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 nonstructural protein
13 (nsp13) stands out as a highly conserved and yet understudied 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, which presents 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 a 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 structural states of the ATP-dependent RNA translocation mechanism.
Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent
populations and support the inchworm mechanism for translocation.
These four states are characterized by different RNA-binding poses
for motifs Ia, IV, and V and
suggest a power stroke-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.