We performed binding free energy calculations using the alchemical approach, as implemented in YANK 0.17.0 (), which is a framework for free energy calculations based on Hamiltonian replica exchange molecular dynamics simulations. After testing different protocols containing manually selected lambda values defining the thermodynamic cycle, we decided to use the 'auto' option provided by YANK to automatically generate an optimized set of lambda values for the WT OPH complex. Then, we used the same protocol to run binding free energy calculations on the mutant forms of OPH. For the complex phase, 43 lambda values were defined for the electrostatic and steric terms and the default value of 1.0 was used for lambda restraints in every thermodynamic state. For the solvent phase, 6 lambda values were specified for the electrostatic and steric contributions. We ran the simulations in an implicit/continuum solvent, namely Onufriev-Bashford-Case (OBC2) GBSA model ). All particle interactions were computed without any cutoff or periodic copes. We applied harmonic restraints to keep the substrate near its binding site within the WT and mutant forms of OPH. These restraints were constant throughout the thermodynamic states.