A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

Abstract

The nucleoprotein (N) of SARS-CoV-2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich domain (SR) that is hyperphosphorylated in infected cells. This modification is thought to regulate function of N, although mechanistic details remain unknown. We use time-resolved NMR to follow local and long-range structural changes occurring during hyperphosphorylation by the kinases SRPK1/GSK-3/CK1, thereby identifying a conformational switch that abolishes interaction with RNA. When 8 approximately uniformly-distributed sites are phosphorylated, the SR domain competitively binds the same interface as single-stranded RNA, resulting in RNA binding inhibition. Phosphorylation by PKA does not prevent RNA binding, indicating that the pattern resulting from the physiologically-relevant kinases is specific for inhibition. Long-range contacts between the RNA-binding, linker and dimerization domains are also abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into recruitment of specific host kinases to regulate viral function.

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2

“…First, mutations can impact the transient helix in the hydrophobic L-rich region of the linker, and, as we have shown previously, promote its helical conformation and self-association into higher oligomeric states (Zhao et al, 2023, 2022). This, in turn, may impact collision frequency or other intra-molecular interactions with folded domains, such as the recently reported intra-molecular contact of the L-rich region to the NTD observed by NMR (Botova et al, 2024). This is reflected in the altered secondary structure observed in CD of N δ and N:G215C, their oligomers observed in SV-AUC, and this would explain the impact of the G215C mutation on the thermal stability reported by intrinsic fluorescence localized to the NTD and CTD.…”

“…Strikingly, point mutations can affect protein properties on all levels of organization, from thermodynamic stability and secondary structure to intra- and inter-molecular interactions, oligomeric state, particle formation, and LLPS. These results must be considered in the context of the highly dynamic nature of N-protein, which is caused by the flexibility of intrinsically disordered domains (Cubuk et al, 2023, 2021; Redzic et al, 2021; Zhao et al, 2021), the NTD and its disordered β-hairpin (Redzic et al, 2021), and the large-scale conformational fluctuations of the N-protein dimer in solution (Botova et al, 2024; Ribeiro-Filho et al, 2022; Różycki and Boura, 2022). High sequence plasticity is accompanied by high plasticity of protein configuration and delicate balances of protein interactions that can be significantly shifted by single mutations with nonlocal effects.…”

“…In SARS-CoV-2 N-protein IDRs, the latter are mediated via weak interactions in transiently folded structures. In addition, the high flexibility of the IDRs and their resulting high intra-chain contact frequencies (Botova et al, 2024; Różycki and Boura, 2022) may magnify non-local consequences of mutations. This endows viral protein IDRs with yet another level of variation of the biophysical phenotype that can impact evolutionary fitness.…”

Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix

“…Phosphorylation of an SR region’s synthetic peptide also exhibited reduced binding to polyU RNA as well as showed more liquid-like droplets ( 50 ). More recently, hyperphosphorylation was reported to promote direct binding of the phosphorylated SR region to the RNA-binding domain of the NTD and thus providing a mechanism for inhibiting RNA binding ( 51 ). The reported work, however, did not address the effect of phosphorylation on the LRH, as we do below.…”

“…These results demonstrate that while phosphorylation of the linker inhibits RNA binding at the SR-region, it does not inhibit overall RNA binding by FL-N presumably because phosphorylation would leave the high affinity NTD-binding site unaltered. A recent preprint reported a direct interaction of the phosphorylated region with the RNA site on the NTD, potentially blocking RNA binding and abolishing binding to FL-N ( 51 ). We see no effect on RNA binding with the GSK-phosphorylated FL-N.…”