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Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

Nature Structural & Molecular Biology volume 29, pages 162–171 (2022)Cite this article

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Abstract

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.

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Fig. 1: Radezolid induces ribosome stalling with alanine in the penultimate position.
Fig. 2: Cryo-EM structures of LZD- and RZD-stalled ribosome complexes.
Fig. 3: Nascent peptide with penultimate alanine stabilizes oxazolidinone binding to the ribosome.
Fig. 4: In the stalled complexes, rRNA nucleotides provide additional contacts with the oxazolidinone antibiotic.
Fig. 5: The Cfr m8A2503 modification reduces RZD-dependent ribosome stalling.
Fig. 6: Model for oxazolidinone context-specific inhibition of translation.

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Data availability

Atomic coordinates for all presented structures have been deposited in the Protein Data Bank and EMDB under the following accession numbers: LZD-SRC, PDB ID 7S1G, EMDB-24800; LZD-70S, PDB ID 7S1H, EMDB-24801; RZD-SRC, PDB ID 7S1I, EMDB-24802; RZD-70S, PDB ID 7S1J, EMDB-24803; RZD-SRC*, PDB ID 7S1K, EMDB-24804. The following datasets were used to generate the presented structures: WT E. coli 50S ribosomal subunit, PDB 6PJ6; ErmBL-stalled ribosome complex, PDB 5JU8.Source data are provided with this paper.

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Acknowledgements

We thank D. Bulkley and G. Gilbert for technical support at the UCSF Center for Advanced CryoEM, which is supported by the National Institutes of Health (S10OD020054 and S10OD021741) and the Howard Hughes Medical Institute (HHMI). Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (U24 GM129541). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank E. Eng, E. Kopylov and the rest of the staff for technical support at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, which is supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539) and by grants from the Simons Foundation (SF349247) and New York State. We acknowledge support from NIAID (R01AI137270 to D.G.F. and F32AI148120 to D.J.L.), a W.M. Keck Foundation Medical Research Grant (to J.S.F. and D.G.F.), a Sanghvi-Agarwal Innovation Award (J.S.F.), UCSF PBBR and Bowes Biomedical Investigator Program awards (D.G.F.), R01 AI 125518 (A.S.M. and N.V.-L.) and R35 GM 127124 (A.S.M.), NSF GRFP (1650113 to K.T.), the UCSF Discovery Fellowship (K.T.) and NIH F32-GM133129 (I.D.Y.).

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Author notes

  1. These authors contributed equally: Kaitlyn Tsai, Vanja Stojković, D. John Lee.

Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

    Kaitlyn Tsai, Vanja Stojković & Danica Galonić Fujimori

  2. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA

    D. John Lee, Iris D. Young & James S. Fraser

  3. Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Teresa Szal, Dorota Klepacki, Nora Vázquez-Laslop & Alexander S. Mankin

  4. Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Teresa Szal, Dorota Klepacki, Nora Vázquez-Laslop & Alexander S. Mankin

  5. Quantitative Biosciences Institute, University of California San Francisco, San Francisco, CA, USA

    James S. Fraser & Danica Galonić Fujimori

  6. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA

    Danica Galonić Fujimori

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Contributions

K.T. performed antibiotic resistance experiments and structural analysis, assisted with model refinement, prepared figures and wrote the manuscript. V.S. prepared ribosome samples, performed model refinement and edited the manuscript. D.J.L. performed cryo-EM analysis, performed model refinement, prepared figures and edited the manuscript. I.D.Y. performed structural analysis, performed model refinement, prepared figures and edited the manuscript. T.S. and D.K. performed in vitro translation experiments. N.V.-L. and A.S.M. interpreted the data and edited the manuscript. J.S.F. and D.G.F. conceived and supervised the research, assisted in data interpretation and edited the manuscript.

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Correspondence to James S. Fraser or Danica Galonić Fujimori.

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Nature Structural & Molecular Biology thanks Daniel Wilson, Marina Rodnina and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Processing workflow, classification tree, FSC curves, and local resolution estimation for each map.

Micrographs were CTF corrected and curated for ice quality, followed by unsupervised particle picking. 2D classification was used to remove residual ice particles, with all others subjected to multi-class 3D classification and refinement approaches to remove particles without tRNA (for SRCs) and select for good particles. Final FSC curves are presented along with center-slab representation of local resolution. All steps were carried out within the cisTEM (v1.0.0-beta) framework with the exception of local resolution estimation, which used the Relion (v3.1.2) implementation.

Extended Data Fig. 2 Confirmation of oxazolidinone-induced ribosome stalling at the Phe5 codon.

(a) Location of the codon-anticodon interaction within the linezolid-stalled 70S ribosome complex. (b) Density for the codon-anticodon interaction is best modeled as UUCmRNA:GAAtRNA-Phe rather than GCAmRNA:UGCtRNA-Ala which would correspond to stalling at the upstream Ala4 codon. The figure was generated from unsharpened maps and the density is contoured at 4σ.

Extended Data Fig. 3 Overview of the binding mode of linezolid and radezolid within the ribosomal PTC.

(a) Structural overlay of linezolid (LZD) bound to a H. marismortui ribosome (PDB: 3CPW) and linezolid-stalled E. coli ribosome complex (LZD-SRC), highlighting binding within the A-site cleft. (b) Overlay of E. coli LZD-SRC and LZD-only bound to a D. radiodurans (PDB: 3DLL) or S. aureus ribosome (PDB: 4WFA). (c) Overlay of E. coli LZD-SRC and MRSA ribosome bound to LZD analog (LZD-5, PDB: 6DDD) or contezolid (PDB: 6WQN). (d) Structural overlay of radezolid (RZD) bound to a MRSA ribosome (PDB: 6WQQ) and radezolid-stalled E. coli ribosome complex (RZD-SRC), highlighting the A-site cleft. (e) Overlay of E. coli RZD-SRC and E. coli ribosome bound to cadazolid (PDB: 6QUL). (f) Overlay of E. coli RZD-SRC and H. marismortui ribosome bound to a biaryl-oxazolidinone (PDB: 3CXC). Insets for panels (e) and (f) highlight the π-stacking interaction between the oxazolidinone D-ring and A2602. All overlays in this figure were generated by alignment of 23S rRNA nucleotides. E. coli numbering is used for all figure panels.

Extended Data Fig. 4 Oxazolidinone binding modes in antibiotic-only and stalled ribosome complexes.

(a) Coordination of a solvent molecule or ion to (a) linezolid (LZD) and surrounding nucleic acids in the linezolid-stalled E. coli ribosome complex (LZD-SRC). Coulomb potential is shown 1.5 Å from the ion and LZD ligand, contoured at 5σ in the unsharpened map, and coordination distances are shown in Ångstroms. (b) Coordination of a solvent molecule or ion to radezolid (RZD) and surrounding nucleic acids in the radezolid-stalled E. coli ribosome complex (RZD-SRC). Coulomb potential is shown 1.5 Å from the ion and RZD ligand, contoured at 5σ in the unsharpened map, and coordination distances are shown in Ångstroms. (c) Comparison of linezolid (LZD) binding modes in the antibiotic-only (LZD-70S, antibiotic in orange) versus LZD-stalled complex (LZD-SRC, antibiotic in yellow). (d) Comparison of radezolid (RZD) binding modes in the antibiotic-only (RZD-70S, antibiotic in teal) versus RZD-stalled complex (RZD-SRC, antibiotic in green). Structural overlays in this figure were performed by aligning the 23S rRNA chain.

Extended Data Fig. 5 In silico analysis of residues at the penultimate position.

Substitution of glycine (Gly), proline (Pro), serine (Ser), threonine (Thr), or valine (Val) for alanine (Ala) at the penultimate position results in either reduced surface complementarity Sc as measured by the sc tool in CCP464 (blue bars) or increased clashscore (orange bars) in all cases except serine, which has two rotamers that slightly improve surface complementarity without clashing. Surface complementarity is calculated between two selections of atoms, the first of which is the sequence of three residues centered on the penultimate position, and the second of which is the linezolid ligand and the three nucleotides close enough to potentially interact either favorably or disfavorably with the peptide sequence in the first selection. Surface complementarity is calculated without hydrogens modeled due to limitations of the CCP4 implementation. Alanine and serine are most highly favored for ligand binding by these metrics.

Extended Data Fig. 6 Stabilization of PTC nucleotides in the oxazolidinone-stalled ribosome complexes.

Direct comparison of nucleotide density in the linezolid-only bound (LZD-70S) and the linezolid-stalled complex (LZD-SRC), displaying m2A2503 as a control nucleotide and dynamic nucleotides U2506 and U2585. Direct comparison of nucleotide density in the radezolid-only bound (RZD-70S) and the radezolid-stalled complex (RZD-SRC), displaying A2503 and dynamic nucleotides U2506, U2585, A2602. Of note, U2506 is modeled in two conformations in LZD/RZD-70S; the relevant conformation for density comparison to SRCs is presented. Coulomb potential density is contoured at 4.0σ in surface representation and 1.0σ in mesh representation from unsharpened cryo-EM density maps. Blue arrows highlight the rRNA bases which exhibit the most prominent changes in density between the antibiotic-only and stalled complexes.

Extended Data Fig. 7 Steric occlusion of A-site tRNA binding by oxazolidinone antibiotics.

Linezolid (LZD, yellow) and radezolid (RZD, green) prevent binding of A-site tRNAs (blue, from PDB: 7K00). Prominent steric clashes between the A-tRNA and LZD C-ring and RZD C/D-ring are highlighted. Structural overlays were performed by alignment of 23S rRNA nucleotides.

Extended Data Fig. 8 LZD and RZD activity against Cfr-modified ribosomes.

(a) Minimum inhibitory concentration of linezolid (LZD) and radezolid (RZD) required to inhibit growth of E. coli BW25113 acrB::kan transformed with either empty pZA plasmid or pZA encoding the evolved Cfr variant CfrV7, which achieves near-complete m8A2503 methylation43. MIC values were determined from two biological replicates. (b) In vitro activity of LZD and RZD against wildtype (WT) and Cfr-modified ribosomes determined by inhibition of sfGFP translation. Percent (%) translation calculated as the percentage of sfGFP translation at the tested antibiotic concentration compared to reactions containing no antibiotic determined from two independent experiments which are plotted as individual data points. N.D. indicates that the IC50 value was not determined. (c) Toeprinting analysis of LZD- and RZD-induced stalling of WT or Cfr-modified ribosomes within the 5’ region of the sfGFP ORF. Drug-specific toeprint bands are indicated by red arrows. ‘None’ designates reactions lacking ribosome-targeting antibiotics. The control antibiotic retapamulin (RET) was used to stall ribosomes at the start codon indicated by the blue arrow51. All antibiotics were added to a final concentration of 50 µM. Toeprinting experiments were performed at least twice with similar results. (d) Overlay of a vacant Cfr-modified E. coli ribosome43 with the RZD-stalled, Cfr-modified E. coli ribosome performed by alignment of 23S rRNA nucleotides 2000–3000. (e) Close-up view of the penultimate alanine, RZD, and C8-methylated A2503 in sphere representation.

Source data

Extended Data Fig. 9 Density for the nascent chain in WT and Cfr-modified RZD-stalled ribosome complexes.

Density comparison for the alanine-containing MFKAF nascent peptide (purple) between RZD-stalled complexes with (a) WT and (b) Cfr-modified ribosome. RZD shown in green and G2505 shown in gray. Coulomb potential density is contoured at 4.0σ in surface representation and 1.0σ in mesh representation from unsharpened cryo-EM density maps and carved at 1.8 Ångstroms from the part of the model shown.

Extended Data Fig. 10 A2062 adopts a lumen conformation in the RZD-stalled complex with a Cfr-modified ribosome.

(a) A2062 adopts both the lumen and the rotated, tunnel wall conformation in the RZD-only bound ribosome. In the rotated wall conformation, A2062 engages in H-bonding interactions with A2503 and RZD. (b) A2062 is only observed in the rotated conformation to H-bond with A2503 and RZD in the WT stalled complex. (c) A2062 is observed in the lumen conformation in the RZD-stalled complex with a Cfr-modified ribosome. Coulomb potential density is contoured at 4.0σ in surface representation and 1.0σ in mesh representation from unsharpened cryo-EM density maps.

Supplementary information

Source data

Source Data Fig. 1

Uncropped toeprinting gel.

Source Data Fig. 5

Uncropped toeprinting gel.

Source Data Extended Data Fig. 8

Numerical data for panel b.

Source Data Extended Data Fig. 8

Uncropped toeprinting gel.

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Tsai, K., Stojković, V., Lee, D.J. et al. Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics. Nat Struct Mol Biol 29, 162–171 (2022). https://doi.org/10.1038/s41594-022-00723-9

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