The substrate specificity of SARS coronavirus 3C-like proteinase

Fan, Keqiang; Ma, Liang; Han, Xiaofeng; Liang, Huanhuan; Wei, Ping; Liu, Ying; Lai, Luhua

doi:10.1016/j.bbrc.2005.02.061

Cited by 90 publications

(124 citation statements)

References 21 publications

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“…In contrast, the activity of the adenovirus proteinase in the absence of a nucleic acid cofactor is very insensitive to ionic strength. It has been postulated that there may be a salt bridge between Glu166 in the SARS CoV main proteinase and the P3 amino acid in the substrate if it is positively charged [25]. Perhaps this is why enzyme activity is so sensitive to ionic strength.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic activity of the SARS coronavirus main proteinase dimer

et al. 2006

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The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH) 2 -Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K m , and is unusually sensitive to ionic strength and reducing agents.

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Section: Discussionmentioning

confidence: 99%

Enzymatic activity of the SARS coronavirus main proteinase dimer

et al. 2006

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“…In addition, SARS-CoV 3CL pro confers absolute specificity for Gln at the P1 position of the peptide substrate (29,40), which requires that the S1 subsite of the substrate-binding pocket should retain a correct conformation to accommodate Gln-P1. The damage of the S1 subsite is likely to entirely abolish the enzymatic activity, as observed in the crystal structure of the inactive protomer obtained at pH 6.0 (PDB code: 1UJ1_B) (5).…”

Section: Discussionmentioning

confidence: 99%

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

Chen

Zhang

et al. 2008

Journal of Biological Chemistry

135

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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.

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“…Here, Q/E represents the mutation of Gln in the extra 8-amino acid peptide (SITSAVLQ) to Glu. Mutation of the critical residue Gln at the P1 position to Glu completely abolishes the hydrolytic activity of SARS 3CL pro (18). 3CLP-Y-His is the truncated protein of His-C-XX(Q/E)-3CLP-Y, which also had a His 6 tag at the C terminus for further protein purification (supplemental Fig.…”

Section: Plasmid Construction Of the Large Fusion Proteins His-c-xx(qmentioning

confidence: 99%

“…For SARS 3CL pro , much has been learned about its catalytic mechanism (17), substrate specificity (18), as well as inhibitor design (19,20). However, few studies have been reported on its maturation mechanism.…”

mentioning

confidence: 99%

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

Teng

et al. 2010

Journal of Biological Chemistry

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The 3C-like proteinase (3CL pro ) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL pro is active. However, as the internally encoded 3CL pro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL pro (XX), SARS 3CL pro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL pro using fluorescence resonance energy transfer. In contrast to the matured 3CL pro , the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL pro in the polyprotein, and a modified model for the 3CL pro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.

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The substrate specificity of SARS coronavirus 3C-like proteinase

Cited by 90 publications

References 21 publications

Enzymatic activity of the SARS coronavirus main proteinase dimer

Enzymatic activity of the SARS coronavirus main proteinase dimer

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

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

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