ABSTRACT
The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.
Subject(s)
COVID-19/virology , Nucleic Acids/metabolism , Nucleocapsid Proteins/metabolism , Protein Interaction Domains and Motifs , SARS-CoV-2/physiology , Binding Sites , DNA/chemistry , DNA/metabolism , Gene Expression Regulation, Viral , Host-Pathogen Interactions , Humans , Hydrogen Bonding , Models, Molecular , Nucleic Acids/chemistry , Nucleocapsid Proteins/chemistry , Protein Binding , RNA/chemistry , RNA/metabolism , Spectrum Analysis , Structure-Activity RelationshipABSTRACT
The nucleocapsid (N) protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. The N protein N-terminal domain (N-NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRSs), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of the severe acute respiratory syndrome coronavirus 2 N-NTD to nonspecific (NS) and TRS dsRNAs. We probed dsRNAs' Watson-Crick basepairing 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 dsTRSs than dsNS. N-NTD dynamics, especially a tweezer-like motion of ß2-ß3 and Δ2-ß5 loops, seems to play a key role in Watson-Crick basepairing 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: 1) 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; and 2) monomeric N-NTD would be active, opening up the possibility of a regulatory dissociation event.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleocapsid Proteins/genetics , Nucleoproteins , RNAABSTRACT
During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid protein is the most abundant in infected cells. It is a multifunctional phosphoprotein involved in the capsid formation, in the modulation and regulation of the viral life cycle. The N-terminal domain of N protein specifically interacts with transcriptional regulatory sequence (TRS) and is involved in the discontinuous transcription through the melting activity of double-stranded TRS (dsTRS).