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

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein-protein interactions are crucial for cellular functions.
• Identifying interaction sites aids in developing inhibitors for therapeutic purposes.

Purpose of the Study:

• To identify peptides representing protein-protein interaction regions using a phage display system.
• To explore the potential of fragmented open reading frame (ORF) libraries for discovering constrained peptides at interaction interfaces.

Main Methods:

• Utilized a Jun-Fos-assisted phage display system.
• Employed affinity selection with polyclonal antibodies against Lac repressor (LacI) and β-lactamase inhibitory protein (BLIP).
• Performed affinity selection with β-lactamase to identify BLIP interaction regions.

Main Results:

• Rapid enrichment of in-frame peptides from LacI and BLIP regions.
• Identified BLIP peptides corresponding to hotspot residues involved in β-lactamase binding.
• Discovered a disulfide-constrained BLIP peptide macrocycle that binds β-lactamase with a dissociation constant (K_D) of approximately 1 μM.

Conclusions:

• Fragmented ORF libraries coupled with phage display and deep sequencing efficiently identify peptides at protein-protein interaction interfaces.
• This approach facilitates the discovery of naturally constrained peptides.
• The findings support the design of therapeutic macrocycles and peptidomimetics to inhibit protein-protein interactions.