Influence of methionine oxidation on protein stability and association studied by free energy simulations

Cellular metabolic systems but also the extracellular environment can generate reactive oxygen species that lead to oxidation of methionine (MET) and interfere with protein folding and protein-protein association. The molecular mechanism of how MET oxidation (MEO) influences conformational stability and binding is not well understood. We employ alchemical free energy simulations to systematically study the influence of MET oxidation on protein-protein binding using the tetramerization domain of the tumor suppression protein p53 as a model system. A single MEO in one tetramerisation domain destabilizes the tetramer by ≈1.1-1.6 kcal/mol depending slightly on the MEO diastereomer. The simulations on double and triple oxidations reveal increased destabilisation (≈ 3-7 kcal/mol) and significant cooperative effects depending on the relative position of the oxidized residues. The MET oxidation effects are of similar magnitude for the change in stability of the human prion protein (HHP) that served as a second model system and also agree with available experimental data. The calculations predict a significant dependence of stability changes on the position of the MEO and also indicate non-additive effects of multiple oxidations which may play a role to protect proteins from oxidative damage and stress. Analysis of the Molecular Dynamics trajectories allowed us to interpret the oxidation effects in molecular detail. The simulation methodology could also serve as a general protocol to analyze single and multiple MET oxidations in other systems and its influence on protein binding and stability.