Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri.

Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-ß-farnesene but M. viciae uses a mixture of (E)-ß-farnesene, (-)-α-pinene, ß-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket.

The RpfC (Rv1884) atomic structure shows high structural conservation within the resuscitation-promoting factor catalytic domain.

The first structure of the catalytic domain of RpfC (Rv1884), one of the resuscitation-promoting factors (RPFs) from Mycobacterium tuberculosis, is reported. The structure was solved using molecular replacement once the space group had been correctly identified as twinned P21 rather than the apparent C2221 by searching for anomalous scattering sites in P1. The structure displays a very high degree of structural conservation with the previously published structures of the catalytic domains of RpfB (Rv1009) and RpfE (Rv2450). This structural conservation highlights the importance of the versatile domain composition of the RPF family.

Crystal structure of reduced MsAcg, a putative nitroreductase from Mycobacterium smegmatis and a close homologue of Mycobacterium tuberculosis Acg.

This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria's adaptation to a dormancy state.

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning.

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis.

Structure of a putative acetyltransferase (PA1377) from Pseudomonas aeruginosa.

Gene PA1377 from Pseudomonas aeruginosa encodes a 177-amino-acid conserved hypothetical protein of unknown function. The structure of this protein (termed pitax) has been solved in space group I222 to 2.25 A resolution. Pitax belongs to the GCN5-related N-acetyltransferase family and contains all four sequence motifs conserved among family members. The beta-strand structure in one of these motifs (motif A) is disrupted, which is believed to affect binding of the substrate that accepts the acetyl group from acetyl-CoA.