Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

Area of Science:

• Biomolecular interactions
• Structural biology
• Molecular self-assembly

Background:

• The adaptor complex 2 (AP-2) is crucial for clathrin-mediated endocytosis, mediating protein-protein interactions.
• AP-2's α-appendage domain possesses two binding grooves, each recognizing distinct peptide motifs with micromolar affinity.
• Understanding the spatial arrangement requirements for AP-2's high-affinity binding is essential for elucidating its function.

Purpose of the Study:

• To investigate the distance-affinity relationships of peptide binding to the AP-2 α-appendage using DNA-programmed spatial screening.
• To determine if specific spatial arrangements of binding motifs are required for enhanced affinity.
• To explore the role of flexibility in DNA-based ligand displays for biomolecular interaction studies.

Main Methods:

• Utilized DNA-based spatial screening with self-assembled peptide-DNA complexes to precisely control ligand display.
• Constructed trimolecular and tetramolecular DNA assemblies to position peptides at defined distances (2-22 nucleotides).
• Measured binding affinities using recombinant AP-2 and native AP-2 in brain extract.

Main Results:

• Cooperative binding between the two sites on the AP-2 α-appendage can enhance affinity up to 40-fold compared to monovalent interactions.
• Flexible DNA duplex segments allowed for less distance-dependent binding, while rigid assemblies showed significant distance sensitivity.
• The flexibility of the DNA scaffold is a critical factor in modulating binding affinity and spatial requirements.

Conclusions:

• The AP-2 α-appendage can achieve high-affinity interactions through cooperative binding, independent of precise peptide spacing when the scaffold is flexible.
• DNA-programmed spatial screening is a powerful tool for characterizing multivalency and optimizing biomolecular interaction studies.
• Findings provide insights into the functional mechanisms of adaptor proteins in endocytosis and protein network organization.