Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance
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 (m8...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-01-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/70017 |
| _version_ | 1811227322560282624 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Kaitlyn Tsai |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T09:40:48Z |
| format | Article |
| id | doaj.art-24316c8437014466b4b990837558a00d |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T09:40:48Z |
| publishDate | 2022-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-24316c8437014466b4b990837558a00d2022-12-22T03:38:04ZengeLife Sciences Publications LtdeLife2050-084X2022-01-011110.7554/eLife.70017Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistanceKaitlyn Tsai0https://orcid.org/0000-0002-0605-2720Vanja Stojković1https://orcid.org/0000-0002-9885-3428Lianet Noda-Garcia2Iris D Young3https://orcid.org/0000-0003-4713-9504Alexander G Myasnikov4Jordan Kleinman5https://orcid.org/0000-0001-7146-824XAli Palla6https://orcid.org/0000-0001-7380-3881Stephen N Floor7https://orcid.org/0000-0002-9965-9694Adam Frost8https://orcid.org/0000-0003-2231-2577James S Fraser9https://orcid.org/0000-0002-5080-2859Dan S Tawfik10https://orcid.org/0000-0002-5914-8240Danica Galonić Fujimori11https://orcid.org/0000-0002-4066-9417Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, United StatesDepartment of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, United StatesDepartment of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, IsraelDepartment of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, United StatesDepartment of Biochemistry and Biophysics, University of California San Francisco, San Francisco, United StatesDepartment of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, United StatesDepartment of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, United StatesHelen 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 StatesDepartment of Biochemistry and Biophysics, University of California San Francisco, San Francisco, United States; Quantitative Biosciences Institute, University of California San Francisco, San Francisco, United StatesDepartment 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 StatesDepartment of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, IsraelDepartment 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 StatesAlteration 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.https://elifesciences.org/articles/70017Cfrdirected evolutionantibiotic resistanceRNA modificationspeptidyl transferase centercryoEM |
| spellingShingle | 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 Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance eLife Cfr directed evolution antibiotic resistance RNA modifications peptidyl transferase center cryoEM |
| title | Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance |
| title_full | Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance |
| title_fullStr | Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance |
| title_full_unstemmed | Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance |
| title_short | Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance |
| title_sort | directed evolution of the rrna methylating enzyme cfr reveals molecular basis of antibiotic resistance |
| topic | Cfr directed evolution antibiotic resistance RNA modifications peptidyl transferase center cryoEM |
| url | https://elifesciences.org/articles/70017 |
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