Structural heterogeneity and dynamics in the apical stem loop of s2m from SARS-CoV-2 Delta by an integrative NMR spectroscopy and MD simulation approach

Figures

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  Figure 1.. Secondary structures of s2m in SCoV-1 (A) [16], SCoV-2 Wuhan (B) [9], and SCoV-2 Delta (C) [17]. Highlighted in pink are the nucleotides that form a nonaloop with a transient G-U base pair in SCoV-2 Delta. Highlighted in blue are the mutated nucleotides from SCoV-1 to SCoV-2: U732-C732; G758-U758. Highlighted in orange are the mutated nucleotides from SCoV-2 Wuhan to SCoV-2 Delta: G742-U742. Nucleotide numbering corresponds to the position in the genome -29 000.

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  Figure 2.. Secondary structure determination and imino proton assignment of s2m Delta_short. (A) 1H, 1H NOESY spectrum of the imino proton region with sequential walk GGUGUGG in black. (B) 1H,15N BEST-TROSY of the base paired region of s2m Delta (sample#2) and s2m Delta_short (sample#3). Nucleotide numbers in grey belong to the full-length construct, nucleotide numbers in magenta belong to the short construct that show a slight chemical shift perturbation, nucleotide numbers in violet belong to both constructs and overlap. (C) Schematic depiction of the 3′-UTR of SCoV-2 with s2m in the HVR highlighted in violet. (D) NMR-determined secondary structures of s2m Delta [17] and s2m Delta_short. Spectra were measured at 283 K.

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  Figure 3.. Analysis of N3-C2 modulation in s2m Delta_short using a 13C, 15N-HSQC experiment with deuterium filter. (A) 13C, 15N-HSQC measured at 308 K with sample#10. Exemplary magnetization transfer shown for CN-HSQC and HCN experiments. (B) 1H,15N plane of HCN experiment to assign N1 chemical shift of uridines measured with sample#3 at 308 K. Dashed lines indicate the N1 chemical shift transfer. (C) Normalized intensity of N3-C2 signals to N3-C2 signal intensity at deuterium filter delay Δ of 0 ms. (D) Projections along the 15N axis with (red) and without delay Δ (black). Projections were scaled to match N1-C2 signal intensity. Arrows indicate signal modulation in N3-C2 correlations for U748, U753, U758, and U742.

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  Figure 4.. 4D HMQC-NOESY-HMQC planes of nucleotides G745-U753 (pink) in s2m Delta_short. (A) In extracted C1’H1’-2D planes of the 4D, a sequential NOESY walk is depicted (pink arrows). Starting from an aromatic signal of a specific nucleotide (i) peaks are visible for the C1’H1’ resonance corresponding to the same nucleotide and to the previous nucleotide (i-1) (blue). By following these connectivities through the planes from 3′ to 5′ direction, a sequential assignment is achieved. Overlay of the 4D HMQC-NOESY-HMQC spectrum (blue, sample#4, Supplementary Table S1) and 1H,13C-HSQC spectrum (black, sample#4, Supplementary Table S1). Both spectra were recorded at 308 K. The resonance assignment of all nucleotides is annotated and the C6H6/C8H8 aromatic chemical shift leads to the shown planes. The respective nucleotide for that plane (pink) is annotated in the lower left corner of each spectrum. Degradation peaks are visible of s2m Delta_short from sample#4 (*). (B) Aromatic 1H,13C-HSQC at 308 K (sample#3, Supplementary Table S1). (C) C1’H1’ region of s2m Delta_short at 308 K (sample#3, Supplementary Table S1).

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  Figure 5.. NMR solution structure of s2m Delta_short RNA. (A) Secondary structure of s2m Delta_short. The loop nucleotides are highlighted in a hetNOE-derived heatmap (red: high, blue: low) from panel (E). (B) Aria-calculated NMR solution structure ensemble of 10 superimposed structures with the lowest χ2 to SAXS data. Loop sequence highlighted in hetNOE derived colours (red: high, blue: low) from panel (E). The overall RMSD was calculated to be 3.60 ± 1.13 Å. (C) Aligned and overlayed loop sequence with an RMSD of 3.31 ± 0.77 Å. (D) Overlaid stem region without G-2-C + 2 base pair and G744-C754 base pair. The stem ensemble of all 20 structures converges with an RMSD of 0.94 ± 0.23 Å (only 10 structures shown). (E) 1H,13C heteronuclear NOE of s2m Delta_short for sugar C1’H1’ resonances (sample#4.5). The RNA sequence is given on the top of each plot with squares indicating base paired regions.

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  Figure 6.. Sugar pucker conformation of s2m Delta_short determined by canonical coordinates, 3J (H, H) coupling constants and CCR at 308 K. (A) Canonical coordinates determined from 13C chemical shift assignments. (B) Determination of pseudorotation phase P from 3J (H1’, H2’), (H3’, H4’), and (H2’, H3’) coupling constants determined via a fw-HCC-TOCSY-CHH-E.COSY experiment. (C) fw-HCC-TOCSY-CHH-E.COSY experiment from A746 signals to determine 3J (H1’, H2’, and H3’, H4’) coupling constants. (D) C3’-endo population distribution of A746 and A749 derived from 3J (H, H) coupling constants. (E) Determination of pseudorotation phase P based on mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\Gamma _{C1\prime H1\prime ,C2\prime H2\prime }^{DD,DD}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\Gamma _{C3\prime…

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  Figure 7.. Structure and implied dynamics of the CCR reweighted ensemble. (A) PC plot with estimated free energies, revealing approximately six densely sampled regions, informing (B) k-means clustering into five CSs with respective centroids (red dots) falling as close as possible to densely sampled structures. (C) RMSF of the terminal loop nucleotides for each CS. (D) Threedimensional structures of CS centroids, proportion of data falling within each CS, and dynamic secondary structures representing the proportion of structures in each CS.

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  Figure 8.. Flipped-back conformation in s2m Delta_short. (A) Overlayed centroid structures of the CCR reweighted ensemble (CS1 to CS5). The dominant dynamic in the data is a flipped back terminal loop (CS4) sampled by only one out of the original 20 MD simulations. (B) Overlayed centroid structure CS4 with apical GNRA-like pentaloop of SCoV-1 (PDB: 1XJR).

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  Figure 9.. Pseudorotation angles and amplitudes of the CCR reweighted ensemble of loop nucleotide G745-U753. The computed mean is given by a red dot while the experimental derived pseudorotation phase from CCRs is given by a yellow triangle for one-state fits. The filled yellow circle is the derived percentage of population distribution from a two-state model with P= 18° for C3’-endo conformation and P= 162° for C2’-endo conformation.

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  Figure 10.. Summary of secondary structures of SCoV-2 Wuhan s2m with experimental depiction of their base pair probability that were reported before. The experimental data include SHAPE in vivo or in vitro, RNA–RNA interaction profiles, DMS in vitro, and RNA sequencing. The structures were combined to be represented using a colour code from light blue (base pair) to dark blue (not base paired) overlayed in their respective secondary structures as well as in table form for better visualization. In dark magenta pentaloop nucleotides are highlighted in the table, light magenta highlights the nonaloop nucleotides. (A) Manfredonia et al. in vitro [27]. (B) Manfredonia et al. in vivo [27]. (C) Huston et al. in vivo [28]. (D) Sun et al. in vivo/ in vitro [29]. (E) Lulla et al. in vitro [30]. (F) Ziv et al. [31]. (G) Zhang et al. A [32]. (H) Zhang et al. B [32]. (I) Manfredonia et al. DMS in vitro [27]. (J) Morandi et al. in vitro [33]. (K) Cao et al. [26]. (L) Ahmed et al. [25]. (M) Rangan et al. [21]. (N) Tengs et al. [14]. (O) Aldhumani et al. [24]. (P) Secondary structure of SCoV-2 Wuhan s2m determined by NMR by Wacker, Weigand et al. [9]. (Q) Secondary structure of SCoV-1 s2m [16].

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  Figure 11.. Cleavage assay with Nsp15 and (A) SCoV-1 s2m, (B) SCoV-2 Wuhan s2m, (C) SCoV-2 s2m Delta, and (D) SCoV-2 s2m Delta_short. A schematic representation of the secondary structure of each construct can be seen next to the respective PAA gel. Nucleotides marked with red correspond to most probable cleavage sites. *: Cleavage products and position in RNAs.