Abstract: bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442358; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 1 2 3 Vitamin C Binds to SARS Coronavirus-2 Main Protease Essential for Viral Replication 4 5 Tek Narsingh Malla1&, Suraj Pandey1&, Luis Aldama2, Dennisse Feliz2, 3, Moraima Noda2, 6 Ishwor Poudyal1, George N. Phillips Jr.4, Emina A. Stojković2*, Marius Schmidt1* 7 8 1 Department of Physics, University of Wisconsin Milwaukee, Milwaukee, WI 53211 USA 9 2 Department of Biology, Northeastern Illinois University, Chicago, IL 60625 USA 10 3 Department of Chemistry, Northeastern Illinois University, Chicago, IL 60625 USA 11 4 Department of BioSciences, Rice University, Houston, TX 77005 USA 12 & 13 *corresponding authors contributed equally 14 15 Abstract 16 There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS- 17 Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity 18 of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as 19 main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing 20 of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in 21 humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in 22 complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we 23 report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the protein's 24 active site at 2.5 Ångstrom resolution. The crystal structure of the Vitamin C 3CLpro complex 25 may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but 26 on related proteases of other infectious viruses. 27 28 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442358; this version posted May 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2 29 a ASC FTE His41 His163 Cys145 subunit A subunit B b FTE c ASC DTT His41 Cys145 Cys145 His163 His41 His163 Figure 1. Vitamin C and DTT bound in the active site of SARS CoV-2 3CLpro. (a) The 3CLpro dimer in the asymmetric unit of the orthorhombic crystals soaked with L-ascorbate. Polder difference electron density (Liebschner et al., 2017) in the active sites is shown in green (contour: 2.5 sigma). (b) The L-ascorbate (ASC) in subunit B. The ASC interacts with the catalytic Cys-145 and is stabilized by a hydrogen bond to His-163. A trifluoroethanol (FTE) is located close to the ASC. (c) Dithiothreitol (DTT) is observed in monoclinic (C2) crystal form. It does not bind to Cys-41. It rather binds to His-41 forming a sulfenamide. Difference electron density maps shown in (a) - (c) are obtained after refining the 3CLpro without the addition of ligand. The ascorbate and DTT explain the additional electron density. 30 The Covid-19 pandemic, caused by a novel severe acute respiratory syndrome (SARS) 31 coronavirus 2, has paralyzed public life..