ABSTRACT
Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance. 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 of the methyltransferase RmtC, we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases. We find that the 30S binding site for these enzymes directly overlaps that of a second family of aminoglycoside-resistance 16S rRNA (m1A1408) methyltransferases, suggesting both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking on the 30S. Within RmtC we define an amino-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contribute to 30S binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the CTD are found to be critical for 16S rRNA modification but do not directly contribute to binding affinity. Thus, our studies 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 potential exploitation of unique aspects of substrate recognition for future therapeutic purposes.
Footnotes
This version of the manuscript has been revised to new preliminary analysis of 30S-methyltransferase interaction using cryo-EM. 2D classifications provide experimental evidence for binding-induced 30S head disorder. Supplemental information also now included to give more detail on protein folding quality control and protein expression in the antibiotic activity assay.
Abbreviations used are
- AME
- aminoglycoside modifying enzyme
- CA-MHB
- cation-adjusted Mueller-Hinton broth
- CTD
- carboxy-terminal domain
- FP
- fluorescence polarization
- h44
- (16S rRNA) helix 44
- MIC
- minimum inhibitory concentration
- NTD
- amino-terminal domain
- Rmt
- (aminoglycoside) resistance methyltransferase
- SAH
- S-adenosylhomocysteine
- SAM
- S-adenosyl-L-methionine
- Ti
- inflection temperature