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

J Biol Chem. 2019 Nov 15;294(46):17642-17653. doi: 10.1074/jbc.RA119.011181. Epub 2019 Oct 8.


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 RmtC at 3.14 Å 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 these enzymes in 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 of m7G1405 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.

Keywords: RNA methylation; RNA methyltransferase; RmtC; aminoglycoside antibiotics; antibiotic resistance; m1A1408; m7G1405; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoglycosides / metabolism
  • Aminoglycosides / pharmacology
  • Anti-Bacterial Agents / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Crystallography, X-Ray
  • Drug Resistance, Bacterial*
  • Humans
  • Methyltransferases / chemistry
  • Methyltransferases / metabolism*
  • Models, Molecular
  • Protein Conformation
  • Proteus Infections / drug therapy
  • Proteus Infections / microbiology
  • Proteus mirabilis / drug effects
  • Proteus mirabilis / enzymology*
  • Proteus mirabilis / metabolism
  • RNA, Ribosomal, 16S / metabolism
  • Ribosome Subunits, Small, Bacterial / chemistry
  • Ribosome Subunits, Small, Bacterial / metabolism*
  • Substrate Specificity


  • Aminoglycosides
  • Anti-Bacterial Agents
  • Bacterial Proteins
  • RNA, Ribosomal, 16S
  • Methyltransferases

Associated data

  • PDB/4OX9
  • PDB/6PQB
  • PDB/3FRH
  • PDB/3LCV
  • PDB/6CN0
  • PDB/4V4Q