Description

The novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus 2019 (COVID-19) epidemic that continues to persist. The viral main protease (Mpro) enzyme plays an important role in mediating replication and transcription of SARS-CoV-2, and as such, is an important drug target that has been studied extensively. The main protease becomes catalytically active when autoprocessing sites at its own N- and C-termini are cleaved, so inhibition of autoprocessing would prevent catalytic activity. In order to gain further insight into the mechanism of autoprocessing by Mpro and possibly suggest novel inhibitory mechanisms, a Glutamine (Q) to Lysine (K) mutation was introduced at amino acid position 306. This prevents autoprocessing, keeping the main protease in its proenzyme form. We then determined the high-resolution structure of the SARS-CoV-2 Mpro proenzyme by x-ray crystallography. X-Ray crystallography was the method used to obtain the structure of the SARS-CoV-2 Mpro, which involves multiple steps. Single crystals were grown using the hanging drop vapor diffusion method, over an optimized reservoir solution containing Tacsimate at pH 6.0. Single crystals were cryo-cooled and shipped to the Stanford Synchrotron Radiation Lightsource, where x-ray diffraction data were collected. The diffraction images were processed in XDS. The phase problem was solved via molecular replacement in Phaser, and model building and refinement were performed using WinCoot and Phenix. The structure of the SARS-CoV-2 Mpro proenzyme form was refined against data at 1.29 Å resolution. Electron density at the N-terminus of the protein confirmed that it was still in the proenzyme form. The crystallographic model of the proenzyme can now be compared against the active form, and it can be further used in studies such as inhibition assays to identify potential drugs that could effectively reduce the symptoms of COVID-19.