Characterization of the Cation Binding Sites in the NCKX2 Na+/Ca2+-K+Exchanger.
NCKX1-5 are proteins involved in K+-dependent Na+/Ca2+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 Ca2+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.
Center for Molecular Simulations, Department of Biological Sciences, University of Calgary , Calgary, AB, Canada T2N 1N4.
Hristina
H
Zhekova
Center for Molecular Simulations, Department of Biological Sciences, University of Calgary , Calgary, AB, Canada T2N 1N4.
Chunfeng
C
Zhao
Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary , Calgary, AB T2N 4N1, Canada.
Paul P M
PP
Schnetkamp
Center for Molecular Simulations, Department of Biological Sciences, University of Calgary , Calgary, AB, Canada T2N 1N4.
Sergei Yu
SY
Noskov
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002412:Cations / Q000378:metabolism*; D018360:Crystallography, X-Ray; D017015:Methanococcales / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013816:Thermodynamics
D019843:Archaeal Proteins; D002412:Cations; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D011188:Potassium; D002118:Calcium