Structural organization and sequence plasticity of N-protein.

(A) Schematics of folded regions (NTD and CTD, rectangles) and disordered regions (N-arm, linker, and C-arm, straight line) along the N-protein sequence. Defining mutations from the Delta-variant are indicated in blue, those from Omicron-variants in magenta. Transient helices in the disordered regions are highlighted, as well as SR-rich and L-rich linker sequences and the C-terminal N3 region. (B) Histogram of the number of distinct amino acid mutations at each position. For clarity and reference to other figures, IDRs are shaded with N-arm highlighted in yellow, linker in magenta, and C-arm in cyan.

Beehive plots showing the distributions of polarity and hydrophobicity of viable N-protein species across the mutant spectrum.

The polarity index (A) and hydrophobicity index (B) was calculated based on amino acid composition for all distinct sequences of N-FL, the folded domains (NTD and CTD), and the IDRs (N-arm, linker, and C-arm). Further subdivisions of the linker into the SR-rich and L-rich regions, and subdivisions of the C-arm into the N3 region and the C-terminal remainder of the C-arm (C-arm1) are indicated in the arrows. Highlighted by horizontal lines are the values for the corresponding peptides from the ancestral sequence Wuhan-Hu-1 (blue), and including the defining mutations of the Delta variant (dotted red) and the Omicron variant (dashed green), respectively. Symbols indicate values for SARS-CoV-2 (ancestral reference, light blue circles), and corresponding peptides from SARS-CoV-1 (red up triangles), MERS (red down triangles), MHV (red squares), human coronavirus NL63 (grey pentagrams), and the bat coronavirus APD51511.1 (grey diamonds).

Beehive plots showing the distributions of charges of viable N-protein species.

(A) Charges were calculated based on the amino acid composition of different N-protein regions as in Figure 2. Highlighted by horizontal lines are the values for the corresponding peptides from the ancestral sequence Wuhan-Hu-1 (blue), and including the defining mutations of the Delta variant (dotted red) and the Omicron variant (dashed green), respectively. Symbols indicate values for SARS-CoV-2 (ancestral sequence, blue circles), SARS-CoV-1 (red up triangles), MERS (red down triangles), MHV (red squares), NL63 (grey pentagrams), and bat coronavirus APD51511.1 (grey diamonds). (B) Same as in (A), with added charges from maximally phosphorylated serine, threonine, and tyrosine residues in the IDRs.

Sequence alignment score of segments from related coronaviruses

Overview of N-protein species compared in biophysical experiments

Thermodynamic stability and structural differences of N-protein reference and mutant species.

(A) Intrinsic fluorescence spectrum of N:D63G in comparison with Nref, showing spectra in triplicate. (B) Differential scanning fluorometry, with the temperature of maximum fluorescence ratio derivative (Ti-values, with an estimated precision 0.3 °C).(C) Circular dichroism spectra of all N-protein species (spectra with error bars are shown in Supplemental Figure S3).

Tertiary and quaternary structure of N-protein species.

(A) Sedimentation coefficient distributions c(s) from SV-AUC experiments show ≈4S dimers and higher oligomers. Data for N:G215C and Nδ are reproduced from (Zhao et al., 2022). (B) Temperature-dependent particle formation reported as average Stokes radius measured by dynamic light scattering.

Protein-protein interactions of N-arm peptide containing the Omicron P13L mutation lead to large structures at high concentrations.

(A) Autocorrelation functions from DLS (A) and sedimentation coefficient distributions from SV-AUC (B) for the ancestral reference Nref:(1-43) (black), N:Δ31-33(1-43) (blue), N:P13L(1-43) (cyan) and N:P13L/Δ31-33(1-43) (identical to the Omicron N-arm, magenta). All peptide concentrations are 400 µM, except for Nref:(1-43) in the SV-AUC experiment which is 275 µM, reproduced from previously reported data (Zhao et al., 2023).

Differences in LLPS propensity of N-protein mutant species.

Optical microscopy images were taken of 10 μM N-protein with 5 μM T40 (except Nδ, which is 4 μM N-protein with 2 μM T40) in LS buffer after incubation for 15 min at room temperature. For N:P13L/Δ31-33 a second image was taken at the 21 min time point highlighting the growth of condensed phases. All scale bars are 10 µm. Histograms of particle areas are in Supplemental Figure S5, and a comparison of two time-points for Nref, N:R203K/G204R and N:P13L/Δ31-33 is provided in Supplemental Figure S6.