A novel auto-cleavage assay for studying mutational effects on the active site of severe acute respiratory syndrome coronavirus 3C-like protease.
The 3C-like protease (3CLpro) of severe acute respiratory syndrome (SARS) has been proposed as an attractive target for drug design. His41 and Cys145 were essential for the active site as the principal catalytic residues. In this study, we mutated the two sites, expressed four resulting mutants in Escherichia coli and characterized. All mutants showed undetectable activity in trans-cleavage assay. In addition, we introduced a 31-mer peptide containing an auto-cleavage site to the N-terminal of the proteases and found the peptide could be cleaved efficiently by 3CLsc itself, but, among the four mutants, only the mutant Cys145-->Ser showed residual activity as detected by the auto-cleavage assay. The data supported the proposition unequivocally that SARS-CoV 3CLpro was a member of serine proteases involving His41 and Cys145 residues at the active site. The auto-cleavage assay also provided a sensitive and reliable compensation to the traditional trans-cleavage assay.
Shan
Yu-Fei
YF
Institute of Biochemistry and Cell biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Li
Shou-Feng
SF
Xu
Gen-Jun
GJ
D001665:Binding Sites; D020134:Catalytic Domain; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics*
D017931:DNA Primers; D010455:Peptides; D014764:Viral Proteins; D012694:Serine; D006639:Histidine; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases