With more than 100 antibacterial drugs at our disposal in the 1980's, the problem of bacterial infection was considered solved. Today, however, most hospital infections are insensitive to several ...classes of antibacterial drugs, and deadly strains of Staphylococcus aureus resistant to vancomycin--the last resort antibiotic--have recently begin to appear. Other life-threatening microbes, such as Enterococcus faecalis and Mycobacterium tuberculosis are already able to resist every available antibiotic. There is thus an urgent, and continuous need for new, preferably large-spectrum, antibacterial molecules, ideally targeting new biochemical pathways. Here we report on the progress of our structural genomics program aiming at the discovery of new antibacterial gene targets among evolutionary conserved genes of uncharacterized function. A series of bioinformatic and comparative genomics analyses were used to identify a set of 221 candidate genes common to Gram-positive and Gram-negative bacteria. These genes were split between two laboratories. They are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallization, X-ray diffraction, structure interpretation, and function prediction. We describe here our strategies for the 111 genes processed in our laboratory. Bioinformatics is used at most stages of the production process and out of 111 genes processed--and 17 months into the project--108 have been successfully cloned, 103 have exhibited detectable expression, 84 have led to the production of soluble protein, 46 have been purified, 12 have led to usable crystals, and 7 structures have been determined.
The Escherichia coli yeaZ gene encodes a 231‐residue protein (Mr = 25 180) that belongs to a family of proteins that are conserved in various bacterial genomes. This protein of unknown function is ...predicted to be a hypothetical protease. The YeaZ protein was overexpressed in E. coli and crystallized at 298 K by the hanging‐drop vapour‐diffusion method. A MAD data set was collected using a gadolinium‐derivative crystal that had been soaked with 0.1 M Gd‐DOTMA. The data set contained data collected to a resolution of 2.7 Å at two wavelengths at the LIII absorption edge of gadolinium, while remote data were collected to a resolution of 2.28 Å. The crystal belonged to the orthorhombic space group P212121, with unit‐cell parameters a = 76.3, b = 97.6, c = 141.9 Å. Phasing using the MAD method confirmed there to be four monomers in the asymmetric unit related by two twofold axes as identified by the self‐rotation function search.
The complete sequence of the largest known double‐stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined Raoult et al. (2004), Science, 306, 1344–1350 and revealed ...numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated Abergel et al. (2005), Acta Cryst. F61, 212–215 both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P213, with unit‐cell parameter 99.425 Å. The self‐rotation function confirms that there are two monomers per asymmetric unit related by a twofold non‐crystallographic axis and that the unit cell thus contains four biological entities.
Purification and crystallization of Kokobera virus helicase De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno ...
Acta crystallographica. Section F, Structural biology and crystallization communications,
March 2007, Letnik:
63, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Kokobera virus is a mosquito‐borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive‐sense ...single‐stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post‐translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C‐terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging‐drop vapour‐diffusion method. The crystals belong to space group P3121 (or P3221), with unit‐cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.
Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C‐terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is ...thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3′ nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid‐binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti‐flaviviral compounds.
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 Å resolution and that were suitable for structure determination.
Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have ...been performed, showing that they are active as ADP‐ribose‐1′′‐phosphatases. Macro domains are also present in a number of positive‐stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS‐CoV) encodes 16 non‐structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS‐CoV nsp3 gene region corresponding to amino acids 182–355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P21, with unit‐cell parameters a = 37.5, b = 55.6, c = 108.9 Å, β = 91.4°, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine‐labelled crystals diffract to 1.8 Å.
The non‐structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine‐specific endoribonuclease. This enzyme plays an ...essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full‐length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N‐terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4132 or P4332, with unit‐cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å.
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