Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial ribosomes by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee apidaecin, traps the release factor (RF) at the ribosome, thereby arresting the ribosomes at stop codons. This study shows that Api137 occupies a second binding site near the exit of the PET and represses protein translation independently of RF1-trapping. Api88, the C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds in a slightly shifted manner to the same regions without RF trapping, presumably by interfering with the translation process. Api88 also binds to a pocket deep within domain III of the 23S rRNA. In conclusion, Api88 and Api137 inhibit ribosomes through different multimodal mechanisms. These mechanisms have likely reduced the emergence of bacterial resistance and contributed to the evolutionary success of this structurally diverse group of PrAMPs, providing a promising pool for drug development efforts.

Tryptic in-gel digests gained from cross-linked and enriched protein mixture were analyzed, whereas a comparison between protein mixtures incubated with and without biotin-SG-Api88(Y7B) was achieved. Samples were analyzed on a nanoACQUITY UPLC system (Waters Corp., Eschborn, Germany) coupled online to an electrospray ionization quadrupole-time-of-flight mass spectrometer (Synapt G2-Si MS, Waters). The acquired data were processed using PEAKS Studio 10.6 Xpro (Bioinformatics Solutions, Waterloo, Canada). Database searches were performed using an E. coli database downloaded from uniprot (March 28, 2020) containing 4392 protein sequences and additionally the sequences of Api88, mCherry, mAzamy, trypsin, and streptavidin, with a false discovery rate of 1% at the peptide level. The data were exported as peptides.pep.xml files and loaded to Skyline (64-bit, version 22.2.0.351, MacCoss Lab Software, Washington, US). A spectral library was built from all identified peptides. Proteins identified in both replicates were considered enriched based on the peak area ratios obtained in 70S ribosome samples UV-irradiated in the presence or absence (control) of biotin-SG-Api88(Y7B).

The 70S ribosome was UV irradiated, either with or without biotin-SG-Api88(Y7B). The protein mixtures were enriched using magnetic beads, and the eluates were separated by SDS PAGE. In-gel digestion of the separated lanes was performed, and the peptides were analyzed by LC-MS. The control (without prAMP) and the ribosome incubated with the peptide (biotin-SG-Api88(Y7B)) were analyzed in duplicates. Skyline Documents: A Main Skyline document was established and further divided into two main projects: _only other proteins (contains all "other" detected proteins except for ribosomal proteins) _Only ribosomal Proteins (contains all detected ribosomal proteins but no others) After detailed inspection of the "_only ribosomal Proteins" Project, it was further narrowed down to the most promising enriched proteins: "_only_candidatesForEnrichedProteins".