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- Coutard, B., et al.
(författare)
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The VIZIER project : Preparedness against pathogenic RNA viruses
- 2008
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Ingår i: Antiviral Research. - : Elsevier BV. - 0166-3542 .- 1872-9096. ; 78:1, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.
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| 4. |
- Fogg, M. J., et al.
(författare)
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Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens
- 2006
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62:10, s. 1196-1207
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Tidskriftsartikel (refereegranskat)abstract
- The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
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| 5. |
- Henriksson, Lena M., et al.
(författare)
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Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis
- 2007
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:27, s. 19905-19916
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Tidskriftsartikel (refereegranskat)abstract
- Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn2+, NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC50 value of 80 nM. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.
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| 6. |
- Ingvarsson, Henrik, et al.
(författare)
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Crystallization of Mycobacterium smegmatis methionyl-tRNA synthetase in the presence of methionine and adenosine
- 2009
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 65:Part 6, s. 618-620
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Tidskriftsartikel (refereegranskat)abstract
- Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni(2+)-affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1 A resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3 A, beta = 124.8 degrees . The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1 A(3) Da(-1). Structure determination is in progress.
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| 7. |
- Jansson, Anna M., et al.
(författare)
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Structure of the methyltransferase domain from the Modoc virus, a flavivirus with no known vector
- 2009
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 65, s. 796-803
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Tidskriftsartikel (refereegranskat)abstract
- The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-l-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.
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