Significance The bacterial ribosome is an important target for antibiotics used to treat infection. However, resistance to these essential drugs can arise through changes in ribosomal RNA (rRNA) modification patterns through the action of intrinsic or acquired rRNA methyltransferase enzymes. How these antibiotic resistance-associated enzymes recognize their ribosomal targets for site-specific modification is currently not well defined. Here, we uncover the molecular basis for large ribosomal (50S) subunit substrate recognition and modification by the Mycobacterium tuberculosis methyltransferase TlyA, necessary for optimal activity of the antitubercular drug capreomycin. From this work, recognition of complex rRNA structures distant from the site of modification and “flipping” of the target nucleotide base both emerge as general themes in ribosome recognition for bacterial rRNA modifying enzymes.

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.

RNA (rRNA) modification is important for correct ribosome assembly, can alter ribosome function, and can confer resistance to clinically important ribosome‐targeting antibiotics. While the activity of most ribosome‐targeting antibiotics is blocked by rRNA methylation, the tuberactinomycin antibiotic capreomycin requires ribose 2′‐OH methylation at C1409 of the 16S rRNA within the small ribosome subunit (30S) and C1920 of the 23S rRNA within the large ribosome subunit (50S). TlyA is the 2′‐O‐methyltransferase that incorporates both modifications using S‐adenosyl‐L‐methionine (SAM) as a methyl group donor. The X‐ray crystal structure of the C‐terminal domain (CTD) of TlyA showed that the domain adopts a Class I methyltransferase fold, while homology modeling suggests the N‐terminal domain (NTD) adopts an S4 ribosomal protein fold. Additionally, the TlyA CTD structural studies revealed that the short interdomain linker was able to adopt two different conformations and was unexpectedly critical for SAM binding within the CTD. This project will test the resulting hypothesis that the interdomain linker acts a “molecular switch,” altering the interaction between the NTD and the CTD to control TlyA activity upon correct recognition of its two structurally distinct substrates. Using a sequence alignment and structure‐guided mutagenesis approach coupled with fluorescence polarization (FP) TlyA‐30S binding and methyltransferase activity assays, the role of the NTD and individual residues in this domain in 30S and 50S binding will be defined. Using complementary studies of TlyA protein dynamics (using HDX‐MS) and structure (using X‐ray crystallography and cryo‐EM), the long‐term goal of this project is to determine TlyA's molecular mechanism of site‐specific ribosome subunit recognition and modification. Defining these molecular details will help complete the picture of TlyA activity and its role in bacterial drug sensitivity or resistance, as well as furthering our general understanding of rRNA modification processes that are prevalent in all domains of life.Support or Funding InformationThis work was supported in part by the Graduate Division of Biological and Biomedical Sciences (GDBBS) of the Laney Graduate School (LGS) of Emory University, NIH grant R01‐AI088025, and the Antibiotic Resistance and Therapeutic Discovery Training Program (ARTDTP) T32‐AI 106699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The tuberactinomycins are a family of cyclic peptide ribosome-targeting antibiotics with a long history of use as essential second-line treatments for drug-resistant tuberculosis. Beginning with the identification of viomycin in the early 1950s, this mini-review briefly describes tuberactinomycin structures and biosynthesis, as well as their past and present application in the treatment of tuberculosis caused by infection with Mycobacterium tuberculosis. More recent studies are also discussed that have revealed details of tuberactinomycin action on the ribosome as well as resistance mechanisms that have emerged since their introduction into the clinic. Finally, future applications of these drugs are considered in the context of their recent removal from the World Health Organization’s List of Essential Medicines.