Mapping Protein–Protein Interaction Interface Peptides with Jun-Fos Assisted Phage Display and Deep Sequencing

Abstract

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 disulfideconstrained macrocycle peptide with a K D 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.

Deep Sequencing of a Systematic Peptide Library Reveals Conformationally‐Constrained Protein Interface Peptides that Disrupt a Protein‐Protein Interaction

“…Thus, the peptides were fused at the C‐terminus of the display protein and displayed on the phage surface via the interaction of Fos‐peptide with Jun‐gene III protein. Three regions of BLIP were enriched after affinity selection of the Jun‐Fos assisted phage display library for binding TEM‐1 β‐lactamase and included BLIP Ala25−Asp49, Lys72−Thr92, and Gly154−Val165 [61] . These regions are also localized at the BLIP‐β‐lactamase binding interface and the synthesized peptides encompassing the regions were shown to bind TEM‐1 β‐lactamase in the low micromolar range; [61] however, they are different regions than those identified here.…”

“…Three regions of BLIP were enriched after affinity selection of the Jun‐Fos assisted phage display library for binding TEM‐1 β‐lactamase and included BLIP Ala25−Asp49, Lys72−Thr92, and Gly154−Val165 [61] . These regions are also localized at the BLIP‐β‐lactamase binding interface and the synthesized peptides encompassing the regions were shown to bind TEM‐1 β‐lactamase in the low micromolar range; [61] however, they are different regions than those identified here. Thus, the peptides selected in the competitive phage display process are likely to depend on the how the library peptides are presented.…”

“…We previously identified BLIP peptides that bind to TEM-1 β-lactamase from a phage display library constructed by randomly fragmenting a plasmid encoding BLIP and the Lac repressor (LacI). [61] This experiment utilized Jun-Fos assisted phage display whereby the M13 gene III capsid protein was fused to the leucine zipper domain of Jun and the randomly fragmented DNA was inserted at the C-terminus of the leucine zipper domain of Fos. [61,62] Thus, the peptides were fused at the C-terminus of the display protein and displayed on the phage surface via the interaction of Fos-peptide with Jun-gene III protein.…”

“…[61] This experiment utilized Jun-Fos assisted phage display whereby the M13 gene III capsid protein was fused to the leucine zipper domain of Jun and the randomly fragmented DNA was inserted at the C-terminus of the leucine zipper domain of Fos. [61,62] Thus, the peptides were fused at the C-terminus of the display protein and displayed on the phage surface via the interaction of Fos-peptide with Jun-gene III protein. Three regions of BLIP were enriched after affinity selection of the Jun-Fos assisted phage display library for binding TEM-1 β-lactamase and included BLIP Ala25À Asp49, Lys72À Thr92, and Gly154À Val165.…”

Mapping human norovirus antigens during infection reveals the breadth of the humoral immune response

“…The inserts with a count of 5 or above were selected for the coverage score determination. Coverage scores were determined using the bedtools genomecov program and were defined as the per-nucleotide number of occurrences for each insert after alignment to the reference genome: pKS-NV68 plasmid for the GI.1 experiments and pKS-SagaFpA50 plasmid for the GII.4 HOV experiments 27 , 28 , 94 . The inserts that are in-frame to GI.1 and GII.4 HOV ORF1 to ORF3 in the pKS-NV68 and pKS-SagaFpA50 plasmids were determined and counted.…”