Molecular Determinants of Ribosome Binding for the Antimicrobial Peptide Pdi1

Abstract

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.