The dimerization domain of SARS-CoV-2 nucleocapsid protein is partially disordered and forms a dynamic high-affinity dimer.

The SARS-CoV-2 nucleocapsid (N) drives the compaction and packaging of the viral genome. Here, we focused on quantifying the mechanisms that control dimer formation utilizing single-molecule Förster resonance energy transfer to investigate the conformations and energetics of the dimerization domain in the context of the full-length protein. Under monomeric conditions, we observed significantly expanded configurations of the dimerization domain (compared to the folded dimer structure), which is consistent with a dynamic conformational ensemble. The addition of unlabeled protein stabilizes a folded dimer configuration with a high mean transfer efficiency, which is in agreement with predictions based on known structures. Dimerization is characterized by a dissociation constant of ~12 nM at 23°C and is driven by strong enthalpic interactions between the two protein subunits, which originate from the coupled folding and binding. We propose that the retained flexibility of the dimer can affect its interaction with RNA and phase separation propensity.