Abstract
In response to the emergence of COVID-19, caused by SARS-CoV-2, there has been a growing interest in understanding the functional mechanisms of the viral proteins to aid in the development of new therapeutics. Non-structural protein 13 (Nsp13) helicase is an attractive target for antivirals because it is essential for viral replication and has a low mutation rate; yet, the structural mechanisms by which this enzyme binds and hydrolyzes ATP to cause unidirectional RNA translocation remain elusive. Using Gaussian accelerated molecular dynamics (GaMD), we generated a comprehensive conformational ensemble of all substrate states along the ATP-dependent cycle. ShapeGMM clustering of the protein yields four protein conformations that describe an opening and closing of both the ATP pocket and RNA cleft. This opening and closing is achieved through a combination of conformational selection and induction along the ATP cycle. Furthermore, three protein-RNA conformations are observed that implicate motifs Ia, IV, and V as playing a pivotal role in an ATP-dependent inchworm translocation mechanism. Finally, based on a linear discriminant analysis of protein conformations, we identify L405 as a pivotal residue for the opening and closing mechanism and propose a L405D mutation as a way of testing our proposed mechanism. This research enhances our understanding of nsp13's role in viral replication and could contribute to the development of antiviral strategies.