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

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. Author summaryThe nucleocapsid (N) protein of the SARS-CoV-2 virus plays an essential role in virus particle assembly as it specifically binds to and wraps the virus genomic RNA into a well-organized structure known as the ribonucleoprotein. Understanding how the N protein wraps around the virus RNA is critical for the development of strategies to inhibit virus assembly within host cells. One of the limitations regarding the molecular structure of the ribonucleoprotein, however, is that the N protein has several unstructured and mobile regions that preclude the resolution of its full atomic structure. Moreover, the N protein can form higher-order oligomers, both in the presence and absence of RNA. Here we employed computational methods, supported by experimental data, to simulate the N protein structural dynamics in the absence and presence of RNA. Our data suggest that the N protein forms structurally dynamic dimers in the absence of RNA, with its structured N- and C-terminal domains oriented in extended conformations. In the presence of RNA, however, the N protein assumes a more compact conformation. Our model for the oligomeric structure of the N protein bound to RNA helps to understand how N protein dimers interact to each other to form the ribonucleoprotein.