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

This study provides the first comprehensive profiling of substrate specificities of 3CL pro from group 1, 2a, and 3 CoVs. We showed that the substrate specificities of these 3CL pro share many similarities to those of 3CL pro from SARS-CoV (group 2b) reported previously by us [26] . Table 2 summarizes the substrate specificities that are common to all 3CL pro . Although the substrate specificities for 3CL pro from different groups of CoVs share a number of similarities, unique substrate preferences were identified in this study. In particular, we showed that only IBV 3CL pro , but not other proteases, prefers P4-Pro (Figure 3 ). To understand the structural basis of this unique substrate preference, we compared the structures of IBV 3CL pro with other coronaviral 3CL pro . We noticed that strand-11 of IBV 3CL pro is positioned further away from the P4 and P5 substrate-binding site compared to other 3CL pro (Figure 4) [31, 32, 33] . This results in a wider substrate-binding pocket in IBV 3CL pro . We further docked the substrate variant A4P into the substrate-binding pocket of IBV 3CL pro . Due to the cyclic structure of Pro residue, the backbone Ø dihedral angle of the P4 residue is restrained to ca. 260u, which causes the substrate peptide to bend towards the strand-11 of 3CL pro . Such conformation of substrate is much better accommodated by IBV 3CL pro , which has a wider substrate-binding pocket near the P4 and P5 positions. This observation justifies why only IBV 3CL pro cleaves P4-Pro efficiently.