Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA

Forsythe, Heather M.; Galvan, Joaquin Rodriguez; Yu, Zhen; Pinckney, Seth; Reardon, Patrick; Cooley, Richard B.; Zhu, Phillip; Rolland, Amber D.; Prell, James S.; Barbar, Elisar

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the SARS-CoV-2 infection cycle: it protects and packages viral RNA in nucleocapsid assembly, interacts with the inner domain of spike protein in virion assembly, binds to structural membrane protein M during virion packaging and maturation, and binds 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 micro molar, but can also form transient interactions with other N proteins facilitating larger oligomers. NMR studies on the ∼100kDa dimeric protein identify a specific domain that binds 1-1000 RNA and show that the N/RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multi-angle 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.