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

Abstract

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

Keywords: AUC; LLPS; NMR; SARS-CoV-2; phosphorylation; protein RNA interactions.

Figure 1

Domain maps of SARS-CoV-2N and constructs used in this work.A, amino acid sequence of SARS-CoV-2N construct spanning the linker region from residues 175 to 245. Residual TEV protease cleavage sites are in orange. The SR-rich region is in bold black, with phosphorylation sites investigated here in bold red. The Leu-rich helix (LRH) spanning residues 216 to 232 is in bold blue. B, domain maps of full-length (FL-N), mutant full-length (muFL-N), shorter constructs N_175–365, N_175–245, and GFP-tagged N_175–245 from top to bottom, respectively. Intrinsically disordered regions are represented by black lines, the structured RNA-binding domain NTD is represented by a dark green rectangle, and the dimerization domain CTD by a dark green oval. The LRH is represented by a small blue rectangle. GSK priming site pSer188 is pointed to by an arrow, and the subsequent phosphorylation sites are indicated by red circles. The muFL-N has a TQT recognition motif for LC8-binding site incorporated to replace the LRH shown as gray rectangle. C, a cartoon depiction of dimeric FL-N showing self-association of LRH- and RNA-binding site of the NTD. CTD, C-terminal domain; GSK, glycogen synthase kinase; LC8, dynein light chain 8; LRH, Leu-rich helix; NTD, N-terminal domain; SR-rich, serine/arginine-rich.

Figure 2

NMR structural characterization of WT N175 to 245 and pSer188 N175 to 245.A, ¹⁵N-HSQC spectrum of 150 μM WT N_175–245 at 10 °C with backbone resonance assignments. B, overlay of ¹⁵N-HSQC spectrum of WT N_175–245 (black) and pSer188 N_175–245 (red), with shifted peaks labeled. C, ¹⁵N nuclear spin relaxation of WT N_175–245 (black) compared to pSer188 N_175–245 (red). The Leu-rich helix is indicated.

Figure 3

Concentration dependence of self-association of WT and pSer188 N175 to 245.A, ¹⁵N-HSQC spectra of WT N_175–245 (left) at 300 μM (red) and 100 μM (black) concentration. Resonances that disappear at higher concentration are labeled. ¹⁵N-HSQC spectra of pSer188 N_175–245 (right) at 300 μM (red) and 200 μM (black) concentration. The same resonances are labeled. Missing peaks for residues 233 to 235 is due to ordered structure in proximity of the self-associating helix. B, sedimentation velocity analytical ultracentrifugation (SV-AUC) of WT-N_175–245 GFP (left) at 50 to 200 μM concentration and SV-AUC of pSer188 N_175–245 GFP (right) at 100 and 200 μM concentration. C, representative sedimentation equilibrium data at three rotor speeds for WT-N_175–245 GFP (left) and for pSer 188 N_175–245 GFP (right). Monomer-dimer equilibrium fits are shown as solid lines. All AUC experiments with N_175–245 were performed with GFP fusion.

Figure 4

Concentration dependence self-association of WT and phosphorylated N175 to 365.A, ¹⁵N-TROSY-HSQC spectra of 50 μM WT N_175–365 (red) compared to WT N_175–245 (black). Resonances of N_175–245 that are not present in N_175–365 are labeled. Resonances that correspond to residual TEV cleavage sites in N_175–245 are indicated by an asterisk. B, ¹⁵N-TROSY-HSQC spectra of 50 μM GSK-hyperphosphorylated N_175–365 (+GSK N_175–365) (red) compared to WT N_175–245 (black). Down-field resonances corresponding to phosphorylated serine are circled. Resonance labeling scheme is the same as in (A). C, SV-AUC of WT-N_175–365 at 100 to 340 μM concentration. D, SV-AUC of +GSK N_175–365 at 200 and 340 μM concentration. E, representative model of proposed tetramerization of N_175–365 due to LRH self-association (dark blue). Tetrameric N_175–365 is in exchange with dimeric N_175–365 as indicated on the right. F, representative model of the effect of hyperphosphorylation of N_175–365 showing the CTD as an intact dimer (green spheres) but with dissociation of the LRH from tetramers and dimers to monomers. CTD, C-terminal domain; GSK, glycogen synthase kinase 3β; LRH, Leu-rich helix.

Figure 5

Phosphorylation modulation of N-RNA interactions.A, peak intensity ratios based on NMR titrations of g(1–1000) RNA into ¹⁵N-labeled N_175–245 (top) and pSer188 N_175–245 (bottom). The protein:RNA ratio varied from 10000:1 to 1000:1. Data are plotted as intensity ratios for peaks with and without RNA. B, EMSA gels for WT N_175–245 (0–30 μM) and pSer188 N_175–245 (0–75 μM) with g(1–1000) RNA (0.5 μM). GSK-3 was used to hyperphosphorylate (+GSK) the pSer188 N_175–245 (−GSK). C, EMSA gels for WT FL-N (0–20 μM) and pSer188 FL-N (0–40 μM) with g(1–1000) RNA (0.5 μM), along with GSK-3 hyperphosphorylation. For (B) and (C), the g(1–1000) RNA migrates as multiple bands because the RNA is not denatured and can adopt secondary structures. D, WT FL-N (left), pSer188 FL-N (middle), and hyperphosphorylated FL-N (+GSK FL-N) (right) liquid-liquid phase separation (LLPS) using fluorescence imaging (top) and bright field (bottom). The g(1–1000) RNA was labeled with cy3 for fluorescence imaging. Images were taken at 40× magnification, and the scale bar represents 50 μm. All LLPS experiments were collected at 37 °C after 2 h of incubation. E, LLPS utilizing the same conditions as (D) for muFL-N, muFL-N with LC8 added, and pSer188 muFL-N with LC8 added (from left to right, respectively). The scale bar represents 100 μm. FL-N, full length N protein; GSK, glycogen synthase kinase 3β; LC8, dynein light chain 8.

Figure 6

Proposed model of how phosphorylation acts as a molecular switch for the role of N from viral packaging to viral replication. FL-N is depicted here using similar illustration as in Figure 1C. (Left) Unphosphorylated FL-N is in a dimer-tetramer equilibrium which shifts to tetramer when bound to RNA. The positive charges in the SR-rich region cause elongation due to charge–charge repulsion. RNA intercalates by binding to the SR region of the linker, resulting in a compacted RNA expected to be most populated in LLPS and in viral packaging. (Middle) A single phosphorylation event (red) introduces negative charges in the SR causing some structural change. RNA remains bound to the NTD but does not bind the linker resulting in reduced compaction. (Right) Four phosphorylation events with GSK-3 causing significant structural changes in the SR-region of GSK hyperphosphorylated FL-N and dissociation of the 4-helix bundle tetramers. The resulting structure of FL-N is a dimer with significant flexibility in the linker after dissociation of the LRH causing significant dispersing of the RNA, consistent with the model expected in viral replication. Two N proteins are shown to illustrate multivalent binding. Phosphates are indicated by red circles, and arginines by circled pluses. RNA is depicted by the dark blue line. FL-N, full length N protein; GSK-3, glycogen synthase kinase 3β; NTD, N-terminal domain; SR-rich, serine/arginine-rich.