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

Abstract

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.

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

“…This is reminiscent of the gain of structure reported by Zeng and co-workers from CD at 55 C (Zeng et al, 2020), and may arise from stabilization of transient helices identified in the disordered regions of SARS-CoV-2 N-protein (Cubuk et al, 2021). Alternatively, it may reflect loss of unusual CD spectral features reported for the NTD (Forsythe et al, 2021). DSF results suggest that these changes also impact the folded domains (where the tyrosine and tryptophan residues exclusively reside).…”

The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs

“…Single-stranded regions spaced between NCAPbound structures provide the conformational flexibility necessary for NCAP proteins to interact with each other favorably in such assemblies. These interactions likely include the previously characterized oligomerization (20,21,24,25) and amyloid-like interactions by the LCD and other IDRs (31,49). These interactions lead to further stabilization of the NCAP-RNA complex, effectively increasing the binding affinity for the longer gRNAs with optimal structural patterns that can recruit larger number of NCAP proteins.…”

“…In the second, any NCAP monomers may dimerize upon binding RNA. RNAfree NCAP has a dimerization constant of 0.4-0.5 µM (21). Binding to the same RNA increases the effective concentration of NCAP monomers to each other.…”

Phylogenomics and population genomics of SARS-CoV-2 in Mexico during the pre-vaccination stage reveals variants of interest B.1.1.28.4 and B.1.1.222 or B.1.1.519 and the nucleocapsid mutation S194L associated with symptoms

“…The SARS-CoV-2 nucleocapsid protein N is a multidomain RNA-binding protein that is found inside the viral envelope and it is required by the virus to maintain its structure and viability once the virus has entered the cell [75, 76]. The mutation we identified, N:S194L, is within the central domain composed of intrinsically disordered regions (IDRs) [75], which are conformationally flexible and promiscuous [77], and are increasingly recognized as important in increased viral transmission [76, 78]. Mutations in positions 193, 197, 203 and 204 of the linker IDR have been mapped to the loops connecting disordered and structured regions [76], implicating these sites in increased transmission.…”