Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

eLife

Alexander G Myasnikov¹  Adam Frost²  Iris D Young³  James S Fraser⁴  Dan S Tawfik⁵  Lianet Noda-Garcia⁵  Kaitlyn Tsai⁶  Vanja Stojković⁶  Ali Palla⁶  Jordan Kleinman⁶  Danica Galonić Fujimori⁷  Stephen N Floor⁸ 

[1] Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, United States;Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, United States;Quantitative Biosciences Institute, University of California San Francisco, San Francisco, United States;Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, United States;Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, United States;Quantitative Biosciences Institute, University of California San Francisco, San Francisco, United States;Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel;Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, United States;Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, United States;Quantitative Biosciences Institute, University of California San Francisco, San Francisco, United States;Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, United States;Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, United States;Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, United States;

关键词: Cfr;    directed evolution;    antibiotic resistance;    RNA modifications;    peptidyl transferase center;    cryoEM;    E. coli;   

DOI  :  10.7554/eLife.70017

来源: eLife Sciences Publications, Ltd

【 摘 要 】

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

【 授权许可】

CC BY