Chapter 4 │ Page 141 Employing MD simulations and umbrella sampling, we aimed to investigate the interaction of MHC-I proteins HLA-Cw4 and HLA-E with their receptors, respectively KIR2DL1 and NKG2A/CD94, and the e ect of NTP-induced oxidative changes thereon. The interaction of both is dominated by polar interactions. Several salt bridges were found to be present in our simulations apart from those reported to be present in the crystal structure of the proteins. Notably, for HLA-E, the most prominent salt bridge (Arg168CD94 – Asp68HLA-E) is not among those present in the crystal structure, although it was previously indicated to be important in other condensed-phase simulations [70]. Meanwhile, some salt bridges important in the crystal structure were not prominent in our simulations, either due to di erent interactions taking the upper hand for the participating amino acids (Asp163CD94 – Arg75HLA-E), or due to the amino acids simply not interacting much when allowed to move over time (Arg137NKG2A – Glu154HLA-E). In the latter case, indeed, it was found experimentally that prohibiting the formation of this salt bridge through mutation did not a ect protein binding [71]. For HLA-Cw4, most salt bridges present in our simulation resulted from promiscuous interactions between the amino acids that form salt bridges in the crystal structure. Oxidation of the protein ligands did not significantly a ect their stability in the simulations. The secondary structure was almost completely retained, and the oxidations did not impact the flexibility of the protein. It has previously been reported that plasma-induced oxidations can a ect the protein structure, or even induce partial denaturation. These e ects were however mostly observed for (small) proteins with solvent-accessible disulfide bridges [29, 31, 72]. Both MHC-I complexes contain disulfide bridges, important for structural stability, but as these are buried inside the protein, they are unavailable to breakage by NTP-induced oxidation. Although the oxidations do cause the ligands to pivot with respect to their receptor (see Figure 3b), the binding domain itself remains nearly una ected, and the calculated binding free energy of the complexes did not change
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