Protein-protein interactions govern many cellular processes, and identifying binding interaction sites on proteins can facilitate the discovery of inhibitors to block such interactions. Here we identify peptides from a randomly fragmented plasmid encoding the β-lactamase inhibitory protein (BLIP) and the Lac repressor (LacI) that represent regions of protein-protein interactions. We utilized a Jun-Fos-assisted phage display system that has previously been used to screen cDNA and genomic libraries to identify antibody antigens. Affinity selection with polyclonal antibodies against LacI or BLIP resulted in the rapid enrichment of in-frame peptides from various regions of the proteins. Further, affinity selection with β-lactamase enriched peptides that encompass regions of BLIP previously shown to contribute strongly to the binding energy of the BLIP/β-lactamase interaction, i.e., hotspot residues. Further, one of the regions enriched by affinity selection encompassed a disulfide-constrained region of BLIP that forms part of the BLIP interaction surface in the native complex that we show also binds to β-lactamase as a disulfide-constrained macrocycle peptide with a KD of ∼1 μM. Fragmented open reading frame (ORF) libraries may efficiently identify such naturally constrained peptides at protein-protein interaction interfaces. With sufficiently deep coverage of ORFs by peptide-coding inserts, phage display and deep sequencing can provide detailed information on the domains or peptides that contribute to an interaction. Such information should enable the design of potentially therapeutic macrocycles or peptidomimetics that block the interaction.
Keywords: Lac repressor; bZIP domain; genomic library; leucine zipper; macrocycle; peptide; phage display; protein−protein interaction; β-lactamase; β-lactamase inhibitory protein.