Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development

The SARS-CoV-2 non-structural protein 14 (NSP14) is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both enzymatic activities appear to be essential for the viral life cycle and thus may be targeted for anti-viral therapeutics. NSP14 forms a stable complex with the SARS-CoV-2 zinc binding protein NSP10, and this interaction greatly enhances the nuclease but not the methyltransferase activity. In this study, we have determined the crystal structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons of this structure with the structure of NSP14/NSP10 complexes solved to date reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including significant movements and helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. Conformational changes are also seen in the MTase active site within a SAM/SAH interacting loop that plays a key role in viral mRNA capping. We have also determined the structure of NSP14 in complex with cap analogue ^7MeGpppG, offering new insights into MTase enzymatic activity. We have used our high resolution crystals to perform X-ray fragment screening of NSP14, revealing 72 hits bound to potential sites of inhibition of the ExoN and MTase domains. These structures serve as excellent starting point tools for structure guided development and optimization of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.