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

View ORCID ProfileKaitlyn Tsai, View ORCID ProfileVanja Stojković, Lianet Noda-Garcia, View ORCID ProfileIris D. Young, View ORCID ProfileAlexander G. Myasnikov, View ORCID ProfileJordan Kleinman, View ORCID ProfileAli Palla, View ORCID ProfileStephen N. Floor, View ORCID ProfileAdam Frost, View ORCID ProfileJames S. Fraser, Dan S. Tawfik, View ORCID ProfileDanica Galonić Fujimori
doi: https://doi.org/10.1101/2021.03.12.435202
Kaitlyn Tsai
1Department of Cellular and Molecular Pharmacology; University of California San Francisco; San Francisco, CA 94158, USA
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Vanja Stojković
1Department of Cellular and Molecular Pharmacology; University of California San Francisco; San Francisco, CA 94158, USA
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Lianet Noda-Garcia
2Department of Biomolecular Sciences, Weizmann Institute of Science; Rehovot, Israel
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Iris D. Young
3Department of Bioengineering and Therapeutic Sciences, University of California San Francisco; San Francisco, CA 94158, USA
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Alexander G. Myasnikov
4Department of Biochemistry and Biophysics, University of California San Francisco; San Francisco, CA 94158, USA
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Jordan Kleinman
1Department of Cellular and Molecular Pharmacology; University of California San Francisco; San Francisco, CA 94158, USA
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Ali Palla
1Department of Cellular and Molecular Pharmacology; University of California San Francisco; San Francisco, CA 94158, USA
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Stephen N. Floor
5Department of Cell and Tissue Biology, University of California San Francisco; San Francisco, CA 94143, USA
6Helen Diller Family Comprehensive Cancer Center, University of California San Francisco; San Francisco, CA 94143, USA
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Adam Frost
4Department of Biochemistry and Biophysics, University of California San Francisco; San Francisco, CA 94158, USA
7Quantitative Biosciences Institute, University of California San Francisco; San Francisco, CA 94158, USA
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James S. Fraser
3Department of Bioengineering and Therapeutic Sciences, University of California San Francisco; San Francisco, CA 94158, USA
7Quantitative Biosciences Institute, University of California San Francisco; San Francisco, CA 94158, USA
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Dan S. Tawfik
2Department of Biomolecular Sciences, Weizmann Institute of Science; Rehovot, Israel
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Danica Galonić Fujimori
1Department of Cellular and Molecular Pharmacology; University of California San Francisco; San Francisco, CA 94158, USA
7Quantitative Biosciences Institute, University of California San Francisco; San Francisco, CA 94158, USA
8Department of Pharmaceutical Chemistry, University of California San Francisco; San Francisco, CA 94158, USA
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  • ORCID record for Danica Galonić Fujimori
  • For correspondence: Danica.Fujimori@ucsf.edu
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ABSTRACT

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 which achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-EM 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.

Competing Interest Statement

The authors have declared no competing interest.

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Posted May 03, 2021.
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Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance
Kaitlyn Tsai, Vanja Stojković, Lianet Noda-Garcia, Iris D. Young, Alexander G. Myasnikov, Jordan Kleinman, Ali Palla, Stephen N. Floor, Adam Frost, James S. Fraser, Dan S. Tawfik, Danica Galonić Fujimori
bioRxiv 2021.03.12.435202; doi: https://doi.org/10.1101/2021.03.12.435202
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Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance
Kaitlyn Tsai, Vanja Stojković, Lianet Noda-Garcia, Iris D. Young, Alexander G. Myasnikov, Jordan Kleinman, Ali Palla, Stephen N. Floor, Adam Frost, James S. Fraser, Dan S. Tawfik, Danica Galonić Fujimori
bioRxiv 2021.03.12.435202; doi: https://doi.org/10.1101/2021.03.12.435202

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