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

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.