Crystallization and preliminary X-ray crystallographic analysis of Ca²⁺-free primary Ca²⁺-sensor of Na⁺/Ca²⁺ exchanger

The plasma-membrane Na⁺/Ca²⁺ exchanger (NCX) regulates intracellular Ca²⁺ levels in cardiac myocytes. Two Ca²⁺-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. The binding of Ca²⁺ to CBD1 results in conformational changes that stimulate exchange to exclude Ca²⁺ ions, whereas CBD2 maintains the structure, suggesting that CBD1 is the primary Ca²⁺-sensor. In order to clarify the structural scaffold for the Ca²⁺-induced conformational transition of CBD1 at the atomic level, X-ray structural analysis of its Ca²⁺-free form was attempted; the structure of the Ca²⁺-bound form is already available. Recombinant CBD1 (NCX1 372–508) with a molecular weight of 16 kDa was crystallized by the sitting-drop vapour-diffusion method at 293 K. The crystals belonged to the hexagonal space group P6₂22 or P6₄22, with unit-cell parameters a = b = 56.99, c = 153.86 Å, β = 120°, and contained one molecule per asymmetric unit (V_M = 2.25 ų Da⁻¹) with a solvent content of about 55% (V_S = 45.57%). Diffraction data were collected within the resolution range 27.72–3.00 Å using an R-AXIS detector and gave a data set with an overall R_merge of 10.8% and a completeness of 92.8%.