Binding free energy calculation is a powerful tool in providing quantitative measurement of protein-ligand interactions (32). The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) approach was used to compute binding free energies, as it is often used to study biomolecular complexes (33). In brief, the binding free energy (ΔG binding) was calculated by the equation listed below using an open source tool called g_mmpbsaΔGbind=GcomplexGproteinGligand=ΔH+ΔGsolTΔS=ΔEMM+ΔGsolTΔS(1)

ΔEMM consists of the electrostatic and van der Waals energies and is the gas-phase interaction energy between protein and ligand. ΔGsol includes the polar solvation free energy and the nonpolar energy—the former is calculated with the generalized Born approximation model and the latter is obtained as a function of the solvent-accessible surface area. TΔS indicates the changing of conformational entropy upon ligand binding. It is acceptable to exclude the entropy contribution (TΔS) in practice if ligands have similar structures and binding modes (34).

Energy decomposition analysis was performed using the program (34). The residue-ligand interaction often contains four parts: van der Waals contribution (ΔGvdw), electrostatic contribution (ΔGele), the polar part of desolvation (ΔGGB), and the nonpolar part of desolvation (ΔGSA)ΔG=ΔGvdw+ΔGele+ΔGsol=ΔGvdw+ΔGele+ΔGGB+ΔGSA(2)