Molecular dynamics simulations were employed to investigate the interaction between Fe(III) and an iron-binding site composed of THR259, ASP252, and GLU261 on the Fc domain of an IgG1. The goal was to provide microscopic mechanistic information for the photochemical, iron-dependent site-specific oxidative fragmentation of IgG1 at THR259 reported in our previous paper. The distance between Fe(III) and residues of interest as well as the binding pocket size was examined for both protonated and deprotonated THR259. The Fe(III) binding free energy (ΔG) was estimated by using an umbrella sampling approach. The pKa shift of the THR259 hydroxyl group caused by the presence of nearby Fe(III) was estimated based on a thermodynamic cycle. The simulation results show that Fe(III) resides inside the proposed binding pocket and profoundly changes the pocket configuration. The ΔG values indicate that the pocket possesses a strong binding affinity for Fe(III). Furthermore, Fe(III) profoundly lowers the pKa value of the THR259 hydroxyl group by 5.4 pKa units.
Keywords: binding free energy; iron-binding pocket; molecular dynamics simulations; monoclonal antibody; pKa shift.