Elucidation of Nonadditive Effects in Protein–Ligand Binding Energies: Thrombin as a Case Study

Accurate predictions
of free energies of binding of ligands to
proteins are challenging partly because of the nonadditivity of protein–ligand
interactions; i.e., the free energy of binding is the sum of numerous
enthalpic and entropic contributions that cannot be separated into
functional group contributions. In principle, molecular simulations
methodologies that compute free energies of binding do capture nonadditivity
of protein–ligand interactions, but efficient protocols are
necessary to compute well-converged free energies of binding that
clearly resolve nonadditive effects. To this end, an efficient GPU-accelerated
implementation of alchemical free energy calculations has been developed
and applied to two congeneric series of ligands of the enzyme thrombin.
The results show that accurate binding affinities are computed across
the two congeneric series and positive coupling between nonpolar R1 substituents and a X = NH3+ substituent
is reproduced, albeit with a weaker trend than experimentally observed.
By contrast, a docking methodology completely fails to capture nonadditive
effects. Further analysis shows that the nonadditive effects are partly
due to variations in the strength of a hydrogen-bond between the X
= NH3+ ligands family and thrombin residue Gly216.
However, other partially compensating interactions occur across the
entire binding site, and no single interaction dictates the magnitude
of the nonadditive effects for all the analyzed protein–ligand
complexes.