Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics. Nsp15 is a uridine specific endoribonuclease present in all coronaviruses. Here, the authors determine the cryo-EM structures of SARS-CoV-2 Nsp15 in the apo and UTP-bound states, which together with biochemical experiments, mass spectrometry and molecular dynamics simulations provide insights into the catalytic mechanism of Nsp15 and its conformational dynamics.

Automated summary

Nsp15 is a uridine specific endoribonuclease present in all coronaviruses. The cryo-EM structures of SARS-CoV-2 Nsp15 in the apo and UTP-bound states, together with biochemical experiments, mass spectrometry, and molecular dynamics simulations, provide insights into the catalytic mechanism of Nsp15 and its conformational dynamics.