Thermodynamic analysis of an entropically driven, high-affinity nanobody-HIV p24 interaction

Area of Science:

• Structural biology
• Computational biophysics
• Immunology

Background:

• Protein-protein interactions are crucial for biological functions.
• Antibody fragments like nanobodies offer high specificity and affinity for molecular studies.
• Understanding the energetic basis of these interactions is key to their specificity and strength.

Purpose of the Study:

• To quantify the entropic and enthalpic contributions to nanobody-protein binding affinity.
• To investigate how amino acid changes impact binding energetics.
• To elucidate the role of water release in driving binding affinity differences.

Main Methods:

• Circular dichroism spectroscopy for qualitative analysis.
• All-atom molecular dynamics simulations.
• Isothermal titration calorimetry, WaterMap, and Free Energy Perturbation for quantitative analysis.

Main Results:

• The affinity difference between high (59H10) and medium (37E7) affinity nanobodies binding to HIV-1 p24 is entropically driven.
• This entropic contribution is attributed to the release of unstable water molecules from the hydrophobic surface of the high-affinity nanobody.
• Amino acid substitutions significantly alter binding energetics, with entropy playing a dominant role.

Conclusions:

• Combined in vitro and in silico approaches effectively separate enthalpy and entropy contributions.
• Differences in water-mediated entropic interactions can account for large variations in binding affinity.
• This study provides insights into the molecular mechanisms governing nanobody-protein interactions and affinity modulation.