Variable Macro X Domain of SARS-CoV-2 Retains the Ability to Bind ADP-ribose

David N. Frick; Rajdeep S. Virdi; Nemanja Vuksanovic; Narayan Dahal; Nicholas R Silvaggi

In contrast to the well-conserved SARS nsp5 protease, nsp12 polymerase and nsp13 helicase enzymes, significantly more differences exist between the nsp3 proteins encoded by SARS-CoV-1 and SARS-CoV-2. The most variation occurs in a domain of Nsp3 domain suspected to bind ADP-ribose, that will be referred to here as the "macro X" domain, to differentiate it from the two downstream "SARS unique macrodomains (SUDs)," which do not bind ADP-ribose. 3 The macro X domain of SARS-CoV-1 is also able to catalyze the hydrolysis of ADPribose 1'' phosphate, albeit at a slow rate. 4 All the above differences preclude the use of the SARS-CoV-1 macro X domain structures as scaffolds to design compounds that might target this nsp3 region in SARS CoV-2, especially in light of the observation that the same nsp3 domain from gamma coronaviruses does not bind ADP-ribose in vitro. 5 Because ADP-ribose binding is a property that could be used to identify possible antiviral agents, the ability of the SARS-CoV-2 macro X domain to bind ADP-ribose was examined using a recombinant purified protein and isothermal titration calorimetry (ITC). We also determined the structure of the SARS-CoV-2 macro X domain in order to examine the biochemical context of ADP-ribose binding and to provide data for rational inhibitor design or in silico screening.