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

The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, has spurred intense research into antiviral therapies. A key target for drug development is the viral main protease, 3CL Mpro, essential for processing viral polyproteins and thus viral replication. 3CL Mpro is a chymotrypsin-like protease that cleaves pp1a and pp1ab polyproteins at multiple sites. Its dependence for viral function and lack of human homolog make it an attractive drug target, despite the lack of FDA-approved inhibitors despite fifteen years of research. The protease comprises three domains: domains I and II (catalytic), and domain III (dimerization). Domain III is crucial for catalytic activity. Unlike many hydrolases, 3CL Mpro utilizes a Cys-His dyad (Cys145 and His41) within an active site cavity for catalysis. This study aimed to determine the atomic details of the SARS-CoV-2 3CL Mpro active site at room temperature to provide a physiologically relevant template for drug design and simulations.

Previous studies have extensively characterized the structure and function of SARS-CoV (and related coronaviruses) 3CL proteases. These studies, often employing low-temperature crystallography, have provided valuable insights into the active site and mechanisms of inhibition. However, the use of low temperatures may not accurately represent the dynamic behavior of the protease at physiological conditions. Therefore, a room-temperature structure was needed to improve the accuracy of drug design efforts.

The researchers cloned, expressed, and purified the SARS-CoV-2 3CL Mpro protein in E. coli. They employed a maltose binding protein (MBP) fusion for purification, followed by cleavage with PreScission protease. Crystallization was achieved using 0.1 M BIS-TRIS pH 6.5, 25% PEG3350. Room-temperature X-ray diffraction data were collected using a Rigaku High Flux HomeLab instrument, and the structure was determined by molecular replacement using a previously solved low-temperature structure as a template (PDB ID 6M03). Refinement was done using Phenix.refine and COOT. Molecular dynamics (MD) simulations (1 µs) were performed using GROMACS 2020 with the CHARMM36m force field to assess protein flexibility.

The room-temperature structure of ligand-free 3CL Mpro revealed a catalytic Cys145 Sγ-His41 Nε2 distance of 3.8 Å, too long for a hydrogen bond. A water molecule (H2Ocat) forms hydrogen bonds with His41, Asp187, and His164, potentially acting as a third catalytic residue. Comparison with low-temperature structures (e.g., PDB ID 6Y2E) showed conformational differences in residues 192-198, particularly a flipped peptide bond in Ala194 at room temperature, resembling inhibitor-bound conformations. Superposition with an inhibitor-bound structure (PDB ID 6LU7) highlighted significant active site plasticity upon ligand binding. The P2 helix (residues 46-50), P5 loop (residues 190-194), and C-terminal tail showed the greatest conformational changes and flexibility, consistent with MD simulation results. These findings suggest that the room-temperature structure provides a more physiologically relevant model for drug design and docking studies.

The observed structural plasticity of the 3CL Mpro active site, particularly at room temperature, has implications for drug design. The differences between room-temperature and low-temperature structures highlight the importance of considering conformational flexibility when screening and designing inhibitors. The identified flexible regions (P2 helix, P5 loop, C-terminal tail) may offer opportunities to design inhibitors that target specific conformations or exploit induced fit mechanisms. The involvement of H2Ocat in the catalytic mechanism suggests potential strategies for targeting this water molecule.

This study provides the first room-temperature X-ray crystal structure of SARS-CoV-2 3CL Mpro, revealing significant active site plasticity. The structure provides a more physiologically relevant template for drug design and molecular docking studies. Future research could focus on further investigation of the role of H2Ocat and exploring the interactions between different inhibitors and the flexible regions of the protease.

The study focuses on the ligand-free form of 3CL Mpro. Further studies incorporating various inhibitors are needed to fully understand the range of conformational changes upon binding. While MD simulations offer insights into flexibility, they have limitations in accurately representing all aspects of protein dynamics.