Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses

Chuck, Chi-Pang; Chow, Hak-Fun; Wan, David Chi-Cheong; Wong, Kam-Bo

In general, the activities of 3CL pro correlate positively with the hydrophobicity and b-sheet propensity of substituting residues at P5 position ( Figure 2 ). The correlations are significant (p,0.05) for group 2a, 2b, and 3 CoVs, but are weaker for group 1 CoV. Like the P3 position, there is no substrate-binding pocket for P5 residue. In the crystal structure of SARS-CoV 3CL pro in complex with a peptide substrate, the P5 residue adopts an extended b-strand conformation to avoid clashing of P5-P6 residues with the protease [31] . Residues with high b-sheet propensity may stabilize the extended conformation at P5 and improve enzyme-substrate interaction. As shown in Figure 1 , a number of substitutions at P5 position resulted in a substrate better than the WT sequence (i.e. with relative activity .1). Consistent with the suggestion that P5 position favors residues with high hydrophobicity and b-sheet Figure 2 . Correlation between 3CL pro activities and structural properties of substituting residues. The relative protease activities of 3CL pro from HCoV-NL63 (shaded, group 1), HCoV-OC43 (white, group 2a), SARS-CoV (black, group 2b) and IBV (grey, group 3), were correlated with structural properties of substituting residue properties, including side chain volume [28] , hydrophobicity [29] and a-helix and b-sheet propensities [30] . propensity, Val-substitution consistently yielded substrates with higher than WT activities for all 3CL pro . On the other hand, negatively charged residues (Asp/Glu) were not favored at P5 position, with significantly lower activities (0.16 to 0.50).