SARS-CoV-2 nucleocapsid (N)-protein is a key component of the SARS-CoV-2 virus and plays a critical role in viral replication by binding to the viral RNA to form a complex that is essential for viral replication and transcription. N-protein is composed of an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a linker region. The NTD is responsible for binding to the viral RNA and is conserved among corona viruses, and the CTD is involved in oligomerization. N-protein exists as a dimer in solution, at concentrations >50nM, although there is some controversy over the exact oligomerization state when bound to RNA or DNA. Here, we use isothermal titration calorimetry (ITC) and sedimentation velocity (SV) to examine N-protein binding to oligo(U)n and oligo(dT)n as a function of length and examine the binding stoichiometries and hydrodynamic properties of the complexes and their thermodynamic parameters. At NaCl ≥100 mM, we find that N-protein binds to oligo(U)n with slightly higher affinity than to oligo(dT)n, although the stoichiometries and energetics for formation of these complexes are similar. We find that binding of N-protein dimers to oligonucleotides with n = 16–24 nucleotides is well described by an overlapping site model with an occluded site size of ∼ 8 nucleotides per subunit. However, the binding is more complicated for longer oligonucleotides. We also report the thermodynamic parameters for this interaction system (ΔG, ΔH, and ΔS) using ITC. Binding is enthalpically driven (ΔH ∼ −13–14 kcal/mol per subunit) and shows low or noncooperative behavior on oligonucleotides that can accommodate more than one dimer. Supported by NIH R35 GM 136632 to TML.
Kozlov et al. (Sun,) studied this question.