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1、Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure Tiancen Hu a,1, Yu Zhanga,1, Lianwei Lia, Kuifeng Wanga, Shuai Chena, Jing Chena, Jianping Dingb, Hualiang Jiang a, Xu Shena, a Drug Discovery and Design Cen
2、ter, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China b State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chi
3、nese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China a b s t r a c ta r t i c l ei n f o Article history: Received 6 February 2009 Returned to author for revision 6 March 2009 Accepted 30 March 2009 Keywords: SARS coronavirus 3C-like protease Dimerization Enzymatic activity Mutation Mo
4、nomer The 3C-like protease of SARS coronavirus (SARS-CoV 3CLpro) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface
5、resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CLpro. S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still
6、 retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-refl ecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structur
7、al analysis. Our work is expected to provide insight on the dimerizationfunction relationship of SARS-CoV 3CLpro. 2009 Elsevier Inc. All rights reserved. Introduction Severe acute respiratory syndrome (SARS) (Stockman et al., 2006) is a highly infectious disease that broke out from November 2002 to
8、July 2003, and it caused a lot of infection cases and deaths (Stockman et al., 2006). SARS coronavirus (SARS-CoV) is responsible for SARS disease (Drosten et al., 2003a, 2003b; Fouchier et al., 2004; Ksiazek et al., 2003; Peiris et al., 2003). The genome of SARS-CoV contains 14 functional open readi
9、ng frames (ORFs) (Thiel et al., 2003). The two large 5-terminal ORFs, 1a and 1b encode two overlapping poly- proteins, pp1a and pp1ab, which have to be cleaved extensively to produce proteins necessary for viral RNA synthesis and genome replication (Anand et al., 2003; Thiel et al., 2003; Ziebuhr et
10、 al., 2000). Such proteolytic processing is performed by two viral proteases, the papain-like cysteine protease (PL2pro) and the chymotrypsin-like protease called 3C-like protease (3CLpro) due toits distantrelationship with the 3C proteases of picornaviruses (Ziebuhr et al., 2000). While PL2prohas e
11、xtra functions like deubiquitination (Barretto et al., 2005, 2006; Lindner et al., 2005, 2007; Ratia et al., 2006; Sulea et al., 2005) and antagonizing type I interferon to counteract innate immunity (Devaraj et al., 2007), 3CLproplays a major role in processing viral polyproteins and controlling th
12、e activities of replication complexes, thus is also called the main protease (Mpro). Meanwhile, it has been also shown to induce mitochondrial-mediated apoptosis (Lai et al., 2007; Lin et al., 2006). Therefore, for its functional indispensability in viral life cycle, SARS-CoV 3CLprohas become an att
13、ractive target for discovering new anti-SARS agents. In crystal structure, SARS-CoV 3CLproforms a dimer with two monomers oriented perpendicular to one another (Yang et al., 2003), similar to the TGEV and HCoV 3CLprostructures (Anand et al., 2002, 2003), in which each monomer contains three domains.
14、 Domains I and II (residues 8101 and residues 102184) each fold into an antiparallel -barrel and together form a chymotrypsin fold respon- sible for catalysis. The substrate-binding site is located in a cleft between these two domains. Domain III (residues 201306) is a globular cluster of fi ve anti
15、parallel -helices connected to the chymotrypsin fold by a long loop (residues 185200) (Anand et al., Virology 388 (2009) 324334 Abbreviations: SARS-CoV, severe acute respiratory syndrome coronavirus; 3CLpro, 3C-like protease; RMSD, root-mean-square deviation; MES, 2-(N-morpholino)ethane- sulfonic ac
16、id; PEG, polyethylene glycol; DTT, dithiothreitol; DMSO, dimethyl sulph- oxide; EDTA, ethylene diaminetetraacetic acid; IPTG, isopropyl -D-thiogalactoside. Coordinates and structure factors of the two SARS-CoV 3CLpromutants have been deposited in the Protein Data Bank with accession number of 3F9E for S139A, and 3F9F, 3F9G, and 3F9H for F140A. Corresponding authors. Fax: +86 21 50806918. E-mail addresses: (H. Jiang), (X. Shen). 1 These two authors contributed equally to this work. 0042-682
