30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC

• Type of Material: Article
• Authors: Pooja Srinivas, Emory University; Meisam Nosrati, Emory University; Natalia Zelinskaya, Emory University; Debayan Dey, Emory University; Lindsay R. Comstock, Wake Forest University; Christine Dunham, Emory University; Graeme Conn, Emory University
• Language: English
• Date: 2023-06-12
• Publisher: PNAS
• Publication Version: Final Published Version
• Copyright Statement: © 2023 the Author(s). Published by PNAS.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): https://doi.org/10.1073/pnas.2304128120
• Title of Journal or Parent Work: Biochemistry
• Volume: 120
• Issue: 25
• Start Page: e2304128120
• Grant/Funding Information: This work was supported by the NIH awards R01-AI088025 (C.M.D. and G.L.C.), T32-AI106699 (P.S.), and T32-GM008602 (P.S.), and the Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award (C.M.D.). This study was also supported by the Robert P. Apkarian Integrated Electron Microscopy Core at Emory University, which is subsidized by the Emory School of Medicine and Emory College of Arts and Sciences. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research.
• Keywords: helix 44; methylation; ribosome; aminoglycoside resistance; methyltransferase
• Research Categories: Chemistry, Biochemistry; Health Sciences, Pharmacology; Biology, Microbiology

Abstract

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine analog to trap the complex in a postcatalytic state to enable determination of a global 3.0 Å cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder-to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to resensitize bacterial pathogens to aminoglycosides.