Multiplex mapping of protein-protein interaction interfaces

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Identifying protein-protein interactions (PPIs) is crucial for understanding cellular functions.
• Existing methods often require specialized equipment or in vitro reconstitution.
• Mapping interaction interfaces, especially for weak or transient complexes, remains challenging.

Purpose of the Study:

• To introduce Split Antibiotic Resistance Complementation (SpARC-map), a novel in vivo method for peptide mapping and PPI interface identification.
• To demonstrate SpARC-map's ability to detect PPIs across a broad affinity range, including weak interactions.
• To validate SpARC-map's utility in identifying functional interfaces and constraining structural predictions.

Main Methods:

• SpARC-map employs in vivo affinity selection within a bacterial host.
• High-throughput DNA sequencing is utilized to infer protein-protein interaction (PPI) interfaces.
• The method relies on routine microbiology techniques, avoiding specialized reagents or in vitro complex reconstitution.

Main Results:

• SpARC-map successfully identified known PPI interfaces in p21-PCNA, p53-MDM2, and MYC-MAX complexes.
• The method revealed potential functional interfaces within the purinosome complex, supporting substrate channeling.
• SpARC-map results were validated using site-specific photocrosslinking and demonstrated utility in constraining machine learning-based structure prediction.

Conclusions:

• SpARC-map is a versatile and sensitive method for identifying protein-protein interaction interfaces in vivo.
• The technique is adaptable for various affinities, multiplexing, and precise background measurement.
• SpARC-map provides valuable insights into complex formation, substrate channeling, and aids in computational structure prediction.