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Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel

Nature Chemical Biology volume 13, pages 1129–1136 (2017)Cite this article

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Abstract

Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms often results in the rediscovery of known compounds, genome-mining programs allow researchers to harness much greater chemical diversity, and result in the discovery of new molecular scaffolds. Here we report the genome-guided identification of a new antibiotic, klebsazolicin (KLB), from Klebsiella pneumoniae that inhibits the growth of sensitive cells by targeting ribosomes. A ribosomally synthesized post-translationally modified peptide (RiPP), KLB is characterized by the presence of a unique N-terminal amidine ring that is essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosomes by interfering with translation elongation. Structural analysis of the ribosome–KLB complex showed that the compound binds in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramin-B. KLB adopts a compact conformation and largely obstructs the tunnel. Engineered KLB fragments were observed to retain in vitro activity, and thus have the potential to serve as a starting point for the development of new bioactive compounds.

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Figure 1: Organization of the biosynthetic gene cluster and production of KLB by the E. coli host.
Figure 2: KLB is an inhibitor of protein synthesis both in vitro and in vivo.
Figure 3: The structure of KLB in complex with 70S ribosome and A- and P-tRNAs.
Figure 4: Occlusion of the nascent peptide exit tunnel by antibiotics.

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Acknowledgements

We thank A. Mankin and T. Florin (University of Illinois at Chicago, Chicago, Illinois, USA) for providing pAM552 plasmids carrying various mutations, as well as for careful reading of the manuscript and valuable suggestions. We are thankful to the members of the K. Severinov, P.V.S., and Y.S.P. laboratories for discussions and critical feedback. We thank R. Roy for critical reading of the manuscript. We also thank I. Kamyshko (MilliporeSigma, Billerica, Massachusetts, USA) for material source consulting and for providing the Supelco C5 HPLC column used for tRNA purification. We thank staff at NE-CAT beamline 24ID-C for help with data collection and freezing of the crystals, especially K. Rajashankar, M. Capel, F. Murphy, I. Kourinov, A. Lynch, S. Banerjee, D. Neau, J. Schuermann, N. Sukumar, J. Withrow, K. Perry, and C. Salbego.

This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences, US National Institutes of Health (P41 GM103403). The Pilatus 6M detector on the 24ID-C beamline is funded by an NIH–ORIP HEI grant (S10 RR029205). This research 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 Illinois State startup funds (to Y.S.P.), the US National Institutes of Health (grant R01 AI117210 to K. Severinov), Skoltech Institutional funds (to K. Severinov), the Russian Foundation for Basic Research (grants 16-04-01100 (to P.V.S.) and 15-34-20139 (to I.A.O.)), the Ministry of Education and Science of the Russian Federation (grant 14.B25.31.0004 to K. Severinov), the Russian Science Foundation (grant 15-15-10017 to M.M. (used for the expression, purification, structure determination, and in vivo activity testing of KLB); grant 14-14-00072 to P.V.S. (used for the toe-printing assays, in vitro activity tests and chemical foot-printing)), the Dynasty Foundation (fellowship to M.M.) and FASIE (grant 9186GU/2015 to D.Y.T.).

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Author notes

  1. Mikhail Metelev and Ilya A Osterman: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Center of Nanobiotechnologies, Peter the Great St.Petersburg Polytechnic University, St. Petersburg, Russia

    Mikhail Metelev, Alexander Yakimov, Irina Utkina, Tatyana Artamonova, Mikhail Khodorkovskii, Andrey L Konevega & Konstantin Severinov

  2. Institute of Antimicrobial Chemotherapy, Smolensk State Medical Academy, Smolensk, Russia

    Mikhail Metelev

  3. Center for Data-Intensive Biomedicine and Biotechnology, Skolkovo Institute of Science and Technology, Moscow, Russia

    Mikhail Metelev, Ilya A Osterman, Dmitry Ghilarov, Irina Utkina, Ekaterina S Komarova, Petr V Sergiev & Konstantin Severinov

  4. Institute of Gene Biology of the Russian Academy of Sciences, Moscow, Russia

    Mikhail Metelev, Dmitry Ghilarov, Marina Serebryakova & Konstantin Severinov

  5. Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

    Ilya A Osterman, Marina Serebryakova & Petr V Sergiev

  6. Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

    Nelli F Khabibullina & Yury S Polikanov

  7. Petersburg Nuclear Physics Institute, NRC Kurchatov Institute, Gatchina, Russia

    Alexander Yakimov, Konstantin Shabalin & Andrey L Konevega

  8. Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia

    Dmitry Y Travin & Ekaterina S Komarova

  9. Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA

    Konstantin Severinov

  10. Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USA

    Yury S Polikanov

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  1. Mikhail Metelev
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Contributions

M.M. and I.U. assembled constructs for the isolation of KLB and derivatives; M.M., I.U., D.G., and D.Y.T. isolated and purified KLB and its derivatives; M.M., D.G., and I.A.O. evaluated bioactivity; D.G. and D.Y.T. selected resistant mutants; A.L.K., N.F.K., and Y.S.P. designed and performed X-ray crystallography experiments; I.A.O., E.S.K., P.V.S., and A.L.K. designed biochemistry and genetic experiments; A.Y. and K. Shabalin performed and analyzed the NMR experiments; and M.M. performed and analyzed the MS experiments, with critical input from M.K., T.A., and M.S. All authors interpreted the results. M.M., Y.S.P., P.V.S., and K. Severinov wrote the manuscript.

Corresponding authors

Correspondence to Konstantin Severinov or Yury S Polikanov.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–11 (PDF 5523 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Data Set 1

Raw data for the luciferase luminescence assay shown in Figure 2b. (XLSX 15 kb)

The KLB functional site in the 70S ribosome. (MOV 28840 kb)

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Metelev, M., Osterman, I., Ghilarov, D. et al. Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel. Nat Chem Biol 13, 1129–1136 (2017). https://doi.org/10.1038/nchembio.2462

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