The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.
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