Single-molecule spectroscopy of the SARS-CoV-2 nucleocapsid protein.

Abstract

The SARS-CoV-2 nucleocapsid (N) protein plays a variety of roles in the viral life cycle including replication, transcription, and genome packaging. N protein contains two folded domains - the RNA-binding domain and the dimerization domain- and three intrinsically disordered regions located at the N and C terminals and in the linker between the folded domains. Here, we harnessed single-molecule spectroscopy and all atom simulations to investigate the conformational ensemble of the protein and its protein-protein and protein-RNA interactions. Single-molecule Foerster Resonance Energy Transfer (FRET) and nanosecond Fluorescence Correlation Spectroscopy (ns-FCS) experiments support that all three disordered regions are flexible and dynamic in the context of the full-length protein. The estimated conformations are in good agreement with all-atom simulations, which reveal transient helicity regions that may act as local binding interfaces for protein-protein or protein-RNA interactions. We further investigate the conformations of the dimerization domain and its dimerization affinity. We have found that the dimerization domain adopts more expanded configurations in its monomeric form, folds upon dimerization, and has a strong dimerization affinity (Kdim = 30 ± 6 nM). Finally, we have started to investigate how disordered regions contribute to N protein binding of RNA. We focused on the interactions with the RNA-binding domain (RBD) and the N-terminal domain (NTD) tail. Our results indicate that the NTD-RBD region has a preferential interaction with single-stranded RNA, forms a dynamic complex with the nucleic acid, and increases the binding affinity of RBD by about 50-fold. Overall, our data support a model where disordered regions are directly involved in modulating intra- and inter-molecular interactions of the protein.