Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA

• Type of Material: Article
• Authors: Jack A. Dunkle, Emory University; Kellie Vinal, Emory University; Pooja M. Desai, Emory University; Natalia Zelinskaya, Emory University; Miloje Savic, Emory University; Dayne M. West, Emory University; Graeme Conn, Emory University; Christine Dunham, Emory University
• Language: English
• Date: 2014-04-29
• Publisher: National Academy of Sciences
• Publication Version: Final Published Version
• Copyright Statement: 2014 National Academy of Sciences
• Final Published Version (URL): http://dx.doi.org/10.1073/pnas.1402789111
• Title of Journal or Parent Work: Proceedings of the National Academy of Sciences
• Volume: 111
• Issue: 17
• Start Page: 6275
• End Page: 6280
• Grant/Funding Information: Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under Contract DE-AC02-06CH11357.; This work is based on research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team (NE-CAT) beamlines, which is supported by NIH-National Center for Research Resources Award RR-15301, and at the Southeastern Regional Collaborative Access Team beamline.; This work was funded by National Institutes of Health (NIH)-National Institute of Allergy and Infectious Diseases Grant R01-AI088025 (to G.L.C.) and, in part, by NIH-National Institute of General Medical Sciences Grant R01-GM093279 (to C.M.D.). C.M.D. is a Pew Scholar in the Biomedical Sciences.
• Keywords: ANTIBIOTIC-RESISTANCE; Science & Technology - Other Topics; RNA METHYLTRANSFERASES; METHYLATION; Multidisciplinary Sciences; Science & Technology; BACTERIA; base flipping; CRYSTAL-STRUCTURE; PURIFICATION; SUBUNIT; BIOGENESIS; CRYSTALLIZATION; antibiotic resistance; STRUCTURAL INSIGHTS; RNA modification
• Research Categories: Health Sciences, Pharmacology; Chemistry, Biochemistry

Abstract

Aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. Here, we report the structural basis for 30S recognition by the novel plasmid-mediated aminoglycoside- resistance rRNA methyltransferase A (NpmA). These studies are supported by biochemical and functional assays that define the molecular features necessary for NpmA to catalyze m1A1408 modification and confer resistance. The requirement for the mature 30S as a substrate for NpmA is clearly explained by its recognition of four disparate 16S rRNA helices brought into proximity by 30S assembly. Our structure captures a "precatalytic state"in which multiple structural reorganizations orient functionally critical residues to flip A1408 from helix 44 and position it precisely in a remodeled active site for methylation. Our findings provide a new molecular framework for the activity of aminoglycoside- resistance rRNA methyltransferases that may serve as a functional paradigm for other modification enzymes acting late in 30S biogenesis.