A novel approach for studying receptor-ligand interactions on living cells surface by using NUS/T1ρ-NMR methodologies...

Area of Science:

• Biophysics
• Structural Biology
• Cellular Biology

Background:

• Studying receptor-ligand interactions on living cells using high-resolution Nuclear Magnetic Resonance (NMR) is challenging due to the short lifespan of cells.
• Existing methods often limit experimental duration to a few hours, hindering detailed structural and dynamic analysis.

Purpose of the Study:

• To develop an on-cell NMR-based approach for investigating the molecular determinants of receptor-ligand recognition mechanisms under native cellular conditions.
• To overcome the limitations of short cell lifetimes in structural biology studies.

Main Methods:

• Combined high-resolution structural and dynamics NMR data with Molecular Dynamics simulations and Molecular Docking.
• Utilized Non-Uniform Sampling (NUS) and T1ρ-NMR techniques to acquire atomic-resolution data within the cellular lifetime.
• Employed 2D NUS [1H-1H] trNOESY spectra for ligand conformational changes and T1ρ-based experiments for epitope mapping.

Main Results:

• Successfully collected high signal-to-noise ratio and high-resolution NMR spectra within the cells' lifespan.
• Characterized the conformational changes of a ligand upon receptor binding and identified the ligand binding epitope.
• Demonstrated the approach's efficacy in studying the αvβ5-integrin and RGDechi15D peptide interaction.

Conclusions:

• The developed on-cell NMR strategy provides an alternative tool for atomic-resolution studies of receptor-ligand recognition on living cell surfaces.
• This method can be valuable for screening drug interactions with therapeutic targets in their native cellular environment.