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

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.

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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.

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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.

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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.

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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)

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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

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