期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 46, 页码 17642-17653出版社
ELSEVIER
DOI: 10.1074/jbc.RA119.011181
关键词
antibiotic resistance; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome; RNA methylation; RNA methyltransferase; aminoglycoside antibiotics; m1A1408; m7G1405; RmtC
资金
- National Institutes of Health [S10 RR25528, S10 RR028976]
- U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]
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 (m(7)G1405) 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 RmtC at 3.14 angstrom resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m(7)G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m(1)A1408) 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 of m(7)G1405 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 high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.
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