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

Nucleotide Binding by the SARS-CoV-2 Macro X domain-Repeated ITC experiments revealed that the purified recombinant protein bound ADP-ribose ( Fig. 3C ) with a dissociation constant of 10 ± 4 µM (uncertainty is the standard deviation of Kd's from independent titrations). To examine binding specificity, similar titrations were repeated with related nucleotides. The SARS-CoV-2 protein bound ADP, cAMP, ATP and ADP-glucose (Fig. 3D ). All nucleotides lacking the ribose moiety bound with similar high affinities, but none bound with an enthalpy change like that seen with ADP-ribose, suggesting specific contacts form between ADP ribose the SARS-CoV-2 protein. Based on what is seen in the SARS-CoV-2 structures below, these contacts likely occur with the conserved D226 and N244 (positions 30 and 43 in the numbering above the alignment in Fig. 2) . None of the other nucleotides bind with an entropic penalty as seen with ADP-ribose, either, suggesting that the ribose moiety becomes structured when bound to the macrodomain.