Alchemical Calculation of Relative Free Energies for Charge-Changing Mutations at Protein-Protein Interfaces Considering Fixed and Variable Protonation States

The calculation of relative free energies (Delta Delta G) for charge-changing mutations at protein-protein interfaces through alchemical methods remains challenging due to variations in the system's net charge during charging steps, the possibility of mutated and contacting ionizable residues occurring in various protonation states, and undersampling issues. In this study, we present a set of strategies, collectively termed TIRST/TIRST-H+, to address some of these challenges. Our approaches combine thermodynamic integration (TI) with the prediction of pK(a) shifts to calculate Delta Delta G values. Moreover, special sets of restraints are employed to keep the alchemically transformed molecules separated. The accuracy of the devised approaches was assessed on a large and diverse data set comprising 164 point mutations of charged residues (Asp, Glu, Lys, and Arg) to Ala at the protein-protein interfaces of complexes with known three-dimensional structures. Mean absolute and root-mean-square errors ranging from 1.38 to 1.66 and 1.89 to 2.44 kcal/mol, respectively, and Pearson correlation coefficients of similar to 0.6 were obtained when testing the approaches on the selected data set using the GPU-TI module of Amber18 suite and the ff14SB force field. Furthermore, the inclusion of variable protonation states for the mutated acid residues improved the accuracy of the predicted Delta Delta G values. Therefore, our results validate the use of TIRST/TIRST-H+ in prospective studies aimed at evaluating the impact of charge-changing mutations to Ala on the stability of protein-protein complexes.

Automated summary

This study presents a set of strategies, termed TIRST/TIRST-H+, to calculate the relative free energies of charge-changing mutations at protein-protein interfaces. The approaches combine thermodynamic integration with pK(a) shifts prediction, and special restraints are used to keep the transformed molecules separated. The accuracy of the methods was validated on a diverse data set, and the inclusion of variable protonation states improved the predicted Delta Delta G values.