50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

• Type of Material: Article
• Authors: Zane T Laughlin, Emory University; Suparno Nandi, Emory University; Debayan Dey, Emory University; Natalia Zelinskaya, Emory University; Marta A Witek, Emory University; Pooja Srinivas, Emory University; Ha An Nguyen, Emory University; Emily G Kuiper, Emory University; Lindsay R Comstock, Wake Forest University; Christine Dunham, Emory University; Graeme Conn, Emory University
• Language: English
• Date: 2022-04-05
• Publisher: NATL ACAD SCIENCES
• Publication Version: Final Published Version
• Copyright Statement: © 2022 the Author(s). Published by PNAS.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1073/pnas.2120352119
• Title of Journal or Parent Work: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• Volume: 119
• Issue: 14
• Start Page: e2120352119
• End Page: e2120352119
• Keywords: STRUCTURAL BASIS; Science & Technology; methyltransferase; 16S; mycobacteria; PROTEIN; RESISTANCE; antibiotic resistance; ribosome; METHYLATION; BACTERIAL RIBOSOME; DNA; Multidisciplinary Sciences; CRYO-EM; Science & Technology - Other Topics; RNA modification; MUTATION; CRYSTAL-STRUCTURE
• Research Categories: Chemistry, Biochemistry

Abstract

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis 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-20-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure 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. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.