Dynamics of the SARS-CoV-2 nucleoprotein N-terminal domain triggers RNA duplex destabilization

Caruso, Ícaro P.; Sanches, Karoline; Da Poian, Andrea T.; Pinheiro, Anderson S.; Almeida, Fabio C.L.

The nucleocapsid (N) protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. N protein N-terminal domain (N-NTD) interacts and melts the double stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs’ Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, triggering melting initiation. dsRNA destabilization driven by N-NTD was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of β2-β3 and α2-β5 loops, seems to play a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTD arms of dimeric N would bind to two different RNA sites, either closely or spatially spaced in the viral genome, in a cooperative manner; (ii) monomeric N-NTD would be active, opening up the possibility of a regulatory dissociation event.