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J. Biol. Chem., Vol. 280, Issue 12, 11520-11527, March 25, 2005

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Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

NEW INSIGHTS INTO CATALYTIC MECHANISM AND CLEAVAGE SPECIFICITY^*

James R. Birtley, Stephen R. Knox¶||, Agnès M. Jaulent¶, Peter Brick, Robin J. Leatherbarrow¶**, and Stephen Curry

From the Biophysics Section, Division of Cell and Molecular Biology, Blackett Laboratory and the ^¶Biological and Biophysical Chemistry Section, Department of Chemistry, Imperial College, South Kensington Campus, Exhibition Road, London SW7 2AZ, United Kingdom

Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C^pro), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C^pro, determined the crystal structure to 1.9-Å resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C^pro adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4–P4') and that FMDV 3C^pro discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C^pro enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C^pro of an extended -ribbon that folds over the substrate binding cleft in other picornavirus 3C^pro structures. Collectively, these results establish a valuable framework for the development of FMDV 3C^pro inhibitors.

Received for publication, November 24, 2004 , and in revised form, January 11, 2005.

We dedicate this publication to David Blow (1931–2004), our long time colleague at Imperial College London, who solved the first protease structure in 1967.

The atomic coordinates and structure factors (code 2bhg) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work is supported by the Fleming Fund (Imperial College) and the BBSRC. 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.

Supported by a Medical Research Council studentship.

^|| Supported by an EPSRC studentship.

^** To whom correspondence may be addressed. Tel. 44-20-7594-5752; Fax: 44-20-7594-1139; E-mail: r.leatherbarrow{at}imperial.ac.uk. To whom correspondence may be addressed. Tel. 44-20-7594-7632; Fax: 44-20-7589-0191; E-mail: s.curry{at}imperial.ac.uk.

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