Profiling of substrate specificity of SARS-CoV 3CL.
BACKGROUND
The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.


RESULTS
To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.


CONCLUSIONS
Our results demonstrated a strong structure-activity relationship between the 3CL(pro) and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.
Chuck
Chi-Pang
CP
School of Biochemical Sciences, The Chinese University of Hong Kong, Sha Tin, Hong Kong, China.
Chong
Lin-Tat
LT
Chen
Chao
C
Chow
Hak-Fun
HF
Wan
David Chi-Cheong
DC
Wong
Kam-Bo
KB
D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases