Deciphering the Multisite Interactions of a Protein and Its Ligand at Atomic Resolution by Using Sensitive...

Area of Science:

• Biophysics
• Structural Biology
• Chemical Biology

Background:

• Analyzing protein-ligand interactions at atomic resolution is challenging due to difficulties in identifying binding sites and their flexibility.
• Paramagnetic metal centers can be used to perturb NMR signals, aiding in the study of binding events.

Purpose of the Study:

• To quantitatively analyze the multisite interactions between hen egg white lysozyme (HEWL) and lanthanide complexes using paramagnetic NMR spectroscopy.
• To investigate the binding affinities and flexibilities at different binding sites at atomic resolution.

Main Methods:

• Sensitive paramagnetic NMR spectroscopy was employed to study the binding of HEWL with lanthanide complexes ([Ln(DPA)3]3-).
• Paramagnetic relaxation enhancement (PRE) was used to detect and characterize binding interactions.
• Atomistic molecular dynamics (MD) simulations were performed to complement experimental findings.

Main Results:

• Paramagnetic NMR revealed four major binding sites for [Ln(DPA)3]3- on HEWL in aqueous solution, differing from previous X-ray crystallography findings.
• Experimental data on varied binding affinities and site flexibilities were consistent with MD simulations.
• The study successfully delineated multisite interactions at atomic resolution.

Conclusions:

• A combined approach of paramagnetic NMR spectroscopy and MD simulations provides a powerful method for characterizing protein-ligand interactions.
• This technique offers atomic-resolution insights into both binding affinity and flexibility at multiple interaction sites.
• The findings advance the understanding of complex protein-ligand dynamics and interactions.