title: The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity.
abstract: We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).
authors.affiliation: Protein Biophysics, Institute for Molecules and Materials, Radboud University Nijmegen, 6525 GA Nijmegen, The Netherlands.
authors.forename: Vincent
authors.initials: V
authors.lastname: Breukels
authors.forename: Albert
authors.initials: A
authors.lastname: Konijnenberg
authors.forename: Sanne M
authors.initials: SM
authors.lastname: Nabuurs
authors.forename: Wouter G
authors.initials: WG
authors.lastname: Touw
authors.forename: Geerten W
authors.initials: GW
authors.lastname: Vuister
mesh_terms: D000818:Animals; D016880:Anisotropy; D001665:Binding Sites; D002021:Buffers; D002118:Calcium / Q000737:chemistry*; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D004285:Dogs; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051580:Neuronal Calcium-Sensor Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D002021:Buffers; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D009587:Nitrogen Isotopes; C101464:frequenin calcium sensor proteins; D008274:Magnesium; D002118:Calcium