Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils.

Cytoplasmic granules of neutrophils store a variety of cationic polypeptides, which exert in vitro a potent antibacterial action and are potentially involved in host defense mechanisms. From an acid extract of bovine neutrophil granules we have purified over 2,000-fold a dodecapeptide exhibiting bactericidal activity against both Escherichia coli and Staphylococcus aureus at 10(-7)-10(-5) M concentration. The purification procedure involved only two steps of ion-exchange and reversed-phase chromatography. The peptide, named bactenecin, has the amino acid sequence, Arg-Leu-Cys-Arg-Ile-Val-Val-Ile-Arg-Val-Cys-Arg, maintained in a cyclic structure by a disulfide bond between the two cysteine residues. Computer modeling of the dodecapeptide resulted in a conformation in which the chain adopts an antiparallel extended structure forming a gamma turn at residue 7

pH dependence of bovine mast cell tryptase catalytic activity and of its inhibition by 4′,6-diamidino-2-phenylindole

AbstractTryptases are oligomeric enzymes localized in the secretory granules of mast cells. Their role(s) in vivo has yet to be clarified and the lack of powerful and specific inhibitors has hampered the comprehension of the biological functions of these enzymes. In this paper, we identify 4′,6-diamidino-2-phenylindole as a potent inhibitor for bovine tryptase. This inhibitory effect and the enzyme catalyzed hydrolysis of the synthetic substrate Boc-Phe-Ser-Arg-methyl-coumarin were investigated in the pH range of 6.0–9.0. On the basis of the pK shifts occurring upon formation of the inhibitor(substrate)/enzyme complexes, some aminoacidic groups are proposed to play a role in such interactions

The High Resolution Structure of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is a bifunctional enzyme involved in the biosynthesis of cell-surface structures, such as blood group antigens. Each subunit in the homodimeric enzyme consists of two domains. The N-terminal domain displays a Rossmann-fold topology and binds the NADP+ coenzyme. The C-terminal domain is held to bind the substrate. The holo-enzyme structure has been refined at 1.45 Å resolution, based on synchrotron data, to a final R-factor of 0.127 (Rfree = 0.167). The refined protein model highlights several residues involved in coenzyme recognition and binding and suggests that the enzyme belongs to the short-chain dehydrogenase protein homology family. Implications of the catalytic mechanism are discussed

Crystallographic Study of Mutant Lysl20Leu Xenopus laevis Cu,Zn Superoxide Dismutase

Theoretical calculations and experimental measurements on the Xenopus laevis Cu,Zn superoxide dismutase (XSODB) wild-type protein and on some of its engineered mutants showed that the electrostatic arrangement around the active site channel plays a fundamental role in determining the catalytic properties of the en-zyme. Lysl20, which lies on the lip of the active site channel, about 11 Å from the catalytic copper ion, influences the enzyme electrostatic environment and binding selectivity. Neutralization of this residue has the effect of decreasing the activity of the enzyme versus the negatively charged substrate. In order to get precise information about the mutated residue and its effects on the structure of the engineered protein, the crystal structure of single site Lysl20Leu mutant XSODB was determined at 2.0 Å resolution, and refined to an R-factor value of 0.181. The structure of Lysl20Leu mutant XSODB is little affected by the amino-acid substitution, suggesting that the main effect of the mutation is perturbation of the electrostatic properties of the SOD catalytic center

Structural determinants in the group III truncated hemoglobin from Campylobacter jejuni.

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 alpha-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-angstrom resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III

Delivery of Suramin as an Antiviral Agent through Liposomal Systems

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Targeting flavivirus RNA dependent RNA polymerase through a pyridobenzothiazole inhibitor

AbstractRNA dependent RNA polymerases (RdRp) are essential enzymes for flavivirus replication. Starting from an in silico docking analysis we identified a pyridobenzothiazole compound, HeE1-2Tyr, able to inhibit West Nile and Dengue RdRps activity in vitro, which proved effective against different flaviviruses in cell culture. Crystallographic data show that HeE1-2Tyr binds between the fingers domain and the priming loop of Dengue virus RdRp (Site 1). Conversely, enzyme kinetics, binding studies and mutational analyses suggest that, during the catalytic cycle and assembly of the RdRp-RNA complex, HeE1-2Tyr might be hosted in a distinct binding site (Site 2). RdRp mutational studies, driven by in silico docking analysis, allowed us to locate the inhibition Site 2 in the thumb domain. Taken together, our results provide innovative concepts for optimization of a new class of anti-flavivirus compounds

Crystallization and preliminary crystallographic studies on the large extracellular domain of human CD81, a tetraspanin receptor for hepatitis C virus

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Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination

SummaryThe sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways