Binding of a pyrimidine RNA base-mimic to SARS-CoV-2 nonstructural protein 9

Figures

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  Figure 1. Identification of residues involved in ssRNA binding and the FR6-binding site in close apposition.A, following peak assignment of the 1H-15N 2D HSQC NMR Nsp9COV19 spectra several peaks are seen to undergo concentration-dependent chemical shift perturbations from their unliganded positions (blue) upon titration of different concentrations of short ssRNA oligomers. B, an approximate KD can thus be obtained for a global shared fitting analysis from the movements of the most perturbed peaks (Val-41, Thr-67, Ile-91, and an unidentified Asn side chain). C, the largest chemical shift perturbations with a white to pink gradient can be mapped onto the crystal structure of Nsp9COV19 solved previously. The positions of the residues used in global fitting are labeled. One subunit of the homodimer is partially transparent. D, chemical structures of guanine, FR6, and uracil.

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  Figure 2. The FR6-induced Nsp9COV19hexamer.A, the arrangement within the FR6-bound hexameric form of Nsp9COV19 is displayed in cartoon representation, one ring of protomer subunits uses green hues and the equivalent symmetry-related protomers use blue ones. In this side view, one-third of the hexamer is in the background. B, a closer view of the aromatic ruler region indicating the binding mode of the tetrameric FR6 stacking and the proximity of the N-terminal latch residues and open AB gate entry to the end-cap channel network. C, cross-sectional view of the three π-π stacks within the hexameric form of Nsp9COV19, the αC-dimer helices are labeled for the six subunits. D, fluorescence polarization anisotropy assays using 17mer PolyU to assess ssRNA binding of different recombinant Nsp9 constructs. Constructs correspond to: gNsp9—protein with an additional GPG stub at the N-terminus; +Nsp9—protein with a free backbone amide at Asn-1 and Nsp9. The plot shows corrected anisotropy for each Nsp9COV19 protein concentration; error bars represent the SD from the mean of triplicate measurements. E, top-down view of the Nsp9COV19 hexamer shows the end-cap channel network along with open and closed…

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  Figure 3. Chemical shift perturbations observed in HSQC spectrum upon ssRNA binding align with latch-binding residues.A, a structural view of the unengaged latch of Nsp9COV19 within its apo state in which the β4–β5 loop is extended, backbone amides of the three most perturbed peaks upon ssRNA-binding are circled. B, the equivalent region of the protein within the N-latch engaged FR6-hexamer is shown for the C protomer. The new hydrogen bonds formed to V41, T67, and I91 in the hexameric state are highlighted. C, for the residues depicted in panels A and B expanded regions of the 1H-15N 2D HSQC spectra following titration of oligonucleotide into Nsp9COV19 are shown. Coloring is consistent with Figure 1C and the direction of perturbation is highlighted. D, the asymmetric form of the Nsp9COV19 hexamer results in open and closed gates to the end-cap channel network via restructuring of the β4-β5 loop over the N-terminal latches (colored gold). Both states have two hydrogen bonds to Val-41. E, coronaviral replication assay comparing the replication rate of a wild-type MERS-CoV virus with two constructs bearing a K58A mutation up to 72 h post infection using a plaque forming unit assay. Sequence…