The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cystine-knot motif.

Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.     

A model of globin evolution

Putative globins have been identified in 426 bacterial, 32 Archaeal and 67 eukaryote genomes. Among these sequences are the hitherto unsuspected presence of single domain sensor globins within Bacteria, Fungi, and a Euryarchaeote. Bayesian phylogenetic trees suggest that their occurrence in the latter two groups could be the result of lateral gene transfer from Bacteria. Iterated psiblast searches based on groups of globin sequences indicate that bacterial flavohemoglobins are closer to metazoan globins than to the other two lineages, the 2-over-2 globins and the globin-coupled sensors. Since Bacteria is the only kingdom to have all the subgroups of the three globin lineages, we propose a working model of globin evolution based on the assumption that all three lineages originated and evolved only in Bacteria. Although the 2-over-2 globins and the globin-coupled sensors recognize flavohemoglobins, there is little recognition between them. Thus, in the first stage of globin evolution, we favor a flavohemoglobin-like single domain protein as the ancestral globin. The next stage comprised the splitting off to single domain 2-over-2 and sensor-like globins, followed by the covalent addition of C-terminal domains resulting in the chimeric flavohemoglobins and globin-coupled sensors. The last stage encompassed the lateral gene transfers of some members of the three globin lineages to specific groups of Archaea and Eukaryotes.     

Destabilization of transthyretin by pathogenic mutations in the DE loop

Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin.     

Nonhistone Scm3 binds to AT-rich DNA to organize atypical centromeric nucleosome of budding yeast

The molecular architecture of centromere-specific nucleosomes containing histone variant CenH3 is controversial. We have biochemically reconstituted two distinct populations of nucleosomes containing Saccharomyces cerevisiae CenH3 (Cse4). Reconstitution of octameric nucleosomes containing histones Cse4/H4/H2A/H2B is robust on noncentromere DNA, but inefficient on AT-rich centromere DNA. However, nonhistone Scm3, which is required for Cse4 deposition in?vivo, facilitates in?vitro reconstitution of Cse4/H4/Scm3 complexes on AT-rich centromere sequences. Scm3 has a nonspecific DNA binding domain that shows preference for AT-rich DNA and a histone chaperone domain that promotes specific loading of Cse4/H4. In live cells, Scm3-GFP is enriched at centromeres in all cell cycle phases. Chromatin immunoprecipitation confirms that Scm3 occupies centromere DNA throughout the cell cycle, even when Cse4 and H4 are temporarily dislodged in S phase. These findings suggest a model in which centromere-bound Scm3 aids recruitment of Cse4/H4 to assemble and maintain an H2A/H2B-deficient centromeric nucleosome.     

TargetMine, an integrated data warehouse for candidate gene prioritisation and target discovery

Prioritising candidate genes for further experimental characterisation is a non-trivial challenge in drug discovery and biomedical research in general. An integrated approach that combines results from multiple data types is best suited for optimal target selection. We developed TargetMine, a data warehouse for efficient target prioritisation. TargetMine utilises the InterMine framework, with new data models such as protein-DNA interactions integrated in a novel way. It enables complicated searches that are difficult to perform with existing tools and it also offers integration of custom annotations and in-house experimental data. We proposed an objective protocol for target prioritisation using TargetMine and set up a benchmarking procedure to evaluate its performance. The results show that the protocol can identify known disease-associated genes with high precision and coverage. A demonstration version of TargetMine is available at http://targetmine.nibio.go.jp/.     

Functional restraints on the patterns of amino acid substitutions: application to sequence-structure homology recognition

Amino acid residues that are involved in functional interactions in proteins have strong evolutionary pressure to remain unchanged and consequently their substitution patterns are different from those that are noninteracting. To characterize and quantify the differences between amino acid substitution patterns due to structural restraints and those under functional restraints, we have made a comparative analysis of families of homologous proteins. Residues classified as having the same amino acid type, secondary structure, accessibility, and side-chain hydrogen bonds are shown to be better conserved if they are close to the active site. We have focused on enzyme families for this analysis since they have functional sites that are easily defined by their catalytic residues. We have derived new sets of environment-specific substitution tables, which we term function-dependent environment-specific substitution tables, where amino acid residues are classified according to their distance from the functional sites. The residues that are within a distance of 9 A from the active site have distinct amino acid substitution patterns when compared to the other sites. The function-dependent environment-specific substitution tables have been tested using the sequence-structure homology recognition program FUGUE and the results compared with the recognition performance obtained using the standard environment-specific substitution tables. Significant improvements are obtained in both recognition performance and alignment accuracy using the function-dependent environment-specific substitution tables (P-value = 0.02, according to the Wilcoxon signed rank test for alignment accuracy). The alignments near the active site are greatly improved with pronounced improvements at lower percentage identities (less than 30%).