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Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it

Abstract

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.

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Fig. 1: T. thermophilus HB27 strain expressing Cfr-like methylase.
Fig. 2: Electron density maps of C2,C8-dimethylated (top) and C2-methylated (bottom) A2503 residue of the 23S rRNA in T. thermophilus 70S ribosome.
Fig. 3: Structure of the Cfr-modified 70S ribosome.
Fig. 4: Structural basis for Cfr-mediated resistance to PTC-acting antibiotics.
Fig. 5: Structure of IBX bound to the Cfr-methylated 70S ribosome.
Fig. 6: Comparison of the structures of TYL bound to the Cfr-modified and WT 70S ribosomes.

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Data availability

Coordinates and structure factors were deposited in the RCSB Protein Data Bank with accession codes: 8G29 for the A2503-C2,C8-dimethylated T. thermophilus 70S ribosome in complex with mRNA, aminoacylated A-site Phe-NH-tRNAPhe, aminoacylated P-site fMet-NH-tRNAiMet and deacylated E-site tRNAPhe; 8G2A for the A2503-C2,C8-dimethylated T. thermophilus 70S ribosome in complex with mRNA, aminoacylated A-site Phe-NH-tRNAPhe, peptidyl P-site fMTHSMRC-NH-tRNAiMet and deacylated E-site tRNAPhe. 8G2B for the A2503-C2,C8-dimethylated T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMet-NH-tRNAiMet, deacylated E-site tRNAPhe and iboxamycin; 8G2C for the A2503-C2,C8-dimethylated T. thermophilus 70S ribosome in complex with mRNA, aminoacylated A-site Phe-NH-tRNAPhe, aminoacylated P-site fMet-NH-tRNAiMet, deacylated E-site tRNAPhe and tylosin; 8G2D for the wild-type T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, deacylated P-site tRNAiMet, deacylated E-site tRNAPhe and tylosin. All previously published structures that were used in this work for structural comparisons were retrieved from the RCSB Protein Data Bank: PDB entries 6XHW, 8CVL, 7LVK, 7RQE, 7S1G, 7S1I, 5DOY, 5VP2, 4V7V, 7RQ8, 1K9M, 1KD1, 1K8A. No sequence data were generated in this study. Analyzed protein sequences are presented with their corresponding accession numbers in the phylogenetic tree (Supplementary Fig. 1) for retrieval from the NCBI protein database. Source data are provided with this paper.

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Acknowledgements

We thank A. Mankin and N. Vazquez-Laslop for providing E. coli SQ171 ΔtolC strains and for valuable discussions. We thank the staff at NE-CAT beamlines 24ID-C and 24ID-E for help with X-ray diffraction data collection, especially M. Capel, F. Murphy, S. Banerjee, I. Kourinov, D. Neau, J. Schuermann, N. Sukumar, A. Lynch, J. Withrow, K. Perry, A. Kaya and C. Salbego. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (NIH) (grant on. P30-GM124165 to NE-CAT). The Eiger 16M detector on 24ID-E beamline is funded by an NIH-ORIP HEI grant (grant no. S10-OD021527 to NE-CAT). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This work was supported by the National Institute of Allergy and Infectious Diseases of the NIH (grant no. R01-AI168228 to A.G.M. and grant no. R21-AI163466 to Y.S.P.), the National Institute of General Medical Sciences of the NIH (grant no. R01-GM094157 to S.T.G. and grant no. R01-GM132302 to Y.S.P.), the National Science Foundation (grant no. MCB-1907273 to Y.S.P.), the USDA National Institute for Food and Agriculture (Hatch Project no. 1016013 to S.T.G.), the Illinois State startup funds (to Y.S.P.), the Swedish Research Council (Ventenskapsrådet) (grant nos. 2019-01085 and 2022-01603 to G.C.A.), the Knut and Alice Wallenberg Foundation (grant no. 2020.0037 to G.C.A.) and the Carl Tryggers Stiftelse för Vetenskaplig Forskning (grant no. CTS19:24 to G.C.A.). K.J.Y.W. was supported by a National Science Scholarship (Ph.D.) from the Agency for Science, Technology and Research (Singapore). The funders had no role in study design, data collection and analysis, decision to publish or manuscript preparation.

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Authors and Affiliations

Authors

Contributions

E.V.A. with help from S.M.B. and M.S.S. constructed the T. thermophilus HB27 strain expressing Cfr-like methylases. G.C.A. performed phylogenetic analysis and identified putative thermostable cfr-like genes. B.I.C.T. and K.J.Y.W. synthesized iboxamycin. E.E.K. prepared ΔrlmN knock-out T. thermophilus HB27 strain. E.V.A. performed the assessment of A2503-C8-methylation using primer extension assay. E.V.A. and E.A.S. grew T. thermophilus cells and purified Cfr-modified 70S ribosomes. E.A.S. and E.V.A. prepared hydrolysis-resistant aminoacyl- and peptidyl-tRNAs. E.V.A. with help from B.I.C.T. and K.J.Y.W. performed microbiological assays. E.V.A., E.A.S. and Y.S.P. designed and performed X-ray crystallography experiments. A.G.M., Y.S.P. and S.T.G. supervised the experiments. All authors interpreted the results. E.V.A., B.I.C.T., K.J.Y.W., A.G.M. and Y.S.P. wrote the manuscript.

Corresponding authors

Correspondence to Andrew G. Myers or Yury S. Polikanov.

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Competing interests

A.G.M. is an inventor in a provisional patent application submitted by the President and Fellows of Harvard College covering oxepanoprolinamide antibiotics described in this work. A.G.M. has filed the following international patent applications: WO/2019/032936 ‘Lincosamide Antibiotics and Uses Thereof’ and WO/2019/032956 ‘Lincosamide Antibiotics and Uses Thereof’. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Comparison of the structures of Cfr-modified and wild-type 70S ribosomes from T. thermophilus and E. coli.

Superpositioning of the previously reported structures of T. thermophilus WT 70S ribosome containing aminoacylated Phe-tRNAPhe in the A site and either fMet-tRNAiMet (a, b; PDB entry 6XHW ref. 24) or fMTHSMRC-peptidyl tRNAiMet (c, d; PDB entry 8CVL ref. 26) in the P site, or E. coli WT 70S ribosome containing radezolid in the A site and fMFKAF-peptidyl-tRNAPhe in the P site (e, f; PDB entry 7S1I ref. 28) with the structures of the same complexes containing Cfr-modified nucleotide A2503 of the 23S rRNA. All structures were aligned based on domain V of the 23S rRNA. (a, c, e) Comparisons of the positions of key 23S rRNA nucleotides around the PTC. (b, d, f) Comparisons of the positions of A- and P-site substrates relative to nucleotides A2062 and A2503. Nucleotides of the Cfr-modified and unmodified ribosomes are shown in blue and light blue, espectively. The Cfr-modified residue A2503 is highlighted in navy blue, with the C8-methyl group shown in orange. E. coli nucleotide numbering is used. H-bonds are shown with dotted lines.

Extended Data Fig. 2 Schematic diagrams of H-bond rearrangement between nucleotides in position 2062 and A2503 of the 23S rRNA upon Hoogsteen base pair formation.

(a) Formation of the symmetric trans A-A Hoogsteen base pair between A2062 and m2A2503 observed in the structures of 70S ribosome. Note that the formation of this base pair requires the N7-atoms of both adenines to be deprotonated in order to serve as H-bond acceptors of the N6-protons of the base-paired nucleotide. (b) The same Hoogsteen base pair is impossible with a guanine nucleotide in position 2062 of the 23S rRNA due to the inability to form an H-bond between O6 of G2062 and N7 of m2A2503.

Extended Data Fig. 3 Electron density maps of 23S rRNA in wild-type and Cfr-modified T. thermophilus 70S ribosome.

2Fo-Fc electron difference Fourier maps (blue mesh) of A2062 and A2503 residues of 23S rRNA in the wild-type (a) or Cfr-modified (b, c) T. thermophilus 70S ribosome carrying aminoacylated Phe-tRNAPhe in the A site and either fMet-tRNAiMet (a, b) or fMTHSMRC-peptidyl-tRNAiMet (c) in the P site. The structure and the electron density map of the wild-type ribosome complex (a) are from PDB entry 6XHW ref. 24. Carbon atoms are colored light blue for the C8-unmethylated A2503 (a) and blue for the Cfr-modified A2503 (b, c); nitrogens are dark blue; oxygens are red.

Extended Data Fig. 4 Interactions of fMTHSMRC-peptidyl-tRNAs with wild-type and Cfr-modified T. thermophilus 70S ribosome.

Close-up views of the aminoacyl and peptidyl moieties of A-site Phe-tRNAPhe and P-site fMTHSMRC-tRNAiMet in the wild-type (a, b; PDB entry 8CVL ref. 26) or Cfr-modified (c, d) T. thermophilus 70S ribosome. H-bonds are shown by black dotted lines. Stacking interactions between the aromatic side chain of His3 of fMTHSMRC-peptidyl-tRNA and A2062 nucleobase of the 23S rRNA are indicated by a black arrow.

Extended Data Fig. 5 Comparison of the structures of WT and Cfr-modified ribosomes from E. coli and T. thermophilus.

(a) Superpositioning of the previous structures of WT 70S ribosomes carrying P-site peptidyl-tRNAs from T. thermophilus (light blue, PDB entry 8CVL ref. 26) and E. coli (teal, PDB entry 7S1I ref. 28). (b) Superpositioning of the new structure of Cfr-modified 70S ribosome carrying P-site peptidyl-tRNAs from T. thermophilus (blue) and E. coli (light teal, PDB entry 7S1K ref. 28).

Extended Data Fig. 6 Comparison of electron density maps of iboxamycin (IBX) in complex with Cfr-modified and wild-type T. thermophilus 70S ribosomes.

2Fo-Fc electron density maps (blue mesh) contoured at 1.0σ of IBX in complex with Cfr-modified (a, b, yellow) or wild-type (c, d, teal) T. thermophilus 70S ribosomes. The C8-methyl group of m2m8A2503 is highlighted in orange. The structure and the electron density map of IBX in complex with wild-type 70S ribosome (c, d) are from PDB entry 7RQ8 ref. 36. Carbon atoms are colored navy blue for the Cfr-modified m2m8A2503 (a, b) and light blue for the WT m2A2503 (c, d); nitrogens are dark blue; oxygens are red.

Extended Data Fig. 7 Comparison of the m2m8A2503 positions in the Cfr-modified ribosome in the presence and absence of iboxamycin.

(a) Superposition of the structures of drug-free Cfr-modified 70S ribosome containing m2m8A2503 residue (shown as navy blue spheres with C8-methyl group highlighted in red) with the structure of ribosome-bound iboxamycin (IBX, yellow). (b) Structure of Cfr-modified 70S ribosome containing m2m8A2503 residue (shown as blue spheres with C8-methyl highlighted in orange) in complex with iboxamycin (IBX, yellow). Note that binding of iboxamycin to the Cfr-modified ribosome causes an ~1 Å shift of the m2m8A2503 residue away from the drug.

Extended Data Fig. 8 Structural basis for the Cfr-mediated resistance to 16-membered macrolides.

(a–c) Chemical structures of tylosin (a), spiramycin (b), and carbomycin (c). (d-f) Superposition of the structures of Cfr-modified T. thermophilus 70S ribosome containing C8-methylated A2503 residue in the 23S rRNA (blue) with the previously reported structures of 16-membered macrolides, such as tylosin (d, green; PDB entry 1K9M ref. 34), spiramycin (e, light teal; PDB entry 1KD1 ref. 34), or carbomycin (f, tealF PDB entry 1K8A ref. 34) in complex with the 50S ribosomal subunit from the archaeon H. marismortui. The degrees of steric overlaps between the C8-methyl group of the m2m8A2503 nucleotide and each PTC-acting drug are shown in yellow. These numbers reflect the distance in Å that the drug and the m2m8A2503 residue need to move away from each other to avoid the steric clash. Note that the C8-methyl group of m2m8A2503 (highlighted in orange) can physically interfere with the binding of 16-membered macrolides.

Extended Data Fig. 9 Structures of tylosin (TYL) bound to the Cfr-modified and WT 70S ribosomes.

(a–f) Electron density map (blue mesh) contoured at 1.0σ of TYL (green or magenta) in complex with the Cfr-modified (a–c) or wild-type (d–f) T. thermophilus 70S ribosome containing m2m8A2503 (dark blue with C8-methyl group highlighted in orange) or m2A2503 (light blue) residues in the 23S rRNA, respectively. The chemical structure of tylosin is shown in panel d. The reactive acetaldehyde group (highlighted in red) at C6 of tylosin’s macrolactone ring forms a covalent bond with the exocyclic N6-amino group of A2062 in the T. thermophilus ribosome. Note that the mycinose moiety at C14 of the macrolactone ring of tylosin is well-resolved in the electron density maps.

Extended Data Fig. 10 Comparisons of A2503 positions in Cfr-modified ribosomes in the presence and absence of iboxamycin or tylosin.

Superposition of the structures of Cfr-modified 70S ribosome both containing m2m8A2503 residue in the presence (blue) and absence (navy blue) of iboxamycin (a, b, yellow) or tylosin (c, d, green). Note that while binding of iboxamycin to the Cfr-modified ribosome causes an ~1 Å shift of m2m8A2503 residue away from the drug, binding of tylosin does not affect the position of the m2m8A2503 residue.

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Supplementary Tables 1–5, Figs. 1 and 2 and references.

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Unprocessed gel for Fig. 1c.

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Unprocessed gel for Fig. 1d.

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Aleksandrova, E.V., Wu, K.J.Y., Tresco, B.I.C. et al. Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it. Nat Chem Biol 20, 867–876 (2024). https://doi.org/10.1038/s41589-023-01525-w

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