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J. Biol. Chem., Vol. 279, Issue 16, 16638-16645, April 16, 2004

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Crystal Structure of Norwalk Virus Polymerase Reveals the Carboxyl Terminus in the Active Site Cleft^*

Kenneth K.-S. Ng, Natalia Pendás-Franco¶, Jorge Rojo¶, José A. Boga||, Àngeles Machín¶, José M. Martín Alonso¶, and Francisco Parra¶

From the Division of Biochemistry, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada and ^¶Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo and ^||Servicio de Microbiología I, Hospital Universitario Central de Asturias, 33006 Oviedo, Spain

Norwalk virus is a major cause of acute gastroenteritis for which effective treatments are sorely lacking. To provide a basis for the rational design of novel antiviral agents, the main replication enzyme in Norwalk virus, the virally encoded RNA-dependent RNA polymerase (RdRP), has been expressed in an enzymatically active form, and its structure has been crystallographically determined both in the presence and absence of divalent metal cations. Although the overall fold of the enzyme is similar to that seen previously in the RdRP from rabbit hemorrhagic disease virus, the carboxyl terminus, surprisingly, is located in the active site cleft in five independent copies of the protein in three distinct crystal forms. The location of this carboxyl-terminal segment appears to interfere with the binding of double-stranded RNA in the active site cleft and may play a role in the initiation of RNA synthesis or mediate interactions with accessory replication proteins.

Received for publication, January 20, 2004 , and in revised form, February 2, 2004.

The atomic coordinates and structure factors (codes 1SH0, 1SH2, and 1SH3) for the two crystal forms of metal-free polymerase as well as for the Mg²⁺-bound form have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This research was supported by a senior research fellowship from the Canadian Institutes for Health Research as well as a Medical Scholar Award and Establishment Grant for medical research from the Alberta Heritage Foundation (to K. K.-S. N.). This work was also supported in part by Grants 01/0976 (to F. P.) and 01/3139 (to J. A. B.) from Fondo de Investigacín Sanitaria, Ministerio de Sanidad y Consumo, Spain. 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.: 403-220-4320; Fax: 403-289-9311; E-mail: ngk{at}ucalgary.ca.

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