Characterization of the Cation Binding Sites in the NCKX2 Na + /Ca 2+ -K + Exchanger.

NCKX1-5 are proteins involved in K ⁺ -dependent Na ⁺ /Ca ²⁺ exchange in various signal tissues. Here we present a homology model of NCKX2 based on the crystal structure of the NCX_Mj transporter found in Methanoccocus jannaschii. Molecular dynamics simulations were performed on the resultant wild-type NCKX2 model and two mutants (D548N and D575N) loaded with either four Na ⁺ ions or one Ca ²⁺ ion and one K ⁺ ion, in line with the experimentally observed transport stoichiometry. The selectivity of the active site in wild-type NCKX2 for Na ⁺ , K ⁺ , and Li ⁺ and the electrostatic interactions of the positive Na ⁺ ions in the negatively charged active site of wild-type NCKX2 and the two mutants were evaluated from free energy perturbation calculations. For validation of the homology model, our computational results were compared to available experimental data obtained from numerous prior functional studies. The NCKX2 homology model is in good agreement with the discussed experimental data and provides valuable insights into the structure of the active site, which is lined with acidic and polar residues. The binding of the potassium and calcium ions is accomplished via Asp 575 and 548, respectively. Mutation of these residues to Asn alters the functionality of NCKX2 because of the elimination of the favorable carboxylate-cation interactions. The knowledge obtained from the NCKX2 model can be transferred to other isoforms of the NCKX family: newly discovered pathological mutations in NCKX4 and NCKX5 affect residues that are involved in ion binding and/or transport according to our homology model.