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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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.
Zhang, Z., Pan, H. X. & Tang, G. L. New insights into bacterial type II polyketide biosynthesis. F1000 Res. 6, 172 (2017).
Wilson, D. N. Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat. Rev. Microbiol. 12, 35–48 (2014).
Agudelo, D., Bourassa, P., Berube, G. & Tajmir-Riahi, H. A. Review on the binding of anticancer drug doxorubicin with DNA and tRNA: structural models and antitumor activity. J. Photochem. Photobiol. B. 158, 274–279 (2016).
Agudelo, D., Bourassa, P., Berube, G. & Tajmir-Riahi, H. A. Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: structural features and biological implications. Int. J. Biol. Macromol. 66, 144–150 (2014).
Agudelo, D., Berube, G. & Tajmir-Riahi, H. A. An overview on the delivery of antitumor drug doxorubicin by carrier proteins. Int. J. Biol. Macromol. 88, 354–360 (2016).
Weber, W., Zahner, H., Siebers, J., Schroder, K. & Zeeck, A. Metabolic products of microorganisms. 175. Tetracenomycin C (author’s translation). Arch. Microbiol. 121, 111–116 (1979).
Anderson, M. G., Khoo, C. L. & Rickards, R. W. Oxidation processes in the biosynthesis of the tetracenomycin and elloramycin antibiotics. J. Antibiot. 42, 640–643 (1989).
Liu, B. et al. Identification of tetracenomycin X from a marine-derived Saccharothrix sp. guided by genes sequence analysis. Acta Pharm. Sinica 49, 230–236 (2014).
Gan, M. et al. Saccharothrixones A-D, tetracenomycin-type polyketides from the marine-derived actinomycete saccharothrix sp. 10-10. J. Nat. Prod. 78, 2260–2265 (2015).
Drautz, H., Reuschenbach, P., Zahner, H., Rohr, J. & Zeeck, A. Metabolic products of microorganisms. 225. Elloramycin, a new anthracycline-like antibiotic from Streptomyces olivaceus. Isolation, characterization, structure and biological properties. J. Antibiot. 38, 1291–1301 (1985).
Rohr, J. & Zeeck, A. Structure-activity relationships of elloramycin and tetracenomycin C. J. Antibiot. 43, 1169–1178 (1990).
Adinarayana, G. et al. Cytotoxic compounds from the marine actinobacterium. Bioorg. Khim 32, 328–334 (2006).
Egert, E., Noltemeyer, M., Siebers, J., Rohr, J. & Zeeck, A. The structure of tetracenomycin C. J. Antibiot. 45, 1190–1192 (1992).
Komarova Andreyanova, E. S. et al. 2-Guanidino-quinazolines as a novel class of translation inhibitors. Biochimie 133, 45–55 (2017).
Ortseifen, V., Kalinowski, J., Puhler, A. & Ruckert, C. The complete genome sequence of the actinobacterium Streptomyces glaucescens GLA.O (DSM 40922) carrying gene clusters for the biosynthesis of tetracenomycin C, 5′-hydroxy streptomycin, and acarbose. J. Biotechnol. 262, 84–88 (2017).
Osterman, I. A. et al. Sorting out antibiotics’ mechanisms of action: a double fluorescent protein reporter for high-throughput screening of ribosome and DNA biosynthesis inhibitors. Antimicrob. Agents Chemother. 60, 7481–7489 (2016).
Pato, M. L. Tetracycline inhibits propagation of deoxyribonucleic acid replication and alters membrane properties. Antimicrob. Agents Chemother. 11, 318–323 (1977).
Frederick, C. A. et al. Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin. Biochemistry 29, 2538–2549 (1990).
Orelle, C. et al. Tools for characterizing bacterial protein synthesis inhibitors. Antimicrob. Agents Chemother. 57, 5994–6004 (2013).
Vester, B. & Douthwaite, S. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob. Agents Chemother. 45, 1–12 (2001).
Jenner, L. et al. Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc. Natl Acad. Sci. USA 110, 3812–3816 (2013).
Polikanov, Y. S. et al. Distinct tRNA accommodation intermediates observed on the ribosome with the antibiotics hygromycinA and A201A. Mol. Cell 58, 832–844 (2015).
Cannone, J. J. et al. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinf. 3, 2 (2002).
Mortison, J. D. et al. Tetracyclines modify translation by targeting key human rRNA substructures. Cell Chem. Biol. 25, 1506–1518 e1513 (2018).
Akulich, K. A. et al. Four translation initiation pathways employed by the leaderless mRNA in eukaryotes. Sci. Rep. 6, 37905 (2016).
Terenin, I. M., Andreev, D. E., Dmitriev, S. E. & Shatsky, I. N. A novel mechanism of eukaryotic translation initiation that is neither m7G-cap-, nor IRES-dependent. Nucleic Acids Res. 41, 1807–1816 (2013).
Metelev, M. et al. Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel. Nat. Chem. Biol. 13, 1129–1136 (2017).
Vazquez-Laslop, N. & Mankin, A. S. How macrolide antibiotics work. Trends Biochem. Sci. 43, 668–684 (2018).
Arenz, S. et al. Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide. Nat. Commun. 5, 3501 (2014).
Arenz, S. et al. A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nat. Commun. 7, 12026 (2016).
Guilfoile, P. G. & Hutchinson, C. R. Sequence and transcriptional analysis of the Streptomyces glaucescens temAR tetracenomycin C resistance and repressor gene loci. J. Bact. 174, 3651–3658 (1992).
Leclercq, R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin. Infect. Dis. 34, 482–492 (2002).
Polikanov, Y. S., Steitz, T. A. & Innis, C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat. Struct. Mol. Biol. 21, 787–793 (2014).
Bischoff, L., Berninghausen, O. & Beckmann, R. Molecular basis for the ribosome functioning as an l-tryptophan sensor. Cell Rep. 9, 469–475 (2014).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Zimin, A. V. et al. Hybrid assembly of the large and highly repetitive genome of Aegilops tauschii, a progenitor of bread wheat, with the MaSuRCA mega-reads algorithm. Genome Res 27, 787–792 (2017).
Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29, 1072–1075 (2013).
Wattam, A. R. et al. Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic Acids Res. 45, D535–D542 (2017).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Aziz, R. K. et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9, 75 (2008).
Overbeek, R. et al. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 33, 5691–5702 (2005).
Zakalyukina, Y. V. et al. Nybomycin-producing Streptomyces isolated from carpenter ant Camponotus vagus. Biochimie 160, 93–99 (2019).
Zaporojets, D., French, S. & Squires, C. L. Products transcribed from rearranged RRN genes of Escherichia coli can assemble to form functional ribosomes. J. Bacteriol. 185, 6921–6927 (2003).
Beckert, B. et al. Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1. Nat. Microbiol 3, 1115–1121 (2018).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).
Moriya, T. et al. High-resolution single particle analysis from electron cryo-microscopy images using SPHIRE. J. Vis. Exp. 16, 55448 (2017).
Natchiar, S. K., Myasnikov, A. G., Kratzat, H., Hazemann, I. & Klaholz, B. P. Visualization of chemical modifications in the human 80S ribosome structure. Nature 551, 472–477 (2017).
Pettersen, E. F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Schuttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D. 60, 1355–1363 (2004).
Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D. 65, 1074–1080 (2009).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. 60, 2126–2132 (2004).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. 66, 213–221 (2010).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. 66, 12–21 (2010).
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983).
Prokhorova, I. V. et al. Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome. Sci. Rep. 6, 27720 (2016).
Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
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.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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 base−pairs 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.
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.
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.
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).
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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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