Protonation states were assessed using H++ 3.2 at pH 7.4. A cubic explicit water box described using the TIP3P model was used to solvated the complex system, which was extended by 10 Å from the solute. An atmosphere of 150 mM NaCl was also included in all simulations. The generated models were parametrized using amber ff1+4SB force fields for protein. Subsequently, the parameter files created by tleap in Amber18 were converted to gromacs format. About, 5000 steps of minimization including 2500 steps of steepest descent minimization and 2500 steps of conjugate gradient minimization were performed to remove bad contacts during the energy minimization phase. Equilibration in NPT ensemble was run at 1.0 bar and 300 K for 500 000 steps at 2 fs/step. Gromacs2020.2 software package was used to run the minimization, equilibration simulations with position constraints (1 kcal/mol/Å 2 ) on protein.