Structural Basis for the Acylation Reaction of Alphacoronavirus 3C-like Protease

Coronavirus 3C-like proteases (3CL^pro) are critical for viral replication and provide targets for antiviral drugs. Using the enteropathogenic alphacoronavirus porcine epidemic diarrhea virus (PEDV) as a model, we determined the crystal structure of an inactive PEDV 3CL^pro variant (C144A) in complex with a peptide of NF-κB essential modulator (NEMO_227-233). Structural characterization showed that the conformational change to PEDV 3CL^pro S1′ pocket conferred tolerance for nonconventional P1′-Val from a NEMO peptide substrate, indicating strong substrate accommodation. Using a combination of classical and quantum mechanics/molecular mechanics simulations, we explored the free-energy landscapes associated with the acylation step of PEDV 3CL^pro with regard to various substrates. The P1′ site plays a key role in the thermodynamics and kinetics of proteolysis, and the S1′ pocket might affect the free-energy cost of the acylation reaction. Our study provides structural insight into coronavirus 3CL^pro-mediated cleavage and will inform the development of anti-coronavirus drugs.