Distant ribose 2'-O-methylation of 23S rRNA helix 69 pre-orders the capreomycin drug binding pocket at the ribosome subunit interface

Abstract

Loss of ribosomal RNA (rRNA) modifications incorporated by the intrinsic methyltransferase TlyA results in reduced sensitivity to tuberactinomycin antibiotics such as capreomycin. However, how rRNA methylation alters drug binding, particularly at the distant but functionally more important site in 23S rRNA helix 69 (H69), is currently unknown. We determined high-resolution cryo-electron microscopy structures of the Mycolicibacterium smegmatis 70S ribosome with or without the two ribose 2'-O-methyl modifications incorporated by TlyA. In the unmodified ribosome, the tip of H69 adopts a more compact conformation, positioning two key nucleotides (A2137 and C2138) such that interactions with capreomycin would be lost and the binding pocket partially occluded. Methylation of 23S rRNA nucleotide C2144 promotes conformational changes that result in a more favorable positioning of C2138 and adoption of a more open conformation to enable capreomycin binding. Molecular dynamics simulations and H69 RNA helical analyses additionally reveal specific propagation of these changes from the site of modification to the H69 tip, allosterically reconfiguring the capreomycin binding site. Methylation of h44 also results in structural rearrangements at the H69-h44 interface to support maintenance of these changes that favor antibiotic binding. This work thus reveals the effect and regulation of distant rRNA methylation on ribosome-targeting antibiotic binding.

Graphical Abstract

Figure 1.

Cryo-EM map and model of the TlyA-methylated and unmethylated H69 within the Msm 70S ribosome. (A) Postprocessed (PHENIX sharpened) map of the methylated 70S ribosome at 3.16 Å resolution. (B) The final model highlighting the location of H69 (boxed region) and indicating the sites of methylation on both subunits. (C) Zoomed-in view of the indicated region, showing the H69 structure with a surrounding map from 50S multibody refinement (DeepEMhancer sharpened, map threshold 0.01; map value range −0.00171 to 1.86). (D) Methylated Cm2144 shown within the map (DeepEMhancer sharpened, map threshold 0.04; map value range −0.00171 to 1.86) supporting the presence of the expected 2′-O-methyl group. (E–H) As for panels (A)–(D) but for the unmethylated Msm 70S ribosome at 3.24 Å resolution, with the unmodified H69 (C2144) highlighted (boxed region). For panels (G) and (H), the map is DeepEMhancer sharpened with a map threshold of 0.013 and 0.042, respectively; map value range −0.00235 to 2.04.

Figure 2.

The nucleotides at the tip of H69 undergo changes in the methylated versus unmethylated ribosome, potentially impacting capreomycin binding. (A) Two orthogonal views of a superimposition of the methylated and unmethylated H69 shown as backbone with phosphates as spheres. Differences in the tip are indicated with a dotted line arrow. Inset: sequence and secondary structure of H69. (B) Residues A2151^Mtb and C2152^Mtb at the tip of H69 in the Mtb 70S ribosome (PDB code 5V93) stabilize capreomycin in its binding pocket via hydrogen bonding interactions (dashed lines). (C) Corresponding view for capreomycin at the H69 tip in the Tth 70S ribosome (PDB code 4V7M). (D, E) in the unmethylated Msm ribosome, A2137 is predicted to clash (boxed regions with atoms shown as semi-transparent spheres) with the modeled capreomycin from both the Mtb and Tth structures, respectively. In contrast, C2138 either weakly (Mtb) or does not interact (Tth) with the antibiotic. (F, G) in the methylated Msm ribosome, movement of C2138 and the tip of H69 partially opens the capreomycin binding pocket and allows the nucleotide to contact the modeled antibiotic. A2137 remains in a position predicted to result in clashes with the bound capreomycin and would thus require further movement promoted by drug binding as part of its mechanism of action in blocking tRNA (transfer RNA) movement.

Figure 3.

C2144 methylation stabilizes H69 and favors capreomycin binding. (A) The components of Mtb 70S ribosome (PDB code 5V93) used for MD simulation (System 1), including H69, h44 and additional 16S rRNA regions, ribosomal proteins, and capreomycin. The unrestrained regions are indicated with transparent spheres. Nucleotide positions are denoted by solid spheres (P atoms) on the rRNA ribbon. (B) Difference RMSF (unmethylated subtracted from methylated) for H69 reveals a decrease in dynamics between the site of modification (Cm2144) and the helix tip. The dotted lines indicate the site of methylation and the H69 tip, as indicated. The vertical shaded regions indicate the restrained regions of H69 and the standard deviation for calculated values is indicated by the shaded region surrounding the plotted data. (C) Plot showing the increase in average angle (indicated on the structure below the plot) between A2137 (N1) and C2138 (N9) in the methylated system compared to the unmethylated system. (D) Calculated capreomycin binding energy is more favorable (lower energy) in the methylated system compared to unmethylated.

Figure 4.

C2144 methylation results in propagation of helical conformational changes to the tip of H69. (A) H69 structure with the site of methylation and the two H69 tip residues critical for capreomycin binding indicated. Base steps for which helical parameters were calculated in 3DNA 2.0 and proposed direction of signal propagation (3′ to 5′) are also shown. (B) Differences between the indicated H69 helical parameters for methylated and unmethylated 70S ribosomes. The left dotted line, left shaded region, and right shaded region indicate the site of methylation (Cm2144), nucleotides between Cm2144 and the helix tip, and the H69 tip, respectively. (C) Comparison of the normalized differences between methylated and unmethylated H69 for the same six helical parameters at each base step. (D) As for panel (C) but from MD simulations of the methylated and unmethylated H69 structures (System 2). All individual helical parameter plots for methylated and unmethylated H69 are shown in Supplementary Fig. S6C and D.

Figure 5.

16S rRNA nucleotides assist in preserving the architecture of the capreomycin binding site. (A) In the unmethylated ribosome (left), 23S rRNA C2138 interacts with 16S rRNA C1392, while in the methylated ribosome (center), these contacts are lost due to the movement of C2138 away from Cm1392. A model combining C2138 as positioned in the unmethylated ribosome and Cm1392 (right) shows that methylation would result in steric clashes (spheres) without this movement of C2138. Methylation of C1392 thus supports the maintenance of C2138 in the more open conformation of the H69 tip. (B) 16S rRNA A1391 and 23S rRNA A2137 are adjacent in the unmethylated ribosome but do not directly interact (left). In the methylated 70S ribosome (center), A2137 moves away from A1391 as the tip of H69 moves upward. A model combining A1391 in the methylated 30S reveals potential clashes with A2137 when positioned as observed in the more compact H69 conformation of the unmethylated 50S subunit (right). (C) In the unmethylated ribosome (left), 23S rRNA A2137 and 16S rRNA A1477 make a π-stacking interaction that is maintained by coordinated rotation of both nucleotides in the methylated ribosome (center). A model combining methylated 30S and unmethylated 50S subunits (right) shows steric clashes would result without the rotations, indicating that these movements maintain the stacking interaction and the more open conformation of H69 in the methylated ribosome.

Figure 6.

The capreomycin binding pocket formation is initiated by methylation and completed by antibiotic binding. (A) The tip of H69 in unmethylated Msm constituted by A2137 and C2138 is proximal to h44 residues C1392 and A1391. A2137 occupies the capreomycin binding pocket and C2138 is unfavorably positioned with respect to an incoming capreomycin antibiotic, reducing its affinity for the ribosome. (B) TlyA-mediated methylation of the 50S results in the transmission of an allosteric signal to the H69 tip, promoting a more open conformation. Within the assembled 70S ribosome, this conformation is preserved by the methyl group of 16S rRNA nucleotide C1392 (shown as a sphere) and the base of A1391. Rotation of A1477 compared to the unmethylated ribosome is also necessary to avoid clashes and thereby assists in maintaining the open conformation. (C) Upon capreomycin binding to the M. tuberculosis 70S ribosome (PDB code 5V93), A2151^Mtb (A2137) flips out of the binding pocket concurrent with a slight rotation of C2152^Mtb (C2138) in H69. h44 nucleotide A1485^Mtb (A1476) and A1486^Mtb (A1477) flip from the antibiotic binding site to accommodate the bound capreomycin. Note that although Mtb has active TlyA^II, the methyl group on Cm1402^Mtb was not modeled (location is indicated by "Methyl").