Fig. 2: The WU-04-resistant mutation M49K perturbs the substrate-binding pocket of SARS-CoV-2 3CLpro.

a–d Alignment of the crystal structures of the WU-04-resistant SARS-CoV-2 3CLpro mutants M49K (a), M49K/M165V (b), M165V (c), and S301P (d) with the crystal structure of the WT 3CLpro (PDB code: 6M03). e Alignment of the crystal structure of the double mutant M49K/S301P with that of the WT 3CLpro in the post-cleavage state (PDB code: 7E5X). The C-terminal tail from another molecule of 3CLpro in the M49K/S301P structure and that in the WT 3CLpro structure were shown as sticks and colored magentas and yellow, respectively. f Alignment of the crystal structure of the double mutant M49K/S301P in complex with WU-04 with the crystal structure of the WT 3CLpro in complex with WU-04 (PDB code: 7EN8). WU-04 in the M49K/S301P structure and that in the WT 3CLpro structure are colored cyan and yellow, respectively. Three regions in the WT 3CLpro, including a short helix where M49 is located (residues 45–51), a short loop (residues 167–171), and the linker connecting domains II and III of 3CLpro (residues 187–196), are colored brown in the crystal structure. Each alignment was carried out by aligning the structure of one protomer of each 3CLpro mutant with the structure of one protomer of the WT 3CLpro using the “align” command in PyMol.