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

The protease activity of 3CL pro was measured by the FRET assay we developed previously [26] . Purified 3CL pro at 0.2 to 2 mM were mixed with 35 mM of the substrate protein in buffer A. Cleavage of the substrate protein leads to a decrease in fluorescence at 530 nm when the reaction mixture was excited at 430 nm. The fluorescence intensity, monitored by EnVision 2101 Multilabel Plate Reader, was fitted to single exponential [31, 32, 33] . The structure of WT substrate (magenta) is derived from crystal structure of SARS-CoV 3CL pro in complex with the autocleavage sequence (TSAVLQQSGFRKM) (PDB: 2Q6G) [31] . The structure of the A4P substrate variant (cyan) was modeled based on the crystal structure of IBV 3CL pro in complex with its own autocleavage sequence (PDB: 2Q6D) [31] . Note that strand-11 of IBV 3CL pro is positioned further away from P4 to P5 positions, resulting in a wider substrate-binding pocket. doi:10.1371/journal.pone.0027228.g004 decay to obtain the observed rate constant (k obs ). The protease activity against variant substrates was normalized against the WT activity to yield the relative activity. The assay was repeated in triplicate.