Cysteine and histidine pKa prediction: alchemical free energy calculations and force field reparameterization

Given the growing interest in designing targeted covalent inhibitors, methods for rapidly and accurately probing pKas—and, by extension, the reactivities—of target cysteines is highly desirable. Complementary to cysteine, histidine is similarly relevant due to its frequent presence in protein active sites and its unique ability to exist in two tautomeric states. Here we demonstrate that non-equilibrium free energy calculations can accurately determine the pKa values of both residues, in many cases outperforming conventional predictors. Importantly, we find that: 1) increasing the van der Waals radii of cysteine's sulfur atom; 2) downscaling the side chain charges of cysteine; and 3) altering the backbone charges of histidine can all significantly improve pKa prediction accuracy. Using the charge-scaled CHARMM36m force field, we achieve an average unsigned error of 1.71 ± 0.23 pK for cysteine and 0.71 ± 0.16 pK for histidine.