Crystallization and preliminary X-ray crystallographic analysis of Ca 2+-free primary Ca2+-sensor of Na+/Ca 2+ exchanger

Abstract

The plasma-membrane Na+/Ca2+ exchanger (NCX) regulates intracellular Ca2+ levels in cardiac myocytes. Two Ca 2+-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 Ca 2+-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 (VM = 2.25 Å3 Da-1) with a solvent content of about 55% (VS = 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%. © International Union of Crystallography 2008.