Molecular Determinants of Ribosome Binding for the Antimicrobial Peptide Pdi1

Type II proline-rich antimicrobial peptides have emerged as promising therapeutic candidates due to their ability to inhibit a previously undrugged stage of bacterial protein synthesis: translation termination. The recent characterization of the potent Type II proline-rich antimicrobial peptide, Pdi1, suggests that it may represent a promising therapeutic candidate; however, no information is available concerning its ribosome-binding activity. The lack of biochemical and biophysical assays to interrogate the binding affinities and mechanism of action of this class of peptides presents a bottleneck in the drug-discovery pipeline. In this study, we report the first characterization of the ribosome-binding kinetics of a Type II proline-rich antimicrobial peptide, for which we applied a biolayer interferometry assay to study the Pdi1-ribosome interaction. Our results reveal slow dissociation kinetics, which indicates prolonged residence time in the ribosomal nascent peptide exit tunnel. Additionally, alanine scanning of the Pdi1 C-terminus revealed that specific residues are critical for ribosome binding and contribute to the rate of Pdi1 dissociation. These findings were validated in cell-based assays where alanine substitutions at these positions reduced antibiotic activity. We also demonstrated that a Pdi1 derivative, which lacks the third-to-last pharmacophoric residue, fails to inhibit translation termination, despite retaining a submicromolar minimum inhibitory concentration. These results demonstrate that select residues in the Pdi1 pharmacophore play a critical role in ribosome binding and dissociation, in addition to inhibiting translation termination.