Antibiotic-resistant bacteria are a global health issue necessitating the development of new effective therapeutics. Proline-rich antimicrobial peptides (PrAMPs), which include oncocins, are an extensively studied class of AMPs that counteract bacterial infection at submicromolar concentrations. Oncocins enter and kill bacteria by inhibiting certain targets rather than by acting through membrane lysis. Although they have recently been reported to bind DnaK and the bacterial ribosome, their mode of inhibition has remained elusive. Here we report the crystal structure of the oncocin derivative Onc112 bound to the Thermus thermophilus 70S ribosome. Strikingly, this 19-residue proline-rich peptide manifests the features of several known classes of ribosome inhibitors by simultaneously blocking the peptidyl transferase center and the peptide-exit tunnel of the ribosome. This high-resolution structure thus reveals the mechanism by which oncocins inhibit protein synthesis, providing an opportunity for structure-based design of new-generation therapeutics.
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We thank P.B. Moore for valuable discussions, suggestions and critical reading of the manuscript. We thank R.L. Grodzicki for preparing the fMet-tRNAfMet. We also thank the staffs of the Advanced Photon Source beamline 24-ID for help with data collection and of the Center for Structural Biology facility at Yale University for computational support. This work was supported by US National Institutes of Health grant GM022778 to T.A.S.
The authors declare no competing financial interests.
Integrated supplementary information
Unbiased difference electron density Fourier map (green) (Fo – Fc, contoured at σ = 2.3) of onc112 (brown) bound in the ribosome peptide exit tunnel. The thirteen amino acid residues of onc112 that were modeled are labeled.
(a) The N-terminal part of onc112 (brown) would collide with the CCA-end of an accommodated tRNA in the A site (grey) (PDB accession 1VY4). Positions of the tRNAfMet bound in the P site of the onc112-70S ribosome complex (green) and the fMet-tRNAfMet (cyan) (PDB accession 1VY4) are shown. (b) The conformation of nucleotide U2585 (2596) in the complex of onc112 bound to the ribosome (light blue), whose usual positioning (grey) would collide with Pro5, is not compatible with the presence of an amino acid moiety attached to the P-site tRNA (cyan) (PDB accession 1VY4).
(a) Interacting atoms are shown in red (oxygen) and blue (nitrogen); hydrogen bonds are indicated by black dashes. The electron density Fourier map (blue), 2Fo – Fc, is contoured at σ=1.0. Distances less than 3.5 Å are shown. (b) The γ-carboxyl group of the Asp2 side chain of onc112 (brown) interacts with the nucleotide G2553 (2564) that otherwise would form a Watson-Crick base pair with the CCA-end (grey) C75 of an accommodated tRNA in the A site (PDB accession 1VY4). The Watson-Crick interactions between C75 and G2553 (2564) are indicated by black dashes.
(a) Nucleotide U2506 (2517) forms stabilizing interactions with the main chain peptide backbone of residues Tyr6 and Leu7 of onc112. (b) Water-mediated hydrogen bonding interaction between the hydroxyl group of Tyr6 and the phosphate oxygen of G2505 (2516). Putative hydrogen bonds between oxygen (red) and nitrogen (blue) atoms are indicated by black dashes.
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Roy, R., Lomakin, I., Gagnon, M. et al. The mechanism of inhibition of protein synthesis by the proline-rich peptide oncocin. Nat Struct Mol Biol 22, 466–469 (2015). https://doi.org/10.1038/nsmb.3031
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