The Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase

The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but non-structural protein 13 (nsp13) stands out as a highly conserved and yet under studied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound presenting a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 μs of Gaussian accelerated molecular dynamics simulation (at least 6 μs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key aspects of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inch-worm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV and V and suggest a powerstroke–like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.