*3.5. MD Simulations*

GROMACS [45] v.2020 beta was utilized to execute the all-atom MD simulations for examining the reliability of the HSA–HpzA docking complex in solvent conditions. The Gromacs parameters for HpzA were generated from the PRODRG server [46] to prepare a protein–ligand complex system. Free HSA and the docked complex were implanted in the simple point charge (SPC) solvent model. HSA and the docked complex, HSA– HpzA, were neutralized with an appropriate number of counter ions (Na<sup>+</sup> and Cl−). The salt concentration was kept at physiological condition, i.e., 0.15 M. Both systems were stipulated on periodic boundary conditions, and the SHAKE algorithm was applied for limiting the movement of all bonds. The energy minimization of both the systems was performed using the steepest descent algorithm with and without solute restraints [18]. Then, 1000 ps simulations were performed in NVT and NPT ensembles at temperature 300 K. To control the temperature and pressure during the simulation, the Berendsen thermostats and barostats were applied. Both the relaxed systems were subjected to a final MD run for 100 ns with a time step of 2 fs [47]. For analysis purposes, RMSD, RMSF, *R*g, SASA, and hydrogen bonding were recorded. The recorded trajectories were checked for the stability of the HSA–HpzA docked complex in comparison with that of the free state of HSA.
