Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography

Abstract

The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL M^pro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL M^pro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

Fig. 1. The three-dimensional structure of 3CL M^pro from SARS-CoV-2.

a One monomer of the dimer is shown as an orange cartoon, while the other monomer is shown as a teal surface with the catalytic site cavity highlighted with water molecules shown as red spheres. b A closeup view of the catalytic site cavity in which the catalytic residues (Cys145 and His41) are highlighted in purple with the residues that flank the cavity highlighted in green with water molecules shown as red spheres.

Fig. 2. The catalytic site of 3CL M^pro from SARS-CoV-2.

Hydrogen bonds are shown as blue dashed lines; the distance between Cys145 and His41 is shown as a black dotted line, the dashed red line indicates a strong C–H…O bond. The 2F_O – F_C electron density map contoured at 1.6 σ level is shown as a violet mesh. All distances are given in Ångstroms.

Fig. 3. Comparison of room-temperature and low-temperature structures.

a A superposition of our room temperature ligand-free structure of 3CL M^pro (magenta) with the ligand-free structure of 3CL M^pro (PDB ID 6Y2E) obtained at 100 K (cyan). b Residues 192–198 in the P5 binding pocket differ in conformation between the room temperature and 100 K structures.

Fig. 4. Comparison of the active site geometries in the ligand-free and ligand-bound structures.

Superposition of the room temperature ligand-free structure of 3CL M^pro (green carbon atoms) with the structure of 3CL M^pro in complex with inhibitor N3 (deep purple, PDB ID 6LU7) from SARS-CoV-2. Upon inhibitor binding, residues Met49, Leu50, and Met165 change their conformations (curved black arrows), whereas the small helix with residues 46–50 and the β-hairpin loop with residues 166–170 move apart, resulting in the loop with residues 190–194 which accommodates the inhibitor’s P5 substituent to shift closer to the β-hairpin loop. All distances are given in Ångstroms.