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J. Biol. Chem., Vol. 283, Issue 1, 554-564, January 4, 2008

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Mutation of Gly-11 on the Dimer Interface Results in the Complete Crystallographic Dimer Dissociation of Severe Acute Respiratory Syndrome Coronavirus 3C-like Protease

CRYSTAL STRUCTURE WITH MOLECULAR DYNAMICS SIMULATIONS^*

Shuai Chen¹, Tiancen Hu¹, Jian Zhang¹, Jing Chen, Kaixian Chen, Jianping Ding, Hualiang Jiang², and Xu Shen³

From the Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203 and the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

SARS-CoV 3C-like protease (3CL^pro) is an attractive target for anti-severe acute respiratory syndrome (SARS) drug discovery, and its dimerization has been extensively proved to be indispensable for enzymatic activity. However, the reason why the dissociated monomer is inactive still remains unclear due to the absence of the monomer structure. In this study, we showed that mutation of the dimer-interface residue Gly-11 to alanine entirely abolished the activity of SARS-CoV 3CL^pro. Subsequently, we determined the crystal structure of this mutant and discovered a complete crystallographic dimer dissociation of SARS-CoV 3CL^pro. The mutation might shorten the -helix A' of domain I and cause a mis-oriented N-terminal finger that could not correctly squeeze into the pocket of another monomer during dimerization, thus destabilizing the dimer structure. Several structural features essential for catalysis and substrate recognition are severely impaired in the G11A monomer. Moreover, domain III rotates dramatically against the chymotrypsin fold compared with the dimer, from which we proposed a putative dimerization model for SARS-CoV 3CL^pro. As the first reported monomer structure for SARS-CoV 3CL^pro, the crystal structure of G11A mutant might provide insight into the dimerization mechanism of the protease and supply direct structural evidence for the incompetence of the dissociated monomer.

Received for publication, June 26, 2007 , and in revised form, October 16, 2007.

The atomic coordinates and structure factors (code 2PWX) 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 by the State Key Program of Basic Research of China (Grants 2004CB58905, 2006AA09Z447, and 2007CB914304), the National Natural Science Foundation of China (Grants 30525024, 20472095, and 20721003), Sino-European Project on SARS Diagnostics and Antivirals (Proposal/Contract 003831), Shanghai Basic Research Project from the Shanghai Science and Technology Commission (Grants 06JC14080 and 03DZ19228), and the Foundation of the Chinese Academy of Sciences (Grant KSCX1-YW-R-18). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1-S3.

¹ These authors contributed equally to this work.

² To whom correspondence may be addressed. Tel./Fax: 86-21-5080-5873; E-mail: hljiang{at}mail.shcnc.ac.cn.

³ To whom correspondence may be addressed. Tel./Fax: 86-21-5080-6918; E-mail: xshen{at}mail.shcnc.ac.cn.

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