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.

ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation.

New therapies are required against Mycobacterium tuberculosis and its cell wall peptidoglycan biosynthesis is a potential therapeutic target. UDP-MurNAc-tripeptide ligase (MurE) is a member of the ATP-dependent ligase family, which incorporate amino acids including meso-diaminopimelic acid (m-DAP) into peptidoglycan during synthesis in a species-specific manner. In the present study, we have cloned, over-expressed, and characterised MurE from M. tuberculosis (Mtb-MurE). The crystal structure has been determined at 3.0A resolution in the presence of the substrate UDP-MurNAc-l-Ala-d-Glu (UAG). The activity of the enzyme was measured through estimating inorganic phosphate released in a non-radioactive high-throughput colourimetric assay. UDP-MurNAc-l-Ala-d-Glu-m-DAP (UMT) formation coupled to inorganic phosphate release was confirmed by HPLC and mass spectrometric analyses. Kinetic constants were determined for a range of natural substrates using optimised conditions. From our findings, it is evident that Mtb-MurE is highly specific in adding m-DAP to UDP-MurNAc-dipeptide and ATP-hydrolysis is an absolute requirement for its activity.

Characterisation of Bombyx mori Odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components.

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1.

Pasteurellaceae ComE1 proteins combine the properties of fibronectin adhesins and DNA binding competence proteins.

A novel fibronectin-binding protein from Pasteurella multocida (PM1665) that binds to the fibronectin type III(9-10) modules via two helix-hairpin-helix motifs has recently been described [1]. This protein shares homology with competence-related DNA-binding and uptake proteins (ComEA and ComE) from Gram-positive and Gram-negative bacteria. Here, we show that recombinant PM1665 (now designated ComE1) also binds to DNA through the same helix-hairpin-helix motifs required for fibronectin-binding. This binding to DNA is non sequence-specific and is confined to double-stranded DNA. We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins. When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA. Inactivation of the gene encoding the ComE1 homologue in Actinobacillus pleuropneumoniae indicates major roles for these proteins in at least two processes: natural transformation, and binding of bacteria to fibronectin.

Novel adhesin from Pasteurella multocida that binds to the integrin-binding fibronectin FnIII9-10 repeats.

Phage display screening with fragmented genomic DNA from the animal pathogen Pasteurella multocida has identified a gene encoding a putative fibronectin binding protein (19). Homologues of this gene (PM1665) are found in all other sequenced members of the Pasteurellaceae. Gene PM1665 has been cloned, and the protein has been expressed. Recombinant PM1665 protein binds to both soluble and immobilized fibronectin and is unique in that it interacts with the integrin-binding fibronectin type III (FnIII) repeats FnIII(9-10) and not, as is the case for almost all other fibronectin adhesins, to the N-terminal type I repeats. Surface plasmon resonance analysis revealed a complex binding mechanism with a K(D) (equilibrium dissociation constant) of 150 nM +/- 70 nM. Bioinformatics analysis suggests that the PM1665 protein contains two helix-hairpin-helix (HhH) motifs, and truncation mutation studies have identified the binding site in the protein as a combination of these two HhH motifs in conjunction with a conserved amino acid motif, VNINTA. We have shown that the PM1665 protein is on the cell surface and that binding of P. multocida to fibronectin is almost completely inhibited by anti-PM1665 antiserum. These results support the hypothesis that the PM1665 protein is a member of a new family of fibronectin binding adhesins that are important in the adhesion of P. multocida to fibronectin.

Exploiting structural principles to design cyclin-dependent kinase inhibitors.

Although cyclin-dependent kinases (CDKs) have been extensively targeted in anti cancer drug design, no CDK inhibitor has yet been approved for use in cancer therapy. While this may in part be because inhibitors clinically evaluated to date have not demonstrated clean inhibition of a single CDK, another contributing factor is an apparent latent functional redundancy in the CDK cell-cycle regulatory system. This further complicates the already challenging goal of targeting CDKs, since it implies that a therapeutically useful inhibitor will have to selectively inhibit more than one CDK family member among the complement of cellular proteins. Despite these difficulties, achieving an appropriate profile of CDK inhibition may yet be possible using ATP-competitive inhibitors, thanks to advances in computational and experimental methods of drug design. However, as an alternative to ATP-competitive inhibitors, inhibitors that interfere with a CDK-specific protein:protein interaction, such as that which occurs at the recruitment site found on several cyclins, may offer a route to a therapeutically useful inhibitory profile.