A core network in the SARS-CoV-2 nucleocapsid NTD mediates structural integrity and selective RNA-binding

Figures

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  Fig. 1. The SARS-CoV-2 nucleocapsid (N) RNA-binding domain NTD and its naturally occurring mutations (nat_mutants). a The N domain architecture including the folded NTD and CTD as indicated, flanked by IDRs. The amino acid numbering is given above, as well as an alternative nomenclature (N1-5). Mutations in the nucleocapsid coding sequence as of May 16, 2024, are depicted according to their respective normalized Shannon entropies57,58. Gray shades highlight IDRs, red and gray boxes the folded NTD and CTD, respectively. The red bar shows the entropy of residue 63 with the highest value inside the NTD (0.46). For the rationale of mutant selection, see the methods section. b NTD NMR structure (PDB 6YI36) with color-coded flexible loop regions (fingers). c Scheme of secondary structure elements (α-helices shown as yellow cylinders, β-strands as purple arrows) shown for the NMR structure boundaries and localization of mutations in green. The primary RNA-binding interface is indicated by a blue box. d WT NTD crystal structure from this study. Residues, mutated in the indicated strains are highlighted in green stick representation (for a comparison with the NMR structure, see Supplementary Fig.…

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  Fig. 2. NTD nat_mutant 3D-folds are identical to WT. a 1H/15N CSD plots for NTD nat_mutants D63G, P67S, and P80R compared to the WT NTD, plotted over the amino acid sequence. Significant CSDs (average + 1SD, threshold indicated by dotted line) are mapped on the surface of the NTD NMR structure (PDB 6YI36) in the respective color. The site of mutation is shown by a red sphere (Cα) and by a red star in the CSD plot. b EMSAs of NTD mutants with a described target RNA, SL4ext (Supplementary Fig. 2c). Protein concentrations are given above. Shown is a representative of two biological replicates (N = 2, see source data for quantification). c Zoom-ins of P67S and WT crystal structures showing the site of mutation. The zoom-in was set to comprise regions of significant CSDs according to panel (a) (see also Supplementary Fig. 2a). d Comparison of MST-derived KD values of WT and three nat_mutants D63G, P67S and P80R for viral Ext RNA (3’-Cy5 labeled, see also source data). The transition point (50% bound) for RNA-binding deficient mutant R107A is indicated by dotted lines (see source data for the full curve). Data are presented as mean values +/− SD from three biological replicates (N = 3), each…

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  Fig. 3. Mutation of Y109 within the RNA-interaction surface diminishes RNA-binding. a Alignment of NTD sequences from Alpha- and Betacoronaviruses, highlighting conserved residues of the RNA-binding interface (blue) and the triplet residues forming a core network (green for Betacoronaviruses, orange for the hydrophobic core in Alphacoronavirus). Sequences are taken from Uniprot81 entries Q6Q1R8 (HCoV-NL63), P15130 (HCoV-229E), K9N4V7 (MERS-CoV), P33469 (HCoV-OC43), Q5MQC6 (HCoV-HKU1), P59595 (SARS-CoV) and P0DTC9 (SARS-CoV-2), respectively. b Electrostatic surface potential of the WT. Y109–involved in RNA-binding–is located in the β-sheet core as visualized by the cartoon representation on the right. c Comparison of MST-derived KD values for the two RNA-binding deficient mutants R107A and Y109A when titrated with viral Ext RNA (3’-Cy5 labeled). The transition point (50% bound) at the KD of WT is indicated by dotted lines (see also source data). Data are presented as mean values +/− SD from three biological replicates (N = 3), each measured in duplicate. d Zoom-in on the site of mutation in our crystal structure of Y109A. The zoom-in area was set to include regions of significant CSDs…

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  Fig. 4. A core network is crucial for the structural integrity of the NTD. a Core network (purple)–as seen in our WT crystal structure–made up of triple residue contacts between Q58, W108 and F171, connecting the NTD N-loop, β-sheet palm and carboxy finger. The core network is expanded by contacts to the β-hairpin residues S105 and P106 (orange – H-bond; gray – vdW; black – vdW/π-π). Note that for the contact to S105, both H-bond and vdW contacts can occur within the four chains of PDB 9EXB. The F171-W108 contact has additional contributions from possible π-π interactions. b Melting temperatures for WT, the two nat_mutants D63G and A119S, and the four des_mutants of the network: W108G, F171G, Q58I and S105I. Data are presented as mean values +/− SD from three biological replicates (N = 3), each measured in duplicate. Individual replicates are shown as circles. c Spectral excerpt showing a comparison of 1H/15N-HSQCs for WT, Q58I and F171G, highlighting the tryptophan sidechain region. The extraordinary shift of W108 is indicated by the arrows. See Supplementary Fig. 2b and Supplementary Fig. 9 for a full spectral overlay. d 1H/15N CSD plots for Q58I and (e) S105I des_mutants vs WT plotted…

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  Fig. 5. The core network is critical for NTD structural integrity and specific RNA-recognition. a Comparison of MST-derived KD values of viral Ext and SL4 RNAs (3’-Cy5 labeled), respectively, when titrated with WT, the two des_mutants Q58I and S105I, or the negative control R107A. The transition point (50% bound) at the KD of NTD_R107A to Ext RNA is shown by dotted lines. Data are presented as mean values +/− SD from three biological replicates (N = 3), each measured in technical duplicates. See Supplementary Fig. 11b and c for details on fitting S105I binding to SL4. b Reduced affinities indicated by fold-change of des_mutant KD values from panel (a) normalized to the WT. Error bars represent standard errors propagated during normalization. Individual replicates are grouped by colored circles representing each biological replicate (yellow – N1, green – N2, and white – N3). c Apparent specificity was determined as the relative affinity increase for Ext RNA (target) versus SL4 RNA (non-target) by quantification of the fold-change of respective KD values. A high value correlates with a strong preference for Ext over SL4. For error bars, see the explanation for panel (b). d Differential…

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  Fig. 6. Summary of SARS-CoV-2 N-NTD mutations and their impact on NTD function and fold. Schematic depiction of the NTD hand-like fold highlighting the expanded network that connects the β-sheet palm (W108), the flexible carboxy finger (F171), the N-loop finger (Q58), and the basic finger (β-hairpin, S105). Herein characterized naturally occurring mutants (nat_mutants) within the NTD are conservative regarding the NTD fold and–except for D63G and P80R–show WT-like RNA-binding (green). The des_mutants R107A and Y109A, as well as nat_mutants D63G and P80R show generally changed RNA-binding affinities (decreased and increased, respectively) despite unchanged fold, in line with an unaffected core network (lilac). Mutations within the (expanded) network destabilize the domain fold (indicated by dotted lines in the schematic depiction) and prevent specific RNA-binding (magenta).