Arnaud Gruez

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-Å 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.

The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases

ABSTRACT The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3Dpol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

A medium-throughput crystallization approach

The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures. Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.

The crystal structure of the Escherichia coli yfdW gene product reveals a New fold of two interlaced rings identifying a wide family of CoA transferases.

Because of its toxicity, oxalate accumulation from amino acid catabolism leads to acute disorders in mammals. Gut microflora are therefore pivotal in maintaining a safe intestinal oxalate balance through oxalate degradation. Oxalate catabolism was first identified in Oxalobacter formigenes, a specialized, strictly anaerobic bacterium. Oxalate degradation was found to be performed successively by two enzymes, a formyl-CoA transferase (frc) and an oxalate decarboxylase (oxc). These two genes are present in several bacterial genomes including that of Escherichia coli. The frc ortholog in E. coli is yfdW, with which it shares 61% sequence identity. We have expressed the YfdW open reading frame product and solved its crystal structure in the apo-form and in complex with acetyl-CoA and with a mixture of acetyl-CoA and oxalate. YfdW exhibits a novel and spectacular fold in which two monomers assemble as interlaced rings, defining the CoA binding site at their interface. From the structure of the complex with acetyl-CoA and oxalate, we propose a putative formyl/oxalate transfer mechanism involving the conserved catalytic residue Asp169. The similarity of yfdW with bacterial orthologs (∼60% identity) and paralogs (∼20–30% identity) suggests that this new fold and parts of the CoA transfer mechanism are likely to be the hallmarks of a wide family of CoA transferases.

Structural and biochemical characterization of free methionine-R-sulfoxide reductase from Neisseria meningitidis.

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys118 is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.

Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

Optimization of crystals from nanodrops: crystallization and preliminary crystallographic study of a pheromone-binding protein from the honeybee Apis mellifera L.

Pheromone-binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in various sensory organs from moths and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. Here, crystals of a PBP (Amel-ASP1) originating from honeybee (Apis mellifera L.) antennae and expressed as recombinant protein using the yeast Pichia pastoris are reported. Crystals of Amel-ASP1 have been obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot under the following conditions: 200 nl of 40 mg ml(-1) protein solution in 10 mM Tris, 25 mM NaCl pH 8.0 was mixed with 100 nl of well solution containing 0.15 M sodium citrate, 1.5 M ammonium sulfate pH 5.5. The protein crystallizes in space group C222(1), with unit-cell parameters a = 74.8, b = 85.8, c = 50.2 A. With one molecule in the asymmetric unit, V(M) is 3.05 A(3) Da(-1) and the solvent content is 60%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). The nanodrop crystallization technique used with a novel optimization procedure made it possible to consume small amounts of protein and to obtain a unique crystal per nanodrop, suitable directly for data collection in-house or at a synchrotron-radiation source.

Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase.

The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 A resolution and that were suitable for structure determination.

The FNR‐like domain of the Escherichia coli sulfite reductase flavoprotein component: crystallization and preliminary X‐ray analysis

The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein subunit was crystallized using the hanging-drop technique, with PEG 4000 as precipitant. The crystals belong to space group P3112 or enantiomorph, with unit-cell parameters a = b = 171.0, c = 152.1 A. A solvent content of 75% was determined by a calibrated tetrachloromethane/toluene gradient which corresponds to three monomers per asymmetric unit. A 3 A resolution native data set was collected at beamline W32 of LURE, Orsay, France.

Structural diversity in the recognition between reduced thioredoxin and its oxidized enzyme partners.

Abstract Thioredoxins (Trx) are ubiquitous proteins that are conserved in all living organisms from archaea to humans. These small proteins display various cellular roles, including functioning as reductases in redox processes. All Trxs share a similar, characteristic three-dimensional fold with the Cys-Pro-Gly-Cys motif that contains both the catalytic and the resolving cysteine (Cys) on the surface of the protein. Reaction of reduced Trx with its oxidized protein partners leads to formation of a transient interdisulfide intermediate. However, the short lifetime of this species hinders the characterization of the stabilizing interactions that occur between the partners. In this short review, the three-dimensional structures of four artificial covalent Trx-protein partner complexes are analyzed. The data show that interprotein stabilization is mainly due to hydrophobic contacts and main-chain hydrogen bonds but that no common recognition motif between Trx and its protein partners can be identified. In two cases, formation of the Trx-partner complex is accompanied by a significant conformational change of the protein target, although in no case does the conformation of Trx change significantly. The absence of a common recognition motif supports the idea that it is difficult to predict with confidence putative oxidized protein substrates of Trx using only soft docking and molecular simulation methods. Instead, biochemical methods including proteomic approaches remain the primary tools to identify novel protein substrates of Trx. The generality and relevance of methods used to identify which of the two Cys of the disulfide-oxidized protein partner forms the transient interdisulfide intermediate with Trx are also discussed.