Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

Abstract

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor

“…In addition, we show that CA inhibited SARS-CoV-2 replication in human lung cells, further suggesting that it may influence N protein-mediated RNA packing in vivo. The latter observation is in line with recently reported models of N protein binding RNA 33 , our 1 H 15 N-HSQC NMR experiments and the N protein crystal structure binding CA, suggesting an allosteric path for N protein RNA binding inhibition by CA. Taken together, our data provide the first evidence of pharmacologically targeting the SARS-CoV-2 N protein with small molecules, thus paving the way for the rational design of new N protein modulators.…”

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor

“…Notably, the binding of CA to the CTD not only alters the topology but also the charge distribution of this region, which might explain why CA disrupts the N protein-RNA1 complex in solution. This hypothesis is additionally supported by a structural model of the SARS-CoV-2 N protein complexed with an RNA molecule, which we have recently reported 40 . This structural model presupposes two possibilities: one in which the CA-binding site fully overlaps with the RNA interaction site (Fig.…”

“…The CTD structure in its apo form shows an electrostatic potential distribution that is conserved amongst all N proteins from the Coronaviridae family 21,37 , with a major positively charged groove located on one side of the dimer surface (Figure 5A). This region, which is composed by Lys256, Lys257, Lys259 Lys261 and Arg262, is thought to contribute to RNA binding [38][39][40] . We observed that the positively charged groove extended towards the CA-binding pocket through Arg259, Arg276, Arg277 and Arg293 (Figure 5B).…”

Structural Dynamics of the SARS-CoV-2 Spike Protein: A 2-Year Retrospective Analysis of SARS-CoV-2 Variants (from Alpha to Omicron) Reveals an Early Divergence between Conserved and Variable Epitopes

“…SARS-CoV-2 evolution has been extensively studied by computational approaches [44,[57][58][59][60][61][62]. Our retrospective analysis of SARS-CoV-2 variants revealed an early divergence between conserved (ADE) and variable (neutralizing) epitopes which is based on their localization on the spike protein.…”