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

ABSTRACT 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.