Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.     

Crystal structure of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture

The bifunctional bacterial enzyme N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against beta-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 A, respectively. The S. pneumoniae GlmU molecule is organized in two separate domains connected via a long alpha-helical linker and associates as a trimer, with the 50-A-long left-handed beta-helix (LbetaH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LbetaH core and exchanged with the beta-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LbetaH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.     

The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 A resolution

Glycoside hydrolases have been classified into over 66 families on the basis of amino acid sequence. Recently a number of these families have been grouped into "clans" which share a common fold and catalytic mechanism [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. Glycoside hydrolase Clan GH-C groups family 11 xylanases and family 12 cellulases, which share the same jellyroll topology, with two predominantly antiparallel beta-sheets forming a long substrate-binding cleft, and act with net retention of anomeric configuration. Here we present the three-dimensional structure of a family 12 endoglucanase, Streptomyces lividans CelB2, in complex with a 2-deoxy-2-fluorocellotrioside. Atomic resolution (1.2 A) data allow clear identification of two distinct species in the crystal. One is the glycosyl-enzyme intermediate, with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the other a complex with the reaction product, 2-deoxy-2-fluoro-beta-D-cellotriose. The active site architecture of the complex provides insight into the double-displacement mechanism of retaining glycoside hydrolases and also sheds light on the basis of the differences in specificity between family 12 cellulases and family 11 xylanases.     

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.     

The auditory system of non-calling grasshoppers (Melanoplinae: Podismini) and the evolutionary regression of their tympanal ears

Reduction of tympanal hearing organs is repeatedly found amongst insects and is associated with weakened selection for hearing. There is also an associated wing reduction, since flight is no longer required to evade bats. Wing reduction may also affect sound production. Here, the auditory system in four silent grasshopper species belonging to the Podismini is investigated. In this group, tympanal ears occur but sound signalling does not. The tympanal organs range from fully developed to remarkably reduced tympana. To evaluate the effects of tympanal regression on neuronal organisation and auditory sensitivity, the size of wings and tympana, sensory thresholds and sensory central projections are compared. Reduced tympanal size correlates with a higher auditory threshold. The threshold curves of all four species are tuned to low frequencies with a maximal sensitivity at 3–5 kHz. Central projections of the tympanal nerve show characteristics known from fully tympanate acridid species, so neural elements for tympanal hearing have been strongly conserved across these species. The results also confirm the correlation between reduction in auditory sensitivity and wing reduction. It is concluded that the auditory sensitivity of all four species may be maintained by stabilising selective forces, such as predation.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

Weighing costs and benefits of mating in bushcrickets (Insecta: Orthoptera: Tettigoniidae), with an emphasis on nuptial gifts,protandry and mate density

ABSTRACT: Sexual selection is a major force driving evolution and is intertwined with ecological factors. Differential allocation of limited resources has a central role in the cost of reproduction. In this paper, I review the costs and benefits of mating in tettigoniids, focussing on nuptial gifts, their trade-off with male calling songs, protandry and how mate density influences mate choice. Tettigoniids have been widely used as model systems for studies of mating costs and benefits; they can provide useful general insights. The production and exchange of large nuptial gifts by males for mating is an important reproductive strategy in tettigoniids. As predicted by sexual selection theory spermatophylax size is condition dependent and is constrained by the need to invest in calling to attract mates also. Under some circumstances, females benefit directly from the nuptial gifts by an increase in reproductive output. However, compounds in the nuptial gift can also benefit the male by prolonging the period before the female remates. There is also a trade-off between adult male maturation and mating success. Where males mature before females (protandry) the level of protandry varies in the direction predicted by sperm competition theory; namely, early male maturation is correlated with a high level of first inseminations being reproductively successful. Lastly, mate density in bushcrickets is an important environmental factor influencing the behavioural decisions of individuals. Where mates are abundant, individuals are more choosey of mates; when they are scarce, individuals are less choosey. This review reinforces the view that tettigoniids provide excellent models to test and understand the economics of matings in both sexes.     

Female bushcrickets fuel their metabolism with male nuptial gifts

In many arthropods, such as bushcrickets, males donate protein-rich nuptial gifts-so-called spermatophores-to females, which females ingest while the sperm enter the female's reproductive tract. Previously, it was shown that females route spermatophore nutrients over the course of hours and days to egg production or body synthesis. We investigated whether female bushcrickets fuel their metabolism with spermatophores immediately after consumption. We fed two male groups diets that were either enriched or depleted in 13C, and then tracked the isotopic changes in exhaled breath in female bushcrickets after spermatophore consumption. Within 3 hours, the stable carbon isotope ratio (delta13C) of female breath converged on the ratio of the male donor of the nuptial gift. This supports the idea that females quickly routed nutrients to metabolism, receiving immediate benefits from spermatophore feeding.     

Review of Biogeography, Host Range and Evolution of Acoustic Hunting in Ormiini (Insecta, Diptera, Tachinidae), Parasitoids of Night-calling Bushcrickets and Crickets (Insecta, Orthoptera, Ensifera)

Interest in parasitoids has grown with the recognition that host-parasitoid systems offer opportunities to examine fundamental questions in behavioural and evolutionary ecology. Tachinid flies of the Ormiini possess a conspicuously inflated prosternal region, enabling them to detect the mating songs of their hosts. This speciality makes them a highly suitable group for studies of adaptive radiation. To emphasise further research in this important group of parasitoids, their biogeography and host species are summarised.The Ormiini are a particularly small group, containing only 68 described species of predominantly tropical, especially neotropical forms. A table of host-parasitoid relations reveals that predominant parasitism is of bushcrickets. The exploitation of cricket songs appears to be a derived pattern that evolved as a host switch some time after the Eocene.Hypotheses concerning fly-host coevolution and the reasons for the development of hearing are discussed, and include the question of mate finding and avoidance of bats as predators.