Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. Typically, such modifications are incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. However, loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis (Mtb) becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. How TlyA accomplishes this feat of dual-target molecular recognition is currently unknown. Here, we report the structure of the Mtb 50S-TlyA subunit complex trapped in a post-catalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. This structure, together with complementary site-directed mutagenesis and methyltransferase functional analyses, reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144 which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. This work reveals critical aspects of substrate recognition by TlyA and suggests that base flipping is likely a common strategy among rRNA methyltransferase enzymes even in cases where the target site is accessible without such structural reorganization.
50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA
