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J. Biol. Chem., Vol. 279, Issue 48, 49755-49761, November 26, 2004
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From the Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp220, Asp318, and Asp319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzyme-bound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.
Received for publication, August 23, 2004
* This work was supported by grants from the Canadian Institutes for Health Research and Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
New Investigator Scholar from the Canadian Institutes for Health Research. To whom correspondence should be addressed: Dépt. de biochimie, Faculté de médecine, Université de Sherbrooke, 3001 12e avenue, Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5227; Fax: 819-564-5340; E-mail: Martin.Bisaillon{at}USherbrooke.ca.
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