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

At the time of writing, we discovered that Michalska et al. of the Center for Structural Genomics of Infectious Diseases (CSGID) deposited coordinates for a very similar construct of the SARS-CoV-2 macro X domain including from E206 to E275 of the nsp3 protein, plus an additional 4 residues at the N-terminus (6VSX; unpublished). Their crystals also allowed binding of ADP-ribose (6W02) and AMP (6W6Y), while ours seemed to be packed too tightly to permit ligands to access the binding site (data not shown). We compared our ultra-high-resolution model of the unliganded protein to the ADP-ribosebound form. The RMSD values for the fitting, done by secondary structure matching (SSM) 12 as implemented in COOT, is 0.59 Å for 165 of 172 Cα atoms. This is very similar to the RMSD values of the free protein (6VXS; 0.66 Å) and the AMP-bound form (6W6Y; 0.50 Å), indicating that there are no large conformational changes that occur upon ligand binding. In fact, the only notable conformational changes occur in three surface-exposed loops in or near the ligand-binding pocket (Fig. 4C) . These loops connect strand β2 with helix α2 (the β2-α2 loop), strand β4 with helix α4 (β4-α4 loop), and strand β5 with helix α5 (β5-α5 loop). The subtle change in conformation of the β4-α4 loop (purple in Fig. 4C ) appears to be the result of crystal contacts and not the direct influence of ADP-ribose binding. The other two loops are more intimately involved in ligand binding. The main chain of the α2-β2 loop rotates 180° in order to allow the amide N atom of G252 to participate in a hydrogen bonding interaction with the 1'-hydroxyl of the ribose moiety of ADPribose. This loop also carries N244, which directly interacts with the ribose. The phenyl ring of F336 in the β5-α5 loop occupies the portion of the binding pocket in the unliganded structure that is occupied by the β-phosphate of ADP-ribose. Thus, without a rearrangement of the β5-α5 loop, ADP-ribose would not be able to bind.