Molecular Characterization of SARS-CoV-2 N Protein Interfaces: Implications for Oligomerization, RNA Binding, and Phase Separation

The SARS-CoV-2 nucleocapsid (N) protein is central to genomic RNA recognition, condensation, and packaging, yet the molecular organization of its multivalent N-N and N-RNA interaction network involved in this process remains unclear. Here, we define the oligomerization and RNA-binding interfaces of the C-terminal domain (CTD) and its flanking intrinsically disordered regions (IDRs), the leucine-rich helix (LH) and the C-terminal IDR (C-IDR), using size-exclusion chromatography (SEC), cross-linking, mutational studies and NMR spectroscopy. We identify discrete oligomerization interfaces within the CTD and C-IDR that drive higher-order assembly, and show, through liquid-liquid phase separation (LLPS) and electron microscopy (EM), that C-IDR residues are essential for RNA-induced condensate formation. Moreover, the mapping of RNA-binding residues highlights Arg277 as a conserved determinant of CTD-RNA recognition. Notably, the two IDRs exert opposing regulatory effects on RNA binding, with the C-IDR enhancing and the LH attenuating CTD-RNA interactions. Together, these findings reveal how cooperative interfaces between the CTD and its flanking IDRs orchestrate N-protein oligomerization and RNA condensate formation and highlight potential intervention sites for disrupting SARS-CoV-2 ribonucleoprotein assembly.