BPS2026 – Molecular dynamics study of binding free energy shift of protein complexes induced by single and double mutations

Protein-protein and protein-ligand interactions promote or inhibit protein functions in living cells and play a key role in many biological processes, including signal transduction, enzyme regulation, and so on. Measurement of binding free energy is essential to quantitatively evaluate the strength of these interactions. In particular, changes in binding free energy induced by amino acid mutations alter the conformational stability of two protein molecules, potentially resulting in functional changes. Compared to protein-ligand binding, calculation of the binding free energy of protein-protein complexes is considerably more complex due to the diverse orientations, large conformational spaces, making the measurement and prediction of their binding free energy more challenging. In this study, we propose a simple procedure to calculate the binding free energy shifts in protein complexes induced by amino acid mutations. We applied this method to the Barnase-Barstar complex using molecular dynamics (MD) simulations combined with alchemical free energy calculations. Our calculation included 10 single mutants and three double mutants. The calculated binding free energy shifts were almost consistent with the experimental results, with the exception of two cases. We showed that our method is effective for predicting binding free energy changes when the amino acid mutations do not induce large-scale conformational changes in the protein complex. The main advantages of our procedure are as follows. (1) There is no need to perform long-time MD simulations for each mutant complex. (2) Only equilibrium sampling of the wild-type complex and monomers are required, significantly reducing computational costs. (3) The method is applicable not only to single or double mutations but also to multiple mutations through an appropriate thermodynamic cycle.