The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

• Published in: Biochemistry (Easton) 2011-10, Vol.50 (41), p.8804
• Main Authors: Breukels, Vincent, Konijnenberg, Albert, Nabuurs, Sanne M, Touw, Wouter G, Vuister, Geerten W
• Format: Article
• Language: English
• Subjects: Animals; Anisotropy; Binding Sites; Buffers; Calcium - chemistry; Calorimetry - methods; Circular Dichroism; Dogs; Kinetics; Magnesium - chemistry; Magnetic Resonance Spectroscopy - methods; Neuronal Calcium-Sensor Proteins - metabolism; Neuropeptides - metabolism; Nitrogen Isotopes - chemistry; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Thermodynamics
• ISSN: 1520-4995
• DOI: 10.1021/bi201134u

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+).