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Structural basis for the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol

Abstract

Ribosome-targeting antibiotics serve as powerful antimicrobials and as tools for studying the ribosome, the catalytic peptidyl transferase center (PTC) of which is targeted by many drugs. The classic PTC-acting antibiotic chloramphenicol (CHL) and the newest clinically significant linezolid (LZD) were considered indiscriminate inhibitors of protein synthesis that cause ribosome stalling at every codon of every gene being translated. However, recent discoveries have shown that CHL and LZD preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms that underlie the context-specific action of ribosome inhibitors are unknown. Here we present high-resolution structures of ribosomal complexes, with or without CHL, carrying specific nascent peptides that support or negate the drug action. Our data suggest that the penultimate residue of the nascent peptide directly modulates antibiotic affinity to the ribosome by either establishing specific interactions with the drug or by obstructing its proper placement in the binding site.

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Fig. 1: Electron density maps of the short tripeptidyl-tRNA analogs bound to the T. thermophilus 70S ribosome in the absence of CHL.
Fig. 2: Comparison of the structures of short tripeptidyl-tRNA analogs reveals no significant differences between each other and with full-length tRNAs.
Fig. 3: Electron density maps of the short tripeptidyl-tRNA analogs bound to the T. thermophilus 70S ribosome in the presence of CHL.
Fig. 4: CHL directly interacts with the stalling peptides MTI and MAI in the T. thermophilus 70S ribosome.
Fig. 5: Schematic diagram illustrating the increased affinity model.

Data availability

Coordinates and structure factors have been deposited in the RCSB Protein Data Bank with the following accession codes: 7RQA for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn and P-site peptidyl-tRNA analog ACCA-ITM; 7RQB for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn and P-site peptidyl-tRNA analog ACCA-IAM; 7RQC for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn and P-site peptidyl-tRNA analog ACCA-IFM; 7RQD for the T. thermophilus 70S ribosome in complex with protein Y, A-site deacylated tRNA analog CACCA, P-site peptidyl-tRNA analog ACCA-ITM and chloramphenicol; 7RQE for the T. thermophilus 70S ribosome in complex with protein Y, A-site deacylated tRNA analog CACCA, P-site peptidyl-tRNA analog ACCA-IAM and chloramphenicol. All previously published structures that were used in this work for structural comparisons were retrieved from the RCSB Protein Data Bank: PDB entries 6XHW, 6WDD, 1VQN, 1VY7, 6TC3, 5NWY, 6ND5, 3CPW. No sequence data were generated in this study.

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Acknowledgements

We thank J. Thaler (Innsbruck) for synthetic support for the RNA-peptide conjugates. We thank A. Mankin for his critical reading of the manuscript, as well as K. Ratia and M. Svetlov for valuable suggestions. We thank the staff at NE-CAT beamlines 24ID-C and 24ID-E for help with data collection and freezing of the crystals, especially M. Capel, F. Murphy, I. Kourinov, A. Lynch, S. Banerjee, D. Neau, J. Schuermann, N. Sukumar, J. Withrow, K. Perry, A. Kaya and C. Salbego. This work is based on 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 (P30-GM124165 to NE-CAT). The Eiger 16M detector on the 24ID-E beamline is funded by an NIH-ORIP HEI grant (S10-OD021527 to NE-CAT). This research has 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 Institutes of Health (R01-GM132302 to Y.S.P.), the National Science Foundation (MCB-1951405 to N.V.-L.), Illinois State startup funds (Y.S.P.) and Austrian Science Fund FWF (P31691 and F8011 to R.M.).

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Authors and Affiliations

Authors

Contributions

L.F. and R.M. synthesized short tripeptidyl-tRNA mimics. D.K. and N.V.-L. performed the toe-printing analysis. E.A.S. and Y.S.P. designed and performed X-ray crystallography experiments. R.M., N.V.-L. and Y.S.P. supervised the experiments. All authors interpreted the results. E.A.S., R.M. and Y.S.P. wrote the manuscript.

Corresponding authors

Correspondence to Ronald Micura or Yury S. Polikanov.

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Nature Structural & Molecular Biology thanks Marat Yusupov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann, Sara Osman and Carolina Perdigoto were the primary editors 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 Chemical synthesis of hydrolysis-resistant tripeptidyl-ACCA conjugates.

(a) Overview of the synthetic pathway. Chemical structure of functionalized solid support 11 (gray sphere represents amino-functionalized polystyrene support (GE Healthcare, Custom Primer SupportTM 200 Amino) used for peptide assembly (Fmoc chemistry) and RNA assembly (2’-O-TOM chemistry), followed by deprotection and purification using anion-exchange chromatography; DMT = 4,4’-dimethoxytrityl, Fmoc = N-(9-fluorenyl)methoxy-carbonyl, TOM [(triisopropylsilyl)oxy]methyl, AE HPLC = anion-exchange high-pressure liquid chromatography. (b) Anion-exchange HPLC profiles of purified ACCA-Ile-Ala-Met, ACCA-Ile-Thr-Met, and ACCA-Ile-Phe-Met conjugates (left) and LC-ESI mass spectra (right). Anion-exchange chromatography conditions: Dionex DNAPac PA-100 (4×250 mm) column; temperature: Flow rate: 1 mL/min; eluent A: 25 mM Tris-HCl (pH 8.0) and 20 mM NaClO4 in 20% aqueous acetonitrile, eluent B: 25 mM Tris-HCl (pH 8.0) and 0.60 M NaClO4 in 20% aqueous acetonitrile; gradient: 0-35% B in A within 28 min; UV detection at λ = 260 nm.

Extended Data Fig. 2 CHL arrests translation at the MTI/MAI tripeptide sequences.

Ribosome stalling in the presence and absence of CHL revealed by reverse-transcription primer-extension inhibition (toe-printing) assay in a cell-free translation system on wild-type osmC mRNA encoding MTI tripeptide at the N-terminus (lanes 1-2) or its mutant versions encoding either alanine (lanes 3-4) or phenylalanine (lanes 5-6) in the 2nd position. Nucleotide sequences of wild-type osmC mRNA and the corresponding amino acid sequence are shown on the left. White arrowhead marks translation start site. Red and blue arrowheads point to the drug-induced arrest sites within the coding sequences of each of the three used mRNAs. Note that due to the large size of the ribosome, the reverse transcriptase used in the toe-printing assay stops 16 nucleotides downstream of the codon located in the P site. Because osmC mRNA template harbors other downstream alanine codons, CHL also caused ribosome stalling at these downstream sites subsequent to the appearance of Ala, Ser, or Thr residues in the penultimate position of the peptide chain (lanes 2, 4, and 6, blue arrowhead). The results of the toe-printing assay confirmed that the presence of the MTI or MAI tripeptide sequences in the P site of the ribosome results in CHL-dependent stalling, whereas MFI tripeptide sequence is not conducive to ribosome stalling and can serve as a negative control. Experiments were repeated twice independently with similar results.

Extended Data Fig. 3 Superpositioning of the structures of short tRNA analogs with the aminoacylated full-length tRNAs.

(a, b, c) Comparisons of the 70S ribosome structures carrying ACC-Pmn (magenta) in the A site and either ACCA-ITM (a, green), or ACCA-IAM (b, blue), or ACCA-IFM (c, teal) short peptidyl tRNA analogs in the P site with the previous structure of ribosome-bound full-length aminoacyl-tRNAs (PDB entry 6ZHW24). All structures were aligned based on domain V of the 23S rRNA. (d, e, f) Comparisons of the positions of key 23S rRNA nucleotides around the PTC in the same structures. Note that there are no substantial differences in the positions of A- or P-site substrates or the PTC nucleotides.

Extended Data Fig. 4 Comparison of the structure of short MTI-tripeptidyl-tRNA analog with the structures of other ribosome-bound peptidyl-tRNAs.

Superpositioning of Thermus themophilus 70S ribosome structure carrying A-site ACC-Pmn (magenta) and P-site ACCA-ITM tripeptidyl-tRNA analog (green) with the previously reported structures of Escherichia coli 70S ribosome in complex with full-length P-site peptidyl tRNA carrying SpeFL (a) or VemP (b) stalling peptides (PDB entry 6TC326 and 5NWY27, respectively). All structures were aligned based on domain V of the 23S rRNA. Note that the full-length peptidyl-tRNAs feature ester bonds between the ribose of A76 nucleotide of tRNA and the peptide moiety. Also, note that the overall path of the MTI tripeptide in our structure is similar to the trajectories of the SpeFL or VemP stalling peptides in the NPET.

Extended Data Fig. 5 Superposition of the ribosome-bound CHL with the structures of ribosome-bound aa-tRNA and peptidyl-tRNA analogs.

(a) Superposition of CHL with the A-site-bound aa-tRNA analog ACC-Pmn. (b) Superposition of CHL with the P-site-bound peptidyl-tRNA analog ACCA-IFM. The structure of CHL is from PDB entry 6ND510. The structures were aligned based on domain V of the 23S rRNA. Note that the side chains of the incoming amino acid in the A site (a) or the penultimate amino acid of the peptide in the P site (b) clash with CHL in its canonical binding site.

Extended Data Fig. 6 Structure of CHL in complex with the 70S ribosome and MTI-tripeptidyl-tRNA analog.

(a) Overview of the CHL binding site (yellow) in the Thermus thermophilus 70S ribosome in complex with the short tripeptidyl-tRNA analogs viewed as a cross-cut section through the nascent peptide exit tunnel. The 30S subunit is shown in light yellow; the 50S subunit is in light blue; ribosome-bound protein Y is colored in green. (b, c) Close-up views of CHL bound in the PTC, highlighting H-bond interactions (dashed lines) and the intercalation of the nitrobenzyl group into the A-site cleft formed by nucleotides A2451 and C2452 of the 23S rRNA. Note that the side chain of the Thr2 residue of the MTI tripeptide directly interacts with the ribosome-bound CHL.

Extended Data Fig. 7 Short MAI and MFI tripeptidyl-tRNA analogs exhibit opposite effects on CHL binding in vitro.

Binding of radioactively labeled [14C]-CHL to the Thermus thermophilus 50S ribosomal subunits (RS) was measured using fragment reaction assay52 in the presence of the indicated A- and P-site substrates. The synthetic oligonucleotide CACCA (analog of the CCA-end of deacylated tRNA) and ACCA-IAM/IFM compounds (analogs of the CCA-ends of the peptidyl-tRNAs carrying MAI or MFI tripeptide moieties) serving as the A- and P-site substrates, respectively, were the same as those used for the structural studies. Error bars represent standard deviations of the mean of two independent measurements.

Extended Data Fig. 8 In silico modeling of MGI-, MCI-, and MVI-tripeptidyl-tRNA analogs in the presence of CHL.

Using the structure of MAI-tripeptidyl-tRNA analog bound to the ribosome in the presence of CHL as a reference, the second alanine residue was mutated either to glycine (a), cysteine (b), or valine (c) and assessed the modeled structures for sterical clashes.

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Syroegin, E.A., Flemmich, L., Klepacki, D. et al. Structural basis for the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol. Nat Struct Mol Biol 29, 152–161 (2022). https://doi.org/10.1038/s41594-022-00720-y

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