ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. In contrast, we found that only the MERS-CoV Mac1 protein bound to poly-ADP-ribose (PAR), and none of these enzymes could hydrolyze PAR. Importantly, using ADP-ribose detecting antibodies and both gel-based assay and novel ELISA assays, we demonstrated highly efficient de-MARylating activity for all 3 CoV Mac1 proteins. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are highly efficient MAR-hydrolases with strikingly similar activity, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.
IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused nearly 350 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We added assays describing the PAR binding and hydrolysis of CoV macrodomains - Fig. 5 and 6 were modified. We also established an ELISA assay to test for deMARylation - added Fig 7. Supplemental files were updated. Paper was modified accordingly.