25Mg2+ NMR experiments

The experiment was performed on an Agilent DD2-600 NMR, equipped with a 10 mm low gamma direct detect probe and low frequency pre-amp. The experimental parameters used were as follows: 20 °C, a Larmor frequency of 36.72 MHz, a spectral width of 2.5 kHz, 1.1 s acquisition time, 0.5 s relaxation time, a 30 µs 90° excitation pulse, and between 128 and 2048 scans depending on the signal-to-noise ratio. For the K_d determination of Mg²⁺ the following protocol was used: A 1 M MgCl₂ solution with 10 mM DyCl₃ in D₂O was used as a reference and was placed in a 5-mm OD tube. A sample of 1.5 mL of the protein solution was placed in a 10-mm OD NMR tube. The reference tube was thoroughly cleaned and inserted into the 10-mm tube and centered with a plastic cap. Titration volumes of 3–37 µL of 4.64 M MgCl₂ were added directly to the 10-mm tube which was then gently mixed using a vortex shaker. The sample was allowed to equilibrate in the magnet for 10 min at the set temperature and the sample was then locked, tuned, gradient shimmed and acquired. To determine K_i associated with Ca²⁺ displacement of Mg²⁺_, 1.3–14.7 µL of 5.0 M CaCl₂ was added directly to the Mg²⁺ saturated receptor dispersion which was then gently mixed using a vortex shaker. The sample was allowed to equilibrate at the set temperature in the magnet for 10 min and then locked, tuned, gradient shimmed and acquired. The K_d and K_i values were determined, using GraphPad Prism 6 software, by fitting the data to the formula, y = A × x/(K_d + x), where x represents the cation concentration.