Abstract
The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.
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Data availability
The uncropped gels, the raw data used for calculating the DS and statistics data for Fig. 2b and Extended Data Fig. 1a are shown in the Source Data file associated with the manuscript. As raw sequencing data for the mutant Dro gene libraries do not represent genomic, RNA-seq or Ribo-seq results, they were not deposited to the public databases but are available from the corresponding authors upon request. Source data are provided with this paper.
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Acknowledgements
We thank T. Koller and D. Wilson (University of Hamburg) for generously sharing their results and the structure of the ribosome–drosocin complex. This work was supported in part by NIH grant R01 AI162961 (to A.S.M., N.V.-L. and Y.S.P.).
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Contributions
R.H., N.V.-L. and A.S.M. conceived the study. K.M. guided and supervised preparation of the endogenously expressed wt Dro and Dro mutant library. D.K. carried out toeprinting and microbiological experiments. A.B. and A.K. synthesized peptides and carried out in vitro translation and MIC testing experiments. I.O. cloned the Dro gene and carried out library screening experiments. K.M. and C.B. analyzed library screening results. C.B. consulted on multiple experiments. W.H. analyzed stop codon readthrough and Dro-resistant mutants. Y.S.P. analyzed structural data. K.M., C.B., A.K., R.H., N.V.-L. and A.S.M. analyzed data. K.M., N.V.-L. and A.S.M. wrote the manuscript.
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Up until 1 year before submission of this manuscript, R.H. served as an advisor for the company EnBiotix, Inc. on a project unrelated to this study. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Dro acts upon translation termination but causes weak translation arrest at UGA stop codons.
a, Inhibition of in vitro GFP translation by synthetic PrAMPs: class-I Oncocin112 (Onc112), class-II Api137, or non-glycosylated Dro (Dro). Bar graphs represent the normalized values of GFP fluorescence from reactions where RF1 was depleted (light grey) or supplemented (dark grey), setting the fluorescence value from reactions with or without RF1 in the absence of PrAMP as 100%. The error bars show standard deviation from the mean in three independent experiments. Significance levels indicated as NS, not significant; *, p-value <0.05; **, p-value < 0.01; ***, value < 0.001 (One-way ANOVA with Tukey’s Multiple Comparison test by GraphPad Prism). b, In vitro toeprinting analysis of the Api137 or Dro-mediated ribosome arrest at the UGA stop codon (red arrowhead) of the model yrbA ORF. The control reaction with no added PrAMPs is labeled as ‘none’. The control antibiotic retapamulin (Ret) stalls ribosomes at the start codon (green arrowhead). Sequencing reactions are labeled as C, U, A, G.
Extended Data Fig. 2 Endogenous expression of Dro in cells grown in rich medium does not prevent cell growth.
Growth of E. coli BL21 cells transformed with pDro[UAG] or pDro[UGA] plasmids on agar lysogeny broth (LB) rich medium supplemented with glucose or L-arabinose. The toxic effect of endogenous expression of Api (in cells transformed with pApi) is shown for comparison. Cells transformed with empty pBAD vector were used as a negative control.
Extended Data Fig. 3 Robustness of experimental data and comparisons of the effect of endogenously expressed Dro and Api variants on cell growth.
a, Correlation of the depletion scores of E. coli clones from the single amino acid Dro mutant libraries generated on the bases of pDro[UAG] or pDro[UGA] expression plasmids. Pearson correlation coefficient (r) is indicated. b, Depletion scores of library clones endogenously expressing single-amino acid Dro variants from the pDro[UGA] plasmids. Coloring is according to the toxic effects (blue - highly toxic, yellow - mildly toxic, salmon - not toxic). The analogous data for the pDro[UAG] library are shown in Fig. 4. c, Similarly calculated depletion scores for endogenously expressed Api mutants from the pApi plasmid using data from the reference7.
Extended Data Fig. 4 Dro variants with single amino acid substitutions retain the ability to arrest ribosomes at stop codons.
Toeprinting analysis of the ribosome arrest of the UAG stop codon of the model yrbA ORF mediated by synthetic non-glycosylated Dro variants. Samples with no added synthetic peptide are labeled as ‘none’. Arrest at stop codons caused by Api137 or at start codons by retapamulin (Ret) are shown as reference. Toeprint bands from ribosomes stalled at start or stop codons are marked by green and red arrowheads, respectively. Shown are representative gels of two independent experiments that produced converging results.
Extended Data Fig. 5 Functionally critical contacts of Dro with the ribosome as revealed by mutational analysis.
The central image depicts the placement of glycosylated Dro in the NPET of the E. coli ribosome50. Functionally critical Dro residues are indicated in salmon; residues that tolerate multiple substitutions are marked in green. a-e, Functionally critical contacts involving Dro residues: a, Arg18, b, Arg15 and Pro16, c, Arg9, d, Arg4, e, Lys2. Panel f shows the ribosomal contacts of the Pro5-Pro8 segment of Dro where most amino acids substitutions do not impair the PrAMP’s activity.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2 combined in a single pdf file.
Source data
Source Data Fig. 1
Uncropped gels for Fig. 1c.
Source Data Fig. 2
Raw data and statistical values for Fig. 2b.
Source Data Fig. 4
Read counts for UAG and UGA codons (Fig. 4).
Source Data Fig. 5
Raw data and statistical values for Fig. 5a.
Source Data Fig. 5
Uncropped gels for Fig. 5a.
Source Data Extended Data Fig. 1
Raw data and statistical values for ED Fig. 1a.
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Mangano, K., Klepacki, D., Ohanmu, I. et al. Inhibition of translation termination by the antimicrobial peptide Drosocin. Nat Chem Biol 19, 1082–1090 (2023). https://doi.org/10.1038/s41589-023-01300-x
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DOI: https://doi.org/10.1038/s41589-023-01300-x
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