Contributions of the N‐terminal intrinsically disordered region of the severe acute respiratory syndrome coronavirus 2 nucleocapsid protein to RNA‐induced phase separation

Figures

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  FIGURE 1. Sequence analysis of the nucleocapsid protein from the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) coronavirus. (a) Domain organization of NSARS‐CoV‐2 with glutamine residues highlighted by magenta bars underneath. (b and c) Prediction of prion‐like domains in the nucleocapsid protein of SARS‐CoV‐2 (b) and the Omicron variant (c) using the PLAAC algorithm. 9 , 10 Background and prion‐like domains are indicated by black and red lines, respectively. The gray rectangle highlights the region with the strongest differences. (d) NSARS‐CoV‐2 mutation and phosphorylation analysis. Amino acid frequency of mutations using 219,909 sequences from the China National Center for Bioinformatics. Mutations present in the Omicron variant of SARS‐CoV‐2 are shown in red. Phosphorylation sites are highlighted in green. Folded domains are highlighted by blue and green boxes.

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  FIGURE 2. The N‐terminal disordered region of the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) promotes liquid–liquid phase separation. (a) Phase diagram of polyU‐induced phase separation of NSARS‐CoV‐2 in 20 mM phosphate buffer, pH 7.5, monitored by solution turbidity at 350 nm. Three independent measurements were averaged. The dashed line marks NSARS‐CoV‐2/polyU‐concentrations at which charge neutralization occurs, when we assume a charge of −1 per phosphate group. Figure adapted from Savastano A, Ibáñez de Opakua A, Rankovic M, Zweckstetter M. Nucleocapsid protein of SARS‐CoV‐2 phase separates into RNA‐rich polymerase‐containing condensates. Nat Commun. 2020;11:6041. (Figure licensed under: http://creativecommons.org/licenses/by/4.0/.) (b) Phase diagram of ΔNTESARS‐CoV‐2 at protein concentration up to 50 μM and in the presence of increasing polyU RNA concentrations measured as turbidity at 350 nm. Conditions of charge compensation are marked by the dashed magenta line. (c) Absorbance at 350 nm of 50 μM full‐length NSARS‐CoV‐2 or of 50 μM ΔNTESARS‐CoV‐2 in the presence of 0.1, 0.2, 0.5, 1, 2 μM polyU RNA. Red circles highlight differences in the…

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  FIGURE 3. Time‐dependent change in diffusion of phase‐separating NSARS‐CoV‐2. (a) Left panel, fluorescence recovery of 30 μM NSARS‐CoV‐2 droplets formed in presence of 0.3 μM polyU measured at early (red) and late (blue) time points. Error bars represent standard deviations for n = 8 and n = 4 for early and late time points, respectively; right panel, representative micrographs showing pre‐ and post‐bleaching. Scale bars = 10 μm. (b) Left panel, fluorescence recovery of 30 μM ΔNTESARS‐CoV‐2 droplets formed in presence of 0.3 μM polyU measured at early (green) and late (orange) time points. Error bars represent standard deviations for n = 8 and n = 4 for early and late time points, respectively; right panel, representative micrographs showing pre‐ and post‐bleaching. Scale bars = 10 μm

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  FIGURE 4. The N‐terminal region of the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) forms a compact structure with transient β‐structure. (a) Superposition of 1H‐15N HSQC spectra of full‐length NSARS‐CoV‐2 in black and the NTE of NSARS‐CoV‐2 in red measured at 5°C with amino‐acid assignments. (b) Order parameter, S2, and secondary structure prediction from the experimental chemical shifts of the NTE peptide of NSARS‐CoV‐2 using the TALOS+ software. 28 The region with transient β‐structure is highlighted in gray. (c) Hydrodynamic radius of the NTE of NSARS‐CoV‐2 determined by NMR (44 residues) and MD simulations (50 residues). The upper (blue) and lower (light gray) curves indicate the estimated values for an IDP and a globular protein, respectively. 29 (d) Representative structures of the NTE of NSARS‐CoV‐2 obtained from six independent MD simulations. The occupancy of the most populated cluster in each of the six MD simulations is indicated on the side.

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  FIGURE 5. RNA‐interaction of the N‐terminal intrinsically disordered region of NSARS‐CoV‐2. (a) Superposition of 1H‐15N HSQC spectra of full‐length NSARS‐CoV‐2 without polyU RNA and in the presence of 1.5 μM polyU. Spectra were measured at 5°C. (b) 1H‐15N HSQC spectra of the NSARS‐CoV‐2‐residue G5 in the presence of 0, 0.05, 0.5, 1.0, and 1.5 μM polyU RNA. (c) Chemical shift perturbation of the NTE upon interaction with 1.5 μM polyU RNA (NTE sequence shown above; unassigned/overlapped residues are displayed in light gray)