Label-Free Mapping of Multivalent Binding Pathways with Ligand-Receptor-Anchored Nanopores.

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Understanding multivalent ligand-receptor interactions is key to biological recognition.
• Label-free, real-time analysis of transient, multistep binding events is challenging.

Purpose of the Study:

• To develop a method for mapping dynamic multivalent interactions at the single-molecule level.
• To elucidate binding pathways and affinities between Omicron spike protein and soluble ACE2.

Main Methods:

• Utilized a ligand-receptor-anchored nanopore system for native protein analysis.
• Employed a four-state Markov chain model for kinetic analysis.
• Performed single-monomeric subunit level real-time kinetic analysis.

Main Results:

• Identified two concentration-dependent binding pathways (sequential and concurrent) for Omicron spike protein (Omicron S) and soluble ACE2 (sACE2).
• Quantified a robust binding affinity between Omicron S1 monomers and sACE2 (-13.1 ± 0.2 kcal/mol).
• Demonstrated that sACE2 binding to Omicron S facilitates subsequent binding steps, resolving previous measurement discrepancies.

Conclusions:

• The nanopore method enables detailed mapping of dynamic multivalent interactions.
• Findings explain Omicron's enhanced infectivity by detailing its robust binding to sACE2.
• Provides insights for drug design targeting viral entry mechanisms.