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. 2023 Apr 5;9(14):eadg6473.
doi: 10.1126/sciadv.adg6473. Epub 2023 Apr 5.

A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins

Huaying Zhao  1 , Di Wu  2 , Sergio A Hassan  3 , Ai Nguyen  1 , Jiji Chen  4 , Grzegorz Piszczek  2 , Peter Schuck  1
Affiliations

A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins

Huaying Zhao et al. Sci Adv. .

Abstract

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

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Figures

Fig. 1.
Fig. 1.. Schematics of N-protein structure and assembly.
(A) N-protein with folded domains (NTD and CTD) and IDRs (N-arm, linker, and C-arm; all IDRs are artificially stretched for clarity). The variability of the amino acid sequence is highlighted through colors indicating for each position the number of distinct mutations contained in the GISAID genomic data base (as of July 2022) (21, 55, 72). (B) Current model for viral assembly: N-protein dimers, noncovalently linked in the CTD (dark squares), undergo a conformational change upon NA binding that allows oligomerization and promotes LLPS through weak protein-protein and/or protein-NA interactions. The dense phase containing genomic RNA (red coil) permits formation of RNPs [Electron Microscopy Data Bank (EMDB) 11868 (16)] leading to viral assembly [EMDB 30430 (17)].
Fig. 2.
Fig. 2.. NLRS peptides self-associate.
(A) Sedimentation coefficient distributions c(s) of NLRS at different concentrations in working buffer (solid lines) show monomer peaks at ≈0.6 S and trimer peaks at ≈1.3 S, the latter fraction increasing with concentration. For typical raw sedimentation boundaries, see fig. S1. For comparison, c(s) distributions in working buffer supplemented with 10% TFE are shown as dashed lines. The inset shows the weight-average sedimentation coefficients sw (circles) with best-fit GMMA models (lines) jointly fitting the sw and θ(222 nm) isotherms (Fig. 3A). A monomer-dimer model yields an effective KD2* = 0.60 mM (black dashed line), a monomer-trimer model yields an effective KD3* = 0.81 mM (red solid line), and a mixed monomer-trimer and monomer-tetramer model yields effective KD3* = 1.6 mM and KD4* = 1.0 mM (blue dotted line). (B) DLS autocorrelation data of NLRS at different concentrations (circles) and best two-species fit (lines) accounting for the z-average peptide diffusion and traces of a ≈15 nm cluster. The top inset presents the average oligomeric state estimated by combining the peptide diffusion coefficient with the oligomer sedimentation coefficient. The bottom inset is the measured temperature dependence of the z-average hydrodynamic radius of NLRS.
Fig. 3.
Fig. 3.. Concentration-dependent helix formation of NLRS.
Shown are CD spectra in units of MRE values θMRE, which directly reflect molecular peptide helicity. (A) Dependence of ellipticity on NLRS concentrations. The inset shows the best fit of θ(222 nm), jointly with the sw isotherm of Fig. 2A, with the GMMA model indicated. The GMMA monomer-trimer model results in best-fit helicities of −4290 and −16,210 deg cm2 dmol−1 for the monomer (red circle) and trimer (red triangle) state, respectively, corresponding to ≈3 helical residues in the NLRS monomer increasing to ≈16 helical NLRS residues per peptide in the trimer. (B) Solvent-induced change in helicity of 0.4 mM NLRS at different concentrations of TFE. The inset shows θ(222 nm) (circles) and the best-fit two-state model (line) resulting in the best-fit ellipticity of the maximally helical state of −14,400 deg cm2 dmol−1 (red open triangle), corresponding to estimated ≈14 helical NLRS residues per peptide chain.
Fig. 4.
Fig. 4.. Schematics of a free energy diagram of NLRS folding and oligomerization.
NLRS folding from the disordered state requires activation energy and is slow, while different oligomeric states of helical NLRS may have similar free energy and coexist in rapid exchange.
Fig. 5.
Fig. 5.. Configuration of a trimeric FL–N-protein predicted by ColabFold.
Three FL-N sequences are predicted to simultaneously have CTD dimer contacts and LRS trimer contacts. The disordered segments are not well predicted and are in quasi-random configuration; therefore, the linker IDR 180-210 and the C-arm are artificially stretched for clarity.
Fig. 6.
Fig. 6.. MD simulations highlight residues that stabilize NLRS oligomers.
Shown are snapshots from the MD simulations for the trimer (A) and tetramer (B). The cores of the oligomers are formed by five nonpolar residues in a well-packed configuration (left column of each panel). More specific interactions stabilizing the complex surfaces are (right column of each panel): a hydrophobic surface cluster formed by four leucine residues, two salt bridges (R226-E231 and K233-E231), and an H-bond interaction between polar (N228) and charged (R226) residues. Helix backbones drawn as gold ribbons; interacting residues rendered as atom-based Van der Waals spheres (nonpolar residues in gray; polar and charged residues in color: white, H; red, O; gray, C; blue, N). G215 is shown in green for reference.
Fig. 7.
Fig. 7.. LRS is conserved in both the mutational landscape of SARS-CoV-2 linker IDR and in alignment with related coronaviruses.
The linker region 181-246 of the Wuhan-Hu-1 SARS-CoV-2 (P0DTC9.1) N-protein is aligned with SARS-CoV (P59595.1), MERS (YP_009047211.1), murine hepatitis virus (NP_045302.1), human coronavirus NL63 (Q6Q1R8.1), and the 229E-related bat coronavirus APD51511.1. Conserved identical residues are highlighted in bold red, and conserved similar residues are highlighted in bold black. Above the aligned sequences are observed distinct mutations of SARS-CoV-2 observed among 3.83 million high-quality sequences uploaded to GISAID between January 2020 and August 2022. The mutations are ordered and colored by number of genomes found to contain that specific mutation: 10 to 100 (light gray), 100 to 1000 (light blue), and >1000 (bold blue). The underlined sequences correspond to trimeric helices predicted by ColabFold.
Fig. 8.
Fig. 8.. Self-association of FL-N and NA-liganded FL-N is mediated by LRS assembly.
SV experiments are carried out with three FL-N protein constructs (cyan): (A) the ancestral reference N-protein, (B) the mutant N:L222P that diminishes LRS folding and assembly, and (C) the double mutant N:L222P/R226P abolishing LRS assembly. Sedimentation coefficient distributions are shown in the presence of 10% TFE (blue), an excess of oligonucleotides T10 (green), and both 10% TFE and T10 (magenta).
Fig. 9.
Fig. 9.. LRS mutants modulate particle formation and LLPS of FL-N.
(A) The onset of particle formation observed by DLS. Z-average hydrodynamic radii as a function of temperature are shown for 3 μM ancestral FL-N (black), the LRS self-association enhancing mutant FL-N:G215C (magenta), and the abrogating mutants FL-N:L222P (cyan) and FL-N:L222P/R226P (blue), all in the absence (solid lines) and presence of 10 μM NA ligand T10 (dashed lines). (B) LLPS was studied by optical microscopy using 5 μM samples of ancestral FL-N (left) or the LRS self-association suppressing mutant FL-N:L222P in the presence of 10 μM T10 in 10.1 mM Na2PO4, 1.8 mM KH2PO4, 2.7 mM KCl, 10 mM NaCl, and 10% polyethylene glycol (pH 7.40) at room temperature. Images shown were taken after 6 hours.
Fig. 10.
Fig. 10.. Related coronaviruses exhibit similar concentration-dependent self-association and linked folding of their LRS.
Peptides aligning with the LRS of SARS-CoV-2 comprising oligomeric helices predicted in ColabFold were created for SARS-CoV, MERS, MHV, human coronavirus NL63, and the 229E-related bat coronavirus APD51511.1. (A) From SV experiments, the weight-average sedimentation coefficient as a function of concentration (symbols), normalized relative to the monomer s-value measured at low concentrations, increases with concentration. (B) From CD experiments, the measured MRE at 222 nm decreases at higher concentration indicating increased folding. For clarity, the symbols show θMRE scaled as fractional change. In both panels, the solid lines depict the global best GMMA fit of a monomer-trimer self-association model to both isotherms of each LRS peptide simultaneously. Best-fit binding parameters are listed in table S1.
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