes coding for cadherin extracellular domains that were re-annotated to 38 genes and represent an expansion of this gene family in comparison to other invertebrate organisms. The majority of Drosophila cadherins show a 1 : 1 Anopheles orthologue, but we have observed a remarkable expansion in some groups in A. gambiae, such as N-cadherins, that were recently shown to have a role in the olfactory system of the fruit fly. In vivo dsRNA silencing of overrepresented genes in A. gambiae and other genes showing expression at critical tissues for parasite infection will likely advance our understanding of the problems of host preference and hostpathogen interactions in this mosquito species.     

Evolution of group size in the superfamily Delphinoidea (Delphinidae, Phocoenidae and Monodontidae): a quantitative comparative analysis

Predictors of group size in Delphinoidea were investigated in a comparative study. Possible predictors included phylogenetic variables, variables of the physical environment, the diet, correlates of predation pressure and life‐history parameters. The strongest predictors were the variables species and subfamily, which explained most of the observed variation in group size. Group size also increased with openness of the habitat and showed a U‐shaped relationship with temperature. In conclusion, phylogeny seems to play the most crucial role in the evolution of group size in Delphinoidea. The simplest interpretation of this result is that group size resulted from a historical (random) process and has only been marginally shaped by direct selection.     

Studying the folding process of the acylphosphatase from Sulfolobus solfataricus. A comparative analysis with other proteins from the same superfamily

The folding process of the acylphosphatase from Sulfolobus solfataricus (Sso AcP) has been followed, starting from the fully unfolded state, using a variety of spectroscopic probes, including intrinsic fluorescence, circular dichroism, and ANS binding. The results indicate that an ensemble of partially folded or misfolded species form rapidly on the submillisecond time scale after initiation of folding. This conformational ensemble produces a pronounced downward curvature in the Chevron plot, appears to possess a content of secondary structure similar to that of the native state, as revealed by far-UV circular dichroism, and appears to have surface-exposed hydrophobic clusters, as indicated by the ability of this ensemble to bind to 8-anilino-1-naphthalenesulfonic acid (ANS). Sso AcP folds from this conformational state with a rate constant of ca. 5 s(-1) at pH 5.5 and 37 degrees C. A minor slow exponential phase detected during folding (rate constant of 0.2 s(-1) under these conditions) is accelerated by cyclophilin A and is absent in a mutant of Sso AcP in which alanine replaces the proline residue at position 50. This indicates that for a lower fraction of Sso AcP molecules the folding process is rate-limited by the cis-trans isomerism of the peptide bond preceding Pro50. A comparative analysis with four other homologous proteins from the acylphosphatase superfamily shows that sequence hydrophobicity is an important determinant of the conformational stability of partially folded states that may accumulate during folding of a protein. A low net charge and a high propensity to form alpha-helical structure also emerge as possibly important determinants of the stability of partially folded states. A significant correlation is also observed between folding rate and hydrophobic content of the sequence within this superfamily, lending support to the idea that sequence hydrophobicity, in addition to relative contact order and conformational stability of the native state, is a key determinant of folding rate.     

The extracellular Leucine-Rich Repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns

Correction to Dolan J, Walshe K, Alsbury S, Hokamp K, O'Keeffe S, Okafuji T, Miller SF, Tear G, Mitchell KJ: The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns. BMC Genomics 2007, 8:320.     

Inhibition of viral group-1 and group-2 neuraminidases by oseltamivir: A comparative structural analysis by the ScrewFit algorithm

The viral surface glycoprotein neuraminidase (NA) allows the influenza virus penetration and the egress of virions. NAs are classified as A, B, and C. Type-A NAs from influenza virus are subdivided into two phylogenetically distinct families, group-1 and group-2. NA inhibition by oseltamivir represents a therapeutic approach against the avian influenza virus H5N1. Here, structural bases for oseltamivir recognition by group-1 NA1, NA8 and group-2 NA9 are highlighted by the ScrewFit algorithm for quantitative structure comparison. Oseltamivir binding to NA1 and NA8 affects the geometry of Glu119 and of regions Arg130-Ser160, Val240-Gly260, and Asp330-Glu382, leading to multiple NA conformations. Additionally, although NA1 and NA9 share almost the same oseltamivir-bound final conformation, they show some relevant differences as suggested by the ScrewFit algorithm. These results indicate that the design of new NA inhibitors should take into account these family-specific effects induced on the whole structure of NAs.