Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex

Nature Structural & Molecular Biology volume 22, pages 470–475 (2015)Cite this article

Subjects

Abstract

The increasing prevalence of multidrug-resistant pathogenic bacteria is making current antibiotics obsolete. Proline-rich antimicrobial peptides (PrAMPs) display potent activity against Gram-negative bacteria and thus represent an avenue for antibiotic development. PrAMPs from the oncocin family interact with the ribosome to inhibit translation, but their mode of action has remained unclear. Here we have determined a structure of the Onc112 peptide in complex with the Thermus thermophilus 70S ribosome at a resolution of 3.1 Å by X-ray crystallography. The Onc112 peptide binds within the ribosomal exit tunnel and extends toward the peptidyl transferase center, where it overlaps with the binding site for an aminoacyl-tRNA. We show biochemically that the binding of Onc112 blocks and destabilizes the initiation complex, thus preventing entry into the elongation phase. Our findings provide a basis for the future development of this class of potent antimicrobial agents.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Onc112-binding site within the exit tunnel of the ribosome.
Figure 2: Interactions between Onc112 and the ribosome.
Figure 3: Onc112 blocks and destabilizes the initiation complex.
Figure 4: Characterization of Onc112, its C-terminally truncated derivatives and its membrane transporter in Gram-negative bacteria.
Figure 5: Mechanism of action and overlap of Onc112 with antibiotics that target the large subunit of the ribosome.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Wang, G. et al. Antimicrobial peptides in 2014. Pharmaceuticals (Basel) 8, 123–150 (2015).

    Article  CAS  Google Scholar 

  2. Casteels, P., Ampe, C., Jacobs, F., Vaeck, M. & Tempst, P. Apidaecins: antibacterial peptides from honeybees. EMBO J. 8, 2387–2391 (1989).

    Article  CAS  Google Scholar 

  3. Li, W. et al. Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria. Amino Acids 46, 2287–2294 (2014).

    Article  CAS  Google Scholar 

  4. Mattiuzzo, M. et al. Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Mol. Microbiol. 66, 151–163 (2007).

    Article  CAS  Google Scholar 

  5. Runti, G. et al. Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7 (1–35). J. Bacteriol. 195, 5343–5351 (2013).

    Article  CAS  Google Scholar 

  6. Hansen, A., Schäfer, I., Knappe, D., Seibel, P. & Hoffmann, R. Intracellular toxicity of proline-rich antimicrobial peptides shuttled into mammalian cells by the cell-penetrating peptide penetratin. Antimicrob. Agents Chemother. 56, 5194–5201 (2012).

    Article  CAS  Google Scholar 

  7. Stalmans, S. et al. Blood-brain barrier transport of short proline-rich antimicrobial peptides. Protein Pept. Lett. 21, 399–406 (2014).

    Article  CAS  Google Scholar 

  8. Otvos, L. et al. Interaction between heat shock proteins and antimicrobial peptides. Biochemistry 39, 14150–14159 (2000).

    Article  CAS  Google Scholar 

  9. Czihal, P. et al. Api88 is a novel antibacterial designer peptide to treat systemic infections with multidrug-resistant Gram-negative pathogens. ACS Chem. Biol. 7, 1281–1291 (2012).

    Article  CAS  Google Scholar 

  10. Knappe, D. et al. Rational design of oncocin derivatives with superior protease stabilities and antibacterial activities based on the high–resolution structure of the oncocin–DnaK complex. ChemBioChem 12, 874–876 (2011).

    Article  CAS  Google Scholar 

  11. Zahn, M. et al. Structural studies on the forward and reverse binding modes of peptides to the chaperone DnaK. J. Mol. Biol. 425, 2463–2479 (2013).

    Article  CAS  Google Scholar 

  12. Zahn, M. et al. Structural identification of DnaK binding sites within bovine and sheep bactenecin Bac7. Protein Pept. Lett. 21, 407–412 (2014).

    Article  CAS  Google Scholar 

  13. Berthold, N. & Hoffmann, R. Cellular uptake of apidaecin 1b and related analogs in Gram-negative bacteria reveals novel antibacterial mechanism for proline-rich antimicrobial peptides. Protein Pept. Lett. 21, 391–398 (2014).

    Article  CAS  Google Scholar 

  14. Krizsan, A. et al. Insect-derived proline-rich antimicrobial peptides kill bacteria by inhibiting bacterial protein translation at the 70 S ribosome. Angew. Chem. Int. Ed. Engl. 53, 12236–12239 (2014).

    Article  CAS  Google Scholar 

  15. Schneider, M. & Dorn, A. Differential infectivity of two Pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera). J. Invertebr. Pathol. 78, 135–140 (2001).

    Article  CAS  Google Scholar 

  16. Arenz, S. et al. Drug sensing by the ribosome induces translational arrest via active site perturbation. Mol. Cell 56, 446–452 (2014).

    Article  CAS  Google Scholar 

  17. Bischoff, L., Berninghausen, O. & Beckmann, R. Molecular basis for the ribosome functioning as an l-tryptophan sensor. Cell Reports 9, 469–475 (2014).

    Article  CAS  Google Scholar 

  18. Shao, S. & Hegde, R.S. Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors. Mol. Cell 55, 880–890 (2014).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Schmeing, T.M., Huang, K.S., Strobel, S.A. & Steitz, T.A. An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA. Nature 438, 520–524 (2005).

    Article  CAS  Google Scholar 

  22. Schuwirth, B.S. et al. Structures of the bacterial ribosome at 3.5 A resolution. Science 310, 827–834 (2005).

    Article  CAS  Google Scholar 

  23. Arenz, S. et al. Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide. Nat. Commun. 5, 3501 (2014).

    Article  Google Scholar 

  24. Knappe, D. et al. Oncocin (VDKPPYLPRPRPPRRIYNR-NH2): a novel antibacterial peptide optimized against Gram-negative human pathogens. J. Med. Chem. 53, 5240–5247 (2010).

    Article  CAS  Google Scholar 

  25. Hartz, D., McPheeters, D.S., Traut, R. & Gold, L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 164, 419–425 (1988).

    Article  CAS  Google Scholar 

  26. Starosta, A.L. et al. Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site. Nucleic Acids Res. 42, 10711–10719 (2014).

    Article  CAS  Google Scholar 

  27. Wilson, D.N. The A–Z of bacterial translation inhibitors. Crit. Rev. Biochem. Mol. Biol. 44, 393–433 (2009).

    Article  CAS  Google Scholar 

  28. Dinos, G. et al. Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding. Mol. Cell 13, 113–124 (2004).

    Article  CAS  Google Scholar 

  29. Vázquez-Laslop, N., Ramu, H., Klepacki, D., Kannan, K. & Mankin, A.S. The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide. EMBO J. 29, 3108–3117 (2010).

    Article  Google Scholar 

  30. Starosta, A.L. et al. Interplay between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition. Chem. Biol. 17, 504–514 (2010).

    Article  CAS  Google Scholar 

  31. Wilson, D.N. Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat. Rev. Microbiol. 12, 35–48 (2014).

    Article  CAS  Google Scholar 

  32. Bulkley, D., Innis, C.A., Blaha, G. & Steitz, T.A. Revisiting the structures of several antibiotics bound to the bacterial ribosome. Proc. Natl. Acad. Sci. USA 107, 17158–17163 (2010).

    Article  CAS  Google Scholar 

  33. Dunkle, J.A., Xiong, L., Mankin, A.S. & Cate, J.H. Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc. Natl. Acad. Sci. USA 107, 17152–17157 (2010).

    Article  CAS  Google Scholar 

  34. Schlünzen, F., Pyetan, E., Fucini, P., Yonath, A. & Harms, J.M. Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin. Mol. Microbiol. 54, 1287–1294 (2004).

    Article  Google Scholar 

  35. Brazier, S.P., Ramesh, B., Haris, P.I., Lee, D.C. & Srai, S.K. Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1. Biochem. J. 335, 375–380 (1998).

    Article  CAS  Google Scholar 

  36. Jean-François, F. et al. Variability in secondary structure of the antimicrobial peptide Cateslytin in powder, solution, DPC micelles and at the air-water interface. Eur. Biophys. J. 36, 1019–1027 (2007).

    Article  Google Scholar 

  37. Jobin, M.L. et al. The enhanced membrane interaction and perturbation of a cell penetrating peptide in the presence of anionic lipids: toward an understanding of its selectivity for cancer cells. Biochim. Biophys. Acta 1828, 1457–1470 (2013).

    Article  CAS  Google Scholar 

  38. Khemtémourian, L., Buchoux, S., Aussenac, F. & Dufourc, E.J. Dimerization of Neu/Erb2 transmembrane domain is controlled by membrane curvature. Eur. Biophys. J. 36, 107–112 (2007).

    Article  Google Scholar 

  39. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006).

    Article  CAS  Google Scholar 

  40. Schmitt, E., Blanquet, S. & Mechulam, Y. Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNAMetf formyltransferase complexed with formyl-methionyl-tRNAMetf. Acta Crystallogr. D Biol. Crystallogr. 55, 332–334 (1999).

    Article  CAS  Google Scholar 

  41. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  42. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  43. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  44. de Bakker, P.I., DePristo, M.A., Burke, D.F. & Blundell, T.L. Ab initio construction of polypeptide fragments: accuracy of loop decoy discrimination by an all-atom statistical potential and the AMBER force field with the Generalized Born solvation model. Proteins 51, 21–40 (2003).

    Article  CAS  Google Scholar 

  45. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  46. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  47. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  Google Scholar 

Download references

Acknowledgements

We thank the staff at the European Synchrotron Radiation Facility (beamline ID-29) for help during data collection and B. Kauffmann and S. Massip at the Institut Européen de Chimie et Biologie for help with crystal freezing and screening. We also thank C. Mackereth for discussions and advice. This research was supported by grants from the Agence Nationale pour la Recherche (ANR-14-CE09-0001 to C.A.I., G.G. and D.N.W.), Région Aquitaine (2012-13-01-009 to C.A.I.), the Fondation pour la Recherche Médicale (AJE201133 to C.A.I.), the European Union (PCIG14-GA-2013-631479 to C.A.I.), the CNRS (C.D.) and the Deutsche Forschungsgemeinschaft (FOR1805, WI3285/4-1 and GRK1721 to D.N.W.). Predoctoral fellowships from the Direction Générale de l'Armement and Région Aquitaine (S. Antunes) and INSERM and Région Aquitaine (A.C.S.) are gratefully acknowledged.

Author information

Author notes

  1. A Carolin Seefeldt, Fabian Nguyen and Stéphanie Antunes: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Européen de Chimie et Biologie, Université de Bordeaux, Pessac, France

    A Carolin Seefeldt, Stéphanie Antunes, Natacha Pérébaskine, K Kishore Inampudi, Céline Douat, Gilles Guichard & C Axel Innis

  2. INSERM U869, Bordeaux, France

    A Carolin Seefeldt, Natacha Pérébaskine, K Kishore Inampudi & C Axel Innis

  3. Department of Biochemistry, Gene Center, University of Munich, Munich, Germany

    Fabian Nguyen, Michael Graf, Stefan Arenz & Daniel N Wilson

  4. Université de Bordeaux, CNRS, Institut Polytechnique de Bordeaux, UMR 5248, Institut de Chimie et Biologie des Membranes et des Nano-objets (CBMN), Pessac, France

    Stéphanie Antunes, Céline Douat & Gilles Guichard

  5. Center for Integrated Protein Science Munich (CiPSM), University of Munich, Munich, Germany

    Daniel N Wilson

Authors
  1. A Carolin Seefeldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fabian Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stéphanie Antunes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Natacha Pérébaskine
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Graf
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefan Arenz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K Kishore Inampudi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Céline Douat
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gilles Guichard
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel N Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. C Axel Innis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.C.S. performed structure solution, model building and analysis. N.P. prepared and crystallized ribosomes. N.P. and C.A.I. collected X-ray crystallography data. F.N. performed growth and in vitro–translation inhibition assays. S. Antunes and C.D. synthesized the peptides and performed NMR, CD and electrospray ionization high-resolution MS experiments. M.G. performed toe-printing assays. S. Arenz performed disome assays. K.K.I. prepared tRNAiMet. G.G., D.N.W. and C.A.I. designed experiments, interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Daniel N Wilson or C Axel Innis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Overlap of Onc112 with nascent polypeptide chains in the ribosome exit tunnel.

Comparison of the binding position of Onc112 (orange) with (a) ErmCL (green), (b) TnaC (blue) and Sec61β (red) nascent chains. In (a)-(c), the CCA-end of the P-tRNA is shown in white and in (b) the two tryptophan molecules are in cyan.

Supplementary Figure 2 Comparison of Tth70S–Onc112 with the DnaK–oncocin complex.

The conformation of residues Lys3–Pro10 of the Oncocin peptide O2 (cyan, VDKPPYLPRPRPPROIYNO–NH2, where O represents ornithine) in complex with DnaK (white surface representation) was compared with residues Val1–Pro12 of Onc112 (orange) from the ribosome-bound Onc112 structure.

Supplementary Figure 3 Conformation of the Onc112 peptide in solution.

Far-UV circular dichroism (CD) spectra of the Onc112 peptide at concentrations ranging from 20 to 200 μM.

Supplementary Figure 4 Inhibitory activity of Onc112 peptide derivatives.

(a-b) Effect of Onc112 (red) and Onc112 derivatives Onc112–L7Cha (blue) and Onc112–D2E (olive) on (a) the overnight growth of E. coli strain BL21(DE3) and (b) the luminescence resulting from the in vitro translation of firefly luciferase (Fluc). In (a), the error bars represent the standard deviation (s.d.) from the mean for a triplicate experiment (n=3). In (b), the experiment was performed in duplicate (n=2). The growth or luminescence measured in the absence of peptide was assigned as 100%.

Supplementary Figure 5 Validation of Onc112 and derivatives.

(a) Electrospray ionization high resolution mass spectrometry (ESI-HRMS) and reverse phase (RP) high performance liquid chromatography (HPLC), and (b) 1H nuclear magnetic resonance (NMR) spectra of the Onc112 peptide. (c-f) ESI-HRMS and RP HPLC of the (c) Onc112–ΔC9, (d) Onc112–ΔC7, (e) Onc112–L7Cha and (f) Onc112–D2E peptides.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 516 kb)

Supplementary Data Set 1

Toe-printing assay performed in the presence of increasing concentrations of Onc112 or several antibiotics, as shown in Figure 3. (PDF 2665 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Seefeldt, A., Nguyen, F., Antunes, S. et al. The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex. Nat Struct Mol Biol 22, 470–475 (2015). https://doi.org/10.1038/nsmb.3034

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3034

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research