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Journal of Biochemistry Advance Access originally published online on January 7, 2008
Journal of Biochemistry 2008 143(4):525-536; doi:10.1093/jb/mvm246
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© 2008 The Japanese Biochemical Society.

Residues on the Dimer Interface of SARS Coronavirus 3C-like Protease: Dimer Stability Characterization and Enzyme Catalytic Activity Analysis

Shuai Chen, Jian Zhang, Tiancen Hu, Kaixian Chen, Hualiang Jiang{dagger} and Xu Shen*

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China

*To whom correspondence should be addressed. Tel: +86-21-50806918, Fax: +86-21-50806918, E-mail: xshen{at}mail.shcnc.ac.cn

{dagger}Correspondence may also be addressed: Tel: +86-21-50805873, Fax: +86-21-50807088, E-mail: hljiang{at}mail.shcnc.ac.cn

Received August 26, 2007; Accepted December 18, 2007


  Abstract

3C-like protease (3CLpro) plays pivotal roles in the life cycle of severe acute respiratory syndrome coronavirus (SARS-CoV) and only the dimeric protease is proposed as the functional form. Guided by the crystal structure and molecular dynamics simulations, we performed systematic mutation analyses to identify residues critical for 3CLpro dimerization and activity in this study. Seven residues on the dimer interface were selected for evaluating their contributions to dimer stability and catalytic activity by biophysical and biochemical methods. These residues are involved in dimerization through hydrogen bonding and broadly located in the N-terminal finger, the {alpha}-helix A' of domain I, and the oxyanion loop near the S1 substrate-binding subsite in domain II. We revealed that all seven single mutated proteases still have the dimeric species but the monomer–dimer equilibria of these mutants vary from each other, implying that these residues might contribute differently to the dimer stability. Such a conclusion could be further verified by the results that the proteolytic activities of these mutants also decrease to varying degrees. The present study would help us better understand the dimerization-activity relationship of SARS-CoV 3CLpro and afford potential information for designing anti-viral compounds targeting the dimer interface of the protease.

Key Words: catalytic mechanism, dimerization-activity relationship, dimer interface, residue–residue interactions, site-directed mutagenesis

Abbreviations: 3Clpro, 3C-like protease; CD, circular dichroism; Dabcyl, 4-[[4-(dimethylamino) phenyl] azo] benzoic acid; EDANS, 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid; FRET, fluorescence resonance energy transfer; SARS-CoV, severe acute respiratory syndrome coronavirus; SEC, size-exclusion chromatography; WT, wild type


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