Journal of structural and functional genomics最新文献

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. Here we identify a Bifidobacterium longum protein with high sequence homology to type II DHQDs but no detectable DHQD activity under standard assay conditions. A crystal structure reveals that the B. longum protein adopts a DHQD-like tertiary structure but a distinct quaternary state. Apparently forming a dimer, the B. longum protein lacks the active site aspartic acid contributed from a neighboring protomer in the type II DHQD dodecamer. Relating to the absence of protein-protein interactions established in the type II DHQD dodecameric assembly, substantial conformational changes distinguish the would-be active site of the B. longum protein. As B. longum possess no other genes with homology to known DHQDs, these findings imply a unique DHQD activity within B. longum.

Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

A high-quality NMR structure of the helicase associated (HA) domain comprising residues 627-691 of the 753-residue protein BVU_0683 from Bacteroides vulgatus exhibits an all α-helical fold. The structure presented here is the first representative for the large protein domain family PF03457 (currently 742 members) of HA domains. Comparison with structurally similar proteins supports the hypothesis that HA domains bind to DNA and that binding specificity varies greatly within the family of HA domains constituting PF03457.

The study of the protein-protein interactions (PPIs) of unique ORFs is a strategy for deciphering the biological roles of unique ORFs of interest. For uniform reference, we define unique ORFs as those for which no matching protein is found after PDB-BLAST search with default parameters. The uniqueness of the ORFs generally precludes the straightforward use of structure-based approaches in the design of experiments to explore PPIs. Many open-source bioinformatics tools, from the commonly-used to the relatively esoteric, have been built and validated to perform analyses and/or predictions of sorts on proteins. How can these available tools be combined into a protocol that helps the non-expert bioinformaticist researcher to design experiments to explore the PPIs of their unique ORF? Here we define a pragmatic protocol based on accessibility of software to achieve this and we make it concrete by applying it on two proteins-the ImuB and ImuA' proteins from Mycobacterium tuberculosis. The protocol is pragmatic in that decisions are made largely based on the availability of easy-to-use freeware. We define the following basic and user-friendly software pathway to build testable PPI hypotheses for a query protein sequence: PSI-PRED → MUSTER → metaPPISP → ASAView and ConSurf. Where possible, other analytical and/or predictive tools may be included. Our protocol combines the software predictions and analyses with general bioinformatics principles to arrive at consensus, prioritised and testable PPI hypotheses.

Aminoacyl-tRNA synthetases (AARSs) are ligases (EC.6.1.1.-) that catalyze the acylation of amino acids to their cognate tRNAs in the process of translating genetic information from mRNA to protein. Their amino acid and tRNA specificity are crucial for correctly translating the genetic code. Glycine is the smallest amino acid and the glycyl-tRNA synthetase (GlyRS) belongs to Class II AARSs. The enzyme is unusual because it can assume different quaternary structures. In eukaryotes, archaebacteria and some bacteria, it forms an α(2) homodimer. In some bacteria, GlyRS is an α(2)β(2) heterotetramer and shows a distant similarity to α(2) GlyRSs. The human pathogen eubacterium Campylobacter jejuni GlyRS (CjGlyRS) is an α(2)β(2) heterotetramer and is similar to Escherichia coli GlyRS; both are members of Class IIc AARSs. The two-step aminoacylation reaction of tetrameric GlyRSs requires the involvement of both α- and β-subunits. At present, the structure of the GlyRS α(2)β(2) class and the details of the enzymatic mechanism of this enzyme remain unknown. Here we report the crystal structures of the catalytic α-subunit of CjGlyRS and its complexes with ATP, and ATP and glycine. These structures provide detailed information on substrate binding and show evidence for a proposed mechanism for amino acid activation and the formation of the glycyl-adenylate intermediate for Class II AARSs.

An efficient semi-automated strategy called PFBD (i.e. Protein Fold from Backbone Data only) has been presented for rapid backbone fold determination of small proteins. It makes use of NMR parameters involving backbone atoms only. These include chemical shifts, amide-amide NOEs and H-bonds. The backbone chemical shifts are obtained in an automated manner from the orthogonal 2D projections of variants of HNN and HN(C)N experiments (Kumar et al., in Magn Reson Chem 50(5):357-363, 2012) using AUTOBA (Borkar et al. in J Biomol NMR 50(3):285-297, 2011); backbone H-bonds are manually derived from constant time long-range 2D-HnCO spectrum (Cordier and Grzesiek in J Am Chem Soc 121:1601-1602, 1999); and amide-amide NOEs are derived from 3D HNCO NOESY experiment which provides NOEs along the direct (1)H dimension that has maximum resolution (Lohr and Ruterjans in J Biomol NMR 9(1):371-388, 1997). All the experiments needed for the execution of PFBD can be recorded and analyzed in about 24-48 h depending upon the concentration of the protein and dispersion of amide cross-peaks in the (1)H-(15)N correlation spectrum. Thus, we believe that the strategy, because of its speed and simplicity will be very valuable in Biomolecular NMR community for high-throughput structural proteomics of small folded proteins of MW < 10-12 kDa, the regime where NMR is generally preferred over X-ray crystallography. The strategy has been validated and demonstrated here on two small globular proteins: human ubiquitin (76 aa) and chicken SH3 domain (62 aa).

The explosion of the size of the universe of known protein sequences has stimulated two complementary approaches to structural mapping of these sequences: theoretical structure prediction and experimental determination by structural genomics (SG). In this work, we assess the accuracy of structure prediction by two automated template-based structure prediction metaservers (genesilico.pl and bioinfo.pl) by measuring the structural similarity of the predicted models to corresponding experimental models determined a posteriori. Of 199 targets chosen from SG programs, the metaservers predicted the structures of about a fourth of them "correctly." (In this case, "correct" was defined as placing more than 70 % of the alpha carbon atoms in the model within 2 Å of the experimentally determined positions.) Almost all of the targets that could be modeled to this accuracy were those with an available template in the Protein Data Bank (PDB) with more than 25 % sequence identity. The majority of those SG targets with lower sequence identity to structures in the PDB were not predicted by the metaservers with this accuracy. We also compared metaserver results to CASP8 results, finding that the models obtained by participants in the CASP competition were significantly better than those produced by the metaservers.

PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.

RhoA, a member of the Rho sub-family of small GTPases, plays a significant signaling role in cell morphogenesis, migration, neuronal development, cell division and adhesion. So far, 4 structures of RhoA:GDP/GTP analogs and 14 structures of RhoA in complex with other proteins have been reported. All RhoA:GDP/GTP analog complexes have been crystallized in primitive lattices and RhoA is monomeric. This is the first time a RhoA:GTP analog complex has been crystallized as a dimer in a centered lattice. The present structure reveals structural differences in the switch-I (residues 28-42) and switch-II (residues 61-66) regions, which play important roles in interactions with downstream targets to transduce signals, when compared to the previously reported structures.

We report the use of anionic (I(-)), cationic (Ba(2+), Cd(2+)) and ionic mixtures (I(-) plus Ba(2+)) for derivatizing liver fatty acid binding protein (LFABP) crystals. Use of cationic and anionic salts in phasing experiments revealed distinct non-overlapping sites for these ions, suggesting exclusive binding regions on LFABP. Interestingly, cations of identical charge and valency (like Ba(2+) and Cd(2+)) bound to distinct pockets on the protein surface. Furthermore, a mixture of salts containing both I(-) and Ba(2+) was very useful in phasing experiments as these oppositely charged ions bound to different regions of LFABP. Our data therefore suggest that cationic and anionic salt mixtures like BaCl(2) with NH(4)I or salts like CdI, BaI where each ion has a significant anomalous signal for a given X-ray wavelength may be valuable reagents for phasing during structure determination.