title: Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors.
abstract: Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.
authors.affiliation: Structure and Biophysics, Discovery Sciences, AstraZeneca Pharmaceuticals, Waltham, MA 02451;
authors.forename: Alexis I
authors.initials: AI
authors.lastname: Cocozaki
authors.affiliation: Department of Physiology and Biophysics and the Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medicine, New York, NY 10065;
authors.forename: Roger B
authors.initials: RB
authors.lastname: Altman
authors.affiliation: Reagents and Assay Development, Discovery Sciences, AstraZeneca Pharmaceuticals, Waltham, MA 02451;
authors.forename: Jian
authors.initials: J
authors.lastname: Huang
authors.affiliation: Infection, Department of Biosciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca Pharmaceuticals, Waltham, MA 02451;
authors.forename: Ed T
authors.initials: ET
authors.lastname: Buurman
authors.affiliation: Structure and Biophysics, Discovery Sciences, AstraZeneca Pharmaceuticals, Waltham, MA 02451;
authors.forename: Steven L
authors.initials: SL
authors.lastname: Kazmirski
authors.affiliation: Reagents and Assay Development, Discovery Sciences, AstraZeneca Pharmaceuticals, Waltham, MA 02451;
authors.forename: Peter
authors.initials: P
authors.lastname: Doig
authors.affiliation: Structure and Biophysics, Discovery Sciences, AstraZeneca Pharmaceuticals, Waltham, MA 02451;
authors.forename: D Bryan
authors.initials: DB
authors.lastname: Prince
authors.affiliation: Department of Physiology and Biophysics and the Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medicine, New York, NY 10065;
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
authors.affiliation: Department of Molecular Cell Biology, University of California, Berkeley, California, CA 94720; Department of Chemistry, University of California, Berkeley, California, CA 94720.
authors.forename: Jamie H D
authors.initials: JH
authors.lastname: Cate
authors.affiliation: Structure and Biophysics, Discovery Sciences, AstraZeneca Pharmaceuticals, Waltham, MA 02451; Andrew.Ferguson@astrazeneca.com.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ferguson
mesh_terms: D000599:Amino Acids, Diamino / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D012270:Ribosomes / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D000599:Amino Acids, Diamino; D000900:Anti-Bacterial Agents; D011500:Protein Synthesis Inhibitors; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C100200:negamycin; D013752:Tetracycline
keywords: antibiotic resistance; negamycin; ribosome structure; tRNA selection; tetracycline