Abstract
As antimicrobial resistance threatens our ability to treat common bacterial infections, new antibiotics with limited cross-resistance are urgently needed. In this regard, natural products that target the bacterial ribosome have the potential to be developed into potent drugs through structure-guided design, provided their mechanisms of action are well understood. Here we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X primarily inhibits peptide bond formation between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3′ adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of tetracenomycin X on the bacterial ribosome and suggests a path forward for the development of novel aromatic polyketide antibiotics.
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Data availability
Cryo-EM maps of the 70S–MAAAPQKC–TcmX complex and the associated molecular models have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, with the accession codes EMD-14956 and PDB 7ZTA, respectively. The original micrograph movies have been deposited in the Electron Microscopy Public Image Archive under accession code EMPIAR-11241. Sequencing data for the iTP-seq experiment have been deposited as a National Center for Biotechnology Information BioProject with the accession code PRJNA838796. Source data are provided with this paper.
Code availability
Scripts used to process iTP-seq data were deposited on GitHub (https://github.com/innislab/ITP-seq).
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Acknowledgements
We thank I. Osterman and D. Wilson for providing TcmX, N. Vázquez-Laslop for providing the JM109 ΔacrA/acrB E. coli strain, A. Malhotra for providing RNase R and S. Blanchard for his comments on an early draft of the paper. We also thank A. Labécot for preliminary experiments. E.C.L. and C.A.I. have received funding for this project from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 724040). C.A.I. is an EMBO YIP (project no. 3869) and has received funding from the Fondation Bettencourt-Schueller. C.A.I. and T.N.P. have received funding for this project from the Agence Nationale de la Recherche (ANR) under the frame of the Joint JPI-EC-AMR Project ‘Ribotarget – Development of novel ribosome-targeting antibiotics’ (grant no. ANR-18-JAM2-0005-02 RIBOTARGET). We thank the cryo-EM facility of the Institut Européen de Chimie et Biologie (Pessac, France) for the data collection time on the Talos Arctica microscope.
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Contributions
C.A.I. designed the study. E.C.L. performed iTP-seq and toeprinting experiments. E.C.L. and T.T.R. processed and analyzed the iTP-seq data. T.N.P. and T.T.R. performed the blue ring assays. E.C.L. prepared complexes for cryo-EM. T.N.P. prepared grids and performed cryo-EM data collection. T.N.P. and C.A.I. processed the cryo-EM data, and E.C.L. and C.A.I. built the model. E.C.L., T.N.P., T.T.R. and C.A.I. wrote the paper.
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Nature Chemical Biology thanks Marina Rodnina, Yury Polikanov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Size distribution of inverse toeprints and diversity of the (NNN)15 library.
a, Number of deep sequencing reads obtained for inverse toeprints of different lengths, plotted as a function of the distance between the start codon and the RNase R cleavage site. Average read numbers for three independent replicates are shown for TcmX-treated (orange) and untreated (blue) samples. Inverse toeprints within a size range featuring a well-defined three-nucleotide periodicity were retained for further analysis (gray area). The iTP-seq experiment consisted of 3 independent replicates for the TcmX-treated condition (2.8 × 106, 2.5 × 106 and 2.3 × 106 reads), and 3 replicates for the untreated condition (2.7 × 106, 2.3 × 106 and 2.6 × 106 reads). On average ~2.5 × 106 reads were obtained per replicate. The error bands show the 95% confidence interval of the mean of the three replicates. b. Nucleotide frequency of the raw reads for the (NNN)15 library, showing a relatively equal proportion of each dNTP for codons 2–15.
Extended Data Fig. 2 Enrichment of two-amino acid motifs in samples treated with TcmX.
Heatmaps showing the enrichment of individual residues at positions −2 to +1, and all possible combinations of motifs consisting of two adjacent amino acids, following treatment with TcmX. Enrichment is defined as log2(FTcmX/Funtreated), where FTcmX is the frequency of occurrence of a given motif in the sample treated with TcmX and Funtreated is its frequency in the untreated sample.
Extended Data Fig. 3 Four-amino acid motifs at position [−2,+1] ending with LK are not significantly enriched following TcmX treatment.
Volcano plot of statistical significance (−log10(p-value)) against enrichment (log2(fold change)) for four-amino acid motifs at position [−2,+1]. Motifs ending in LK are shown in yellow and the two LK-containing motifs described in Ref. 6 (FVLK/TILK) are labeled in bold. P-values were calculated by DESeq2 (Ref. 33) using a one-sided Wald test, without correction for multiple-testing.
Extended Data Fig. 4 Ability of different PQKC variants to induce TcmX-dependent stalling.
Volcano plots of statistical significance (−log10(p-value)) against enrichment (log2(fold change)) for four-amino acid motifs at position [−2,+1]. Single amino acid variants of the PQKC motif are shown in red, XQKX motifs are in blue, PQXC or PXKC motifs are in orange and PXXC motifs are in yellow. P-values were calculated by DESeq2 (Ref. 33) using a one-sided Wald test, without correction for multiple-testing.
Extended Data Fig. 5 Codon composition does not have a major influence on TcmX-dependent ribosome stalling at QK motifs.
Violin plot showing the enrichment of various three-amino acid motifs at positions [−2,0] following treatment with TcmX. Each bar represents a motif variant with a distinct codon composition.
Extended Data Fig. 6 Overview of cryo-EM data processing.
Flowchart showing the workflow used to process cryo-EM data for the 70S–MAAAPQKC–TcmX complex in Relion v4.0 (ref. 41). The final reconstruction could be refined to an overall resolution of 2.7 Å, assessed using a Fourier shell correlation (FSC) cutoff of 0.143.
Extended Data Fig. 7 Quality of the 70S–MAAAPQKC–TcmX reconstruction.
a, Cryo-EM density map obtained for a 3D reconstruction of the 70S–MAAAPQKC–TcmX complex in Relion v4.0 (ref. 41) and auto-sharpened with Phenix v1.20.1 (ref. 45), filtered and colored by local resolution estimation values in Chimera X v.1.3 (ref. 49). b, Cross-section of the same map showing the E, P and A sites of the ribosome. c, Isolated ligand densities in the E, P and A sites, filtered and colored by local resolution. d, The same isolated densities shown in c, displayed as a transparent surface and fitted with molecular models of the E-, P- and A-site tRNAs, mRNA, TcmX and the nascent peptide. e, Model to map correlation curve calculated for the 70S–MAAAPQKC–TcmX structure. f, Representative cryo-EM densities of three different tRNA modifications within the A- and P-site tRNAs.
Extended Data Fig. 8 Comparison of 70S–MAAAPQKC–TcmX with earlier structures of the ribosome in complex with TcmX or other antibiotics.
a,b, Structural comparison of the peptidyl transferase center and TcmX binding site in 70S–MAAAPQKC–TcmX (white) and in complexes between E. coli 70S (gray, PBD 6Y69) or human 80 S (pale green, PDB 6Y6X) ribosomes and TcmX (Ref. 6). c, d, Displacement of the 3’ end of peptidyl-tRNA (dark blue) by TcmX (c) or Blasticidin S (PDB 6B4V, Ref. 23). e, f, Displacement of the 3’ end of aminoacyl-tRNA (light blue) by (e) Hygromycin A (PDB 5DOY, Ref. 25) or (f) A201A (PDB 4Z3S, Ref. 25).
Extended Data Fig. 9 Effect of TcmX on the accommodation of the 3’ end of aminoacyl-tRNA, on 23S rRNA residue 2602 and on the N-terminus of ribosomal protein bL27.
a–d, Structural comparison of 23S rRNA nucleotides and tRNAs within the A site (a, b) and P site (c, d) regions of the peptidyl transferase center in the uninduced (Ref. 21) and induced (pre-attack) (Ref. 22) states (PDB 1VQ6 and 1VY4, respectively) (a, c), and the uninduced and TcmX-inhibited states (b, d). In the 70S–MAAAPQKC–TcmX structure, 23S rRNA residues U2506, G2583 and U2584 are in their induced conformation, but 23S rRNA nucleotide U2585 and nucleotide A76 of the P-site tRNA remain in the uninduced conformation, indicating partial aminoacyl-tRNA accommodation into the peptidyl transferase center. e, Sequestration of residue A76 of the P-site tRNALys within the tunnel by TcmX causes the preceding residue C75 to shift, which in turn leads to a conformational change in the peptidyl transferase center resulting in the stacking of the base of 23S rRNA residue A2602 against the imidazole ring of residue His-3 of ribosomal protein bL27.
Extended Data Fig. 10 Possible way forward for the design of TcmX derivatives with increased specificity for the bacterial ribosome.
a, The O-methyl group attached to the D ring of TcmX could be used as a starting point to ‘grow’ the drug molecule towards a cavity lined with the backbone phosphate groups of 23S rRNA residues G2582 and G2608 in the E. coli ribosome (white). b, TcmX derivatives with suitable side chains attached to this position may retain activity against the bacterial ribosome while no longer binding to the human ribosome (pale green, PDB 6Y6X), where this cavity is blocked by 28S rRNA residue U1591.
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Supplementary Information
Supplementary Tables 1 and 2.
Source data
Source Data Fig. 1 and Extended Data Figs. 3 and 4
Statistical source data for Fig. 1 and Extended Data Figs. 3 and 4.
Source Data Fig. 2
Uncropped toeprinting gel and agar plates.
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Leroy, E.C., Perry, T.N., Renault, T.T. et al. Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs. Nat Chem Biol 19, 1091–1096 (2023). https://doi.org/10.1038/s41589-023-01343-0
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DOI: https://doi.org/10.1038/s41589-023-01343-0
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