Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition

• Type of Material: Article
• Authors: Meisam Nosrati, Emory University; Debayan Dey, Emory University; Atousa Mehrani, Florida State University; Sarah E. Strassler, Emory University; Natalia Zelinskaya, Emory University; Eric D. Hoffer, Emory University; Scott M. Stagg, Florida State University; Christine Dunham, Emory University; Graeme Conn, Emory University
• Language: English
• Date: 2019-11-15
• Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
• Publication Version: Final Published Version
• Copyright Statement: © 2019 Nosrati et al.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.RA119.011181
• Title of Journal or Parent Work: JOURNAL OF BIOLOGICAL CHEMISTRY
• Volume: 294
• Issue: 46
• Start Page: 17642
• End Page: 17653
• Grant/Funding Information: The Southeast Regional Collaborative Access Team is supported by its member institutions and Equipment Grants S10 RR25528 and S10 RR028976 from the National Institutes of Health. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract W-31-109-Eng-38.; This work was supported by National Institutes of Health Grants R01-AI088025 (to G. L. C.) and T32-GM008367 and Postdoctoral Fellowship DEY18F0 from the Cystic Fibrosis Foundation (to D. D.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
• Keywords: RNA methyltransferase; RmtC; m1A1408; 16S RIBOSOMAL-RNA; CATALYTIC-ACTIVITY; ribosome; antibiotic resistance; STRUCTURAL BASIS; CONTRIBUTES; SELECTIVITY; Life Sciences & Biomedicine; ANTIBIOTICS; Science & Technology; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); M(1)A1408 METHYLTRANSFERASE; MECHANISMS; Biochemistry & Molecular Biology; ENTEROBACTERIACEAE; RNA methylation; aminoglycoside antibiotics; METHYLATION; m7G1405
• Research Categories: Biology, Molecular; Biology, Genetics; Chemistry, Biochemistry

Abstract

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtCat 3.14Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for theseenzymesin the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features ofm7G1405 methyltransferase-substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future highresolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.