1. The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity
- Author
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Sanne M. Nabuurs, Geerten W. Vuister, Vincent Breukels, Wouter G. Touw, and Albert Konijnenberg
- Subjects
Circular dichroism ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Neuronal Calcium-Sensor Proteins ,chemistry.chemical_element ,Plasma protein binding ,Calcium ,Buffers ,Calorimetry ,Bio-Organic Chemistry ,Biochemistry ,Protein structure ,Dogs ,Animals ,Magnesium ,Binding site ,Binding Sites ,Nitrogen Isotopes ,Chemistry ,Circular Dichroism ,Neuropeptides ,Nuclear magnetic resonance spectroscopy ,Protein Structure, Tertiary ,Crystallography ,Kinetics ,Domain (ring theory) ,Anisotropy ,Thermodynamics ,Biophysical Chemistry ,Binding domain ,Protein Binding - 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+).
- Published
- 2011