The Second Ca2+-Binding Domain of NCX1 Binds Mg2+ with High Affinity

We report the effects of binding of Mg2+ to the second Ca2+-binding domain (CBD2) of the sodium calcium exchanger. CBD2 is known to bind two Ca2+ ions using its Ca2+-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg2+ at both sites, but with significantly different affinities. The results from Mg2+ Ca2+ competition experiments show that Ca2+ can replace Me2+ from site I, but not site II, and that Mg2+ binding affects the affinity for Ca2+. Furthermore, thermal unfolding circular dichroism data demonstrate that Mg2+ binding stabilizes the domain. NMR chemical shift perturbations and N-15 relaxation data reveal that Mg2+-bound CBD2 adopts a state intermediate between the apo and fully Ca2+-loaded forms. Together, the data show that at physiological Mg2+ concentrations CBD2 is loaded with Mer preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca2+.