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

Abstract

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates.

Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

“…The urea-treated N eluted as a major peak with a higher elution volume compared with the untreated protein (Figure 1A) and showed a molecular mass (87,2 ± 3.9 kDa) and hydrodynamic radius (9.9 ± 1.3 nm) determined by SEC-MALS and DLS, respectively, consistent with a dimer in solution (Figure 1, C and D). These results are in line with literature data showing that the N protein readily oligomerizes into dimers 34,35 . The urea-treated N showed a circular dichroism profile comparable to that of the protein treated with RNase A (Figure 1E), indicating that the urea treatment did not substantially alter the protein secondary structure.…”

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

“…Based on the literature of SARS-CoV-1 N-protein RBD2 crystal structure (Chen et al, 2007), we hypothesized that RBD2-del region may stabilize the formation of higher order oligomers of N-protein. To address if RBD2-del mutation was destabilizing the formation of N-protein dimers (the reported oligomerization state of N-protein in the absence of nucleic acid (Zeng et al, 2020; Zhao et al, 2021a) we performed mass photometry. We observed that, consistent with previous studies, wildtype N-protein forms a dimer ( Fig.…”

“…N-protein dsRNA interaction is unlikely to be observed in reconstitution experiments conducted with less physiological, unstructured RNA (poly U for example). RBD2 also seems to regulate the dimerization domain of N-protein (Zhao et al, 2021a). RBD2 dimerization is highly dependent on the salt concentration with only physiological salt concentrations (150 +/-∼30) allowing for LCST behavior (Iserman et al, 2020; Lu et al, 2021; Zhao et al, 2021a).…”

“…RBD2 also seems to regulate the dimerization domain of N-protein (Zhao et al, 2021a). RBD2 dimerization is highly dependent on the salt concentration with only physiological salt concentrations (150 +/-∼30) allowing for LCST behavior (Iserman et al, 2020; Lu et al, 2021; Zhao et al, 2021a). Lower salt results in an increase in N-protein dimerization domain interactions (Jack et al, 2020) which might increase the required total solution concentration of N-protein for phase separation, thus also increasing the temperature boundary of the LCST behavior.…”

“…It is highly likely that post-translational modifications (PTMs) play a huge role not only in RBD-2 binding RNA but also dimerization and LCST behavior. This may begin to explain why those labs which purify N-protein from mammalian cells did not observe LCST while those which purify N-protein from bacterial sources did (Iserman et al, 2020; Jack et al, 2020; Zhao et al, 2021a). As packaged N-protein is specifically free of post-translational modifications (Fung and Liu, 2018; Wu et al, 2009), we hold that the LCST behavior is likely still relevant for packaging with other N-protein compartments such as those regulating viral RNA transcription and translation being far more likely candidates to be regulated by PTMs (particularly those droplets that form outside double membrane vesicles associated with packaging).…”