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 (SAM) analog to trap the complex in a post-catalytic state to enable determination of an overall 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 re-sensitize bacterial pathogens to aminoglycosides.
Significance Increasing prevalence of bacterial antibiotic resistance threatens our ability to treat bacterial infections and with it, many other facets of modern healthcare. For the ribosome-targeting aminoglycoside antibiotics, diverse pathogenic bacteria have acquired ribosomal RNA (rRNA) methyltransferase enzymes that confer exceptionally high-level resistance through site-specific modification of the drug binding site. Here, we define the molecular basis for ribosomal substrate recognition and modification by an enzyme (RmtC) representing the most clinically prevalent methyltransferase family. Specifically, RmtC exploits a conserved rRNA surface for binding and induces significant disruption of the rRNA structure to capture the target nucleotide for modification via a “base flipping” mechanism. These insights also present a platform for methyltransferase inhibitor development to extend usefulness of aminoglycoside antibiotics.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Competing Interest Statement: The authors declare no competing interests.
Text and figure updates to provide additional detail on cryo-EM structure determination. Additional reorganization of figures and tables between main manuscript and supporting information.
