Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel

Abstract

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

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Fig. 1: TcmX inhibits protein synthesis and not DNA metabolism.
Fig. 2: Binding site of TcmX on the bacterial and human ribosomes.
Fig. 3: Cytotoxicity and inhibition of eukaryotic translation by TcmX.
Fig. 4: TcmX inhibits translation elongation in a template-dependent fashion.

Data availability

The cryo-EM and associated molecular models for the TcmX-Eco70S and TcmX-Hsa80S ribosome complexes are available from the EMDB (EMD-10705 and EMD-10709) and PDB (ID 6Y69 and PDB 6Y6X), respectively. The complete genome sequence of Amycolatopsis sp. A23 has been deposited in the European Nucleotide Archive with the accession number GCA_902497555.1. Source data are provided with this paper.

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Acknowledgements

We thank A. Mankin (University of Illinois at Chicago, Chicago, Illinois) for providing pAM552 and pLK35 plasmids carrying various mutations. We are grateful to O. Saveliev for the expert technical assistance in NMR measurements. This work was supported by Russian Science Foundation grant no. 18-44-04005 (to I.A.O., used for microbiological, biochemical and structural study of TcmX action), 19-14-00115 (to V.I.P., used for NMR studies) and the Deutsche Forschungsgemeinschaft grant no. WI3285/6-1 (to D.N.W.), the Russian Foundation for Basic Research grant no. 19-34-51021 (to I.A.O., used for the expression, purification and structure determination of TcmX) and the Moscow State University development program PNR 5.13 (O.A.D.). The work of A.L.O., S.A.L and J.E.Z. on genome analysis and comparative genomics was supported by the Laboratory Funding Initiative at Sanford Burnham Prebys Medical Discovery Institute (to A.L.O.).

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Contributions

I.A.O., D.A.L., D.I.S., T.P.M., Y.V.Z., V.N.T. and M.V.B. isolated and purified TcmX. I.A.O., V.N.T. and V.I.P. determined structure of TcmX. I.A.O., T.P.M., E.S.K., D.A.L., D.I.S. and D.A.S. evaluated bioactivity. K.A.L. and S.E.D. analyzed inhibitory activity of TcmX in mammals. I.A.O. and E.S.K. selected resistant mutants. M.W., R. Buschauer and J.C. determined cryo-EM structures of the TcmX-Eco70S and TcmX-Hsa80S ribosome complexes, respectively. S.A.L., J.E.Z. and A.L.O. sequenced and analyzed genome of Amycolatopsis sp. A23. I.A.O., P.V.S. A.A.B. and O.A.D. designed biochemistry and genetic experiments. Y.S.P. designed and performed X-ray crystallography experiments. All authors, including R. Beckmann, interpreted the results. I.A.O., P.V.S. and D.N.W. wrote the manuscript.

Corresponding authors

Correspondence to Ilya A. Osterman or Daniel N. Wilson or Petr V. Sergiev.

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Extended data

Extended Data Fig. 1 Modeling of TcmX into DNA and onto the 30S subunit.

a, Structure of doxorubicin (Dox, green) intercalating with dsDNA (PDB 1D12). b,c, Alignment of TcmX (blue) onto Dox (green) based on the planar rings C-D illustrates that the methoxy groups attached to ring A of TcmX would clash with the basepairs and prevent efficient intercalation with DNA. d, Transverse section of reconstructed map of Thermus thermophilus 70 S (30 S, yellow; 50 S, grey) with the Tet (green) binding site at the decoding site relative to P-site tRNA (orange) (PDB 4V9A)21. e, Alignment of TcmX (blue) onto Tet (green) (PDB 4V9A)21 based on rings C and D. f, Based on the alignments from (e), TcmX clashes with G1054 (top panel) and the backbone of U1196-G1198 (bottom panel). g,h, Two views showing the tetracyclic antibiotic elloramycin (cyan) aligned onto the TcmX (blue) binding site of the Eco70S based on rings A-D.

Extended Data Fig. 2 Interaction schemes of Tetracenomycin X and Tetracycline.

a,b, Interaction scheme of Tetracenomycin X (TcmX) on the large subunit of the E. coli and H. sapiens ribosomes, respectively. c, Interaction scheme of Tetracycline (Tet) on decoding site of the small subunit of the T. thermophilus 70 S ribosome21, with red crosses marking interactions that TcmX cannot form when docked into the Tet binding site.

Extended Data Fig. 3 Cryo-EM structure of the TcmX-Eco70S complex.

a, Sorting scheme for cryo-EM data; after initial picking, 818,287 particles were subjected to 2D-Classification, of which 548,675 particles were used for 3D-classification. Particles were sorted into five distinctive classes: non-aligning, 50 S, 100 S, 70 S with 100S-substoichiometric density, and clean 70 S particles. The latter were picked for further refinement (29.34%, 161,915 particles). After CTF-refinement and Bayesian Polishing, a final overall resolution of 2.89 Å was achieved. b, Fourier-Shell-Correlation (FSC 0.143) curve of the final reconstruction, with the resolution at FSC = 0.143 indicated with a dashed line. c, Overview and (d) transverse section of cryo-EM map filtered and coloured according to local resolution. e,f, Electron density for TcmX with Mg1 and Mg2 and (g) the interacting nucleotides of the 23 S rRNA.

Extended Data Fig. 4 Conservation of the TcmC binding site.

a, In wild-type U2609 basepairs with A752; (b-d) in silico mutations of TcmX resistant mutations (U2609G, U2609C and U2609A) which can no longer basepair with A752 and due to possible clashing have to adopt a different conformation. e, In wild-type U1752 base-pairs with U2586, while in the resistance mutations (f-i) the previously base-paired nucleotides will have to adapt a different conformation to avoid clashing, possibly obstructing the TcmX binding site. j, In E. coli (grey) TcmX stacks upon a U–U basepair U1782-U2586; in T. thermophilus (light blue) (PDB 4V9A)21 it is a C–C base-pair C 1814:C2599. k, Bacterial 23 S (above) and eukaryotic 28 S (below) rRNA alignments of select organisms within the vicinity of the U1782 and U2586 (E. coli numbering) in eubacteria and U3644 and U4532 (H. sapiens numbering).

Extended Data Fig. 5 Processing and analysis of H. sapiens-TcmX cryo-EM structure.

a, Sorting scheme for cryo-EM data; after Initial picking, 836,588 particles were subjected to 2D-Classification, of which 461,131 particles were used for 3D-classification. Particles were sorted into four classes: low resolution particles, 80 S with E-Site, and two distinct 80 S with E-Site and eEF2. All but low-resolution particles were picked for further refinement (65.7%, 302,737 particles). After CTF-refinement and focused refinement using a 60 S mask, a final overall resolution of 2.76 Å was achieved. b, Fourier-Shell-Correlation (FSC 0.143) of the final reconstruction, with the resolution at FSC = 0.143 indicated with a dashed line. c, Overview and transverse section of cryo-EM map filtered and coloured according to local resolution. d, Electron density (mesh) and molecular model for the TcmX (blue) binding site on the human 80 S ribosome (28 S rRNA nucleotides shown in grey sticks Mg1 and Mg2 as blue spheres). e, Overview of the putative Tet-analog binding sites on the Hsa80S ribosome at the terminal loop of H89 (binding site 1, red) and within the exit tunnel (binding site 2, pink)24 relative to the binding site of TcmX (blue). f, Zoom of the relative location of TcmX (blue) to 28 S rRNA nucleotides identified in binding site 2 (magenta).

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Supplementary Tables 1–5, Notes 1–3 and reference.

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Osterman, I.A., Wieland, M., Maviza, T.P. et al. Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel. Nat Chem Biol 16, 1071–1077 (2020). https://doi.org/10.1038/s41589-020-0578-x

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