Chapter 4 │ Page 121 To elucidate the e ect of oxidation on the binding of the investigated complexes, we performed non-reactive MD simulations. The simulations were performed with the GROMACS software (version 2020.2) [39], employing the GROMOS 54A7 force field [40]. The crystal structures of both the NKG2A/CD94 – HLA-E complex and the KIR2DL1 – HLA-Cw4 complex were obtained from the Protein Data Bank (ID: 3CDG [41] and 1IM9 [42], respectively). Force field parameters for the oxidized amino acids were obtained from [43]. Each model system was prepared by placing the protein complex in a cubic simulation box with periodic boundary conditions applied in all Cartesian directions, and solvating in SPC water [44] containing a concentration of 150 mM NaCl. A minimum distance of 1 nm was ensured between the protein and the box sides to adhere to the minimum image criterion. The system was energy minimized using the steepest descent algorithm, followed by a 2 ns equilibration in the canonical (NVT) ensemble (i.e. a constant number of particles (N), volume (V) and temperature (T)) while applying position restraints with a force constant of 10 000 kJ·mol-1·nm-2 to the heavy atoms of the proteins. Afterwards, a series of equilibrations, totaling 10 ns, was performed in the isothermal-isobaric (NPT) ensemble (i.e. with a constant pressure (P) instead of a constant volume) while enforcing decreasingly strong position restraints (10 000 kJ·mol-1·nm-2 for 2 ns, 1000 kJ·mol-1·nm-2 for 2 ns, 200 kJ·mol-1·nm-2 for 6 ns). In this way, the complexes were gradually equilibrated. The equilibrations were performed at 310 K and 1.0 bar to mimic the conditions the proteins would experience in the body, employing the vrescale thermostat [45] with a coupling constant of 0.1 ps and the Parrinello-Rahman barostat [46] with a compressibility and coupling constant of 4.5 × 10-5 bar-1 and 2 ps, respectively. Electrostatics in the system were treated using the reaction field (RF) method [47], while using a cuto distance of 1.4 nm for the van der Waals and Coulomb interactions. All simulations were performed with a time step of 2 fs. A
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