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J. Biol. Chem., Vol. 280, Issue 18, 18202-18210, May 6, 2005

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Crystal Structures of the RNA-dependent RNA Polymerase Genotype 2a of Hepatitis C Virus Reveal Two Conformations and Suggest Mechanisms of Inhibition by Non-nucleoside Inhibitors^*

Bichitra K. Biswal, Maia M. Cherney, Meitian Wang, Laval Chan, Constantin G. Yannopoulos, Darius Bilimoria, Olivier Nicolas, Jean Bedard, and Michael N. G. James, Canada Research Chair in Protein Structure and Function¶

From the Canadian Institutes of Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and ViroChem Pharma Incorporated, Laval, Quebec H7V 4A7, Canada

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus (HCV) from two crystal forms have been determined. Similar to the three-dimensional structures of HCV polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains. The main structural differences between the molecules in the two crystal forms lie at the interface of the fingers and thumb domains. The relative orientation of the thumb domain with respect to the fingers and palm domains and the -flap region is altered. Structural analysis reveals that the NS5B polymerase in crystal form I adopts a "closed" conformation that is believed to be the active form, whereas NS5B in crystal form II adopts an "open" conformation and is thus in the inactive form. In addition, we have determined the structures of two NS5B polymerase/non-nucleoside inhibitor complexes. Both inhibitors bind at a common binding site, which is nearly 35 Å away from the polymerase active site and is located in the thumb domain. The binding pocket is predominantly hydrophobic in nature, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions. Inhibitors can only be soaked in crystal form I and not in form II; examination of the enzyme-inhibitor complex reveals that the enzyme has undergone a dramatic conformational change from the form I (active) complex to the form II (inactive).

Received for publication, November 29, 2004 , and in revised form, February 23, 2005.

The atomic coordinates and structure factors (codes 1YUY, 1YV2, 1YVZ, and 1YVX) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by funding from the Alberta Heritage Foundation for Medical Research for the purchase of area detector facilities and Canadian Institutes of Health Research for operating expenses. 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.

^¶ To whom correspondence should be addressed. Tel.: 780-492-4550; Fax: 780-492-0886; E-mail: Michael.james{at}ualberta.ca.

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