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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 Å3 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%.
The Na+/Ca2+ exchanger (NCX) is a plasma-membrane protein that extrudes one intracellular Ca2+ ion in exchange for three extracellular Na+ ions and regulates intracellular Ca2+ levels in many mammalian cell types (Blaustein & Lederer, 1999 ; Philipson & Nicoll, 2000 ; Shigekawa & Iwamoto, 2001 ). The activity of NCX is modulated by the binding of Ca2+ to a high-affinity regulatory site on an intracellular loop portion of the protein; the regulatory Ca2+ is not transported but potentially activates the exchange activity. Two Ca2+-binding domains (CBD1 and CBD2) in the intracellular loop have been identified as being responsible for regulation of activity (Philipson et al., 2002 ; Hilge et al., 2006 ). NMR studies (Hilge et al., 2006 ) have shown that the Ca2+-free (apo) structure of CBD1 differs significantly from that of the Ca2+-bound state, while CBD2 maintains its structural integrity in the Ca2+-free state. A fluorescence resonance energy-transfer study indicated that binding elicits a conformational change in CBD1 (Ottolia et al., 2004 ). These observations indicate that CBD1 is the primary Ca2+-sensor.
The crystal structure of Ca2+-bound CBD1 (Nicoll et al., 2006 ) revealed a novel Ca2+-binding site consisting of four Ca2+ ions arranged in a tight planar cluster, which may represent a general platform for Ca2+-sensing. The structure of Ca2+-free CBD1 is not available at present. Therefore, the X-ray structural analysis of the Ca2+-free CBD1 of NCX1, one of three mammalian isoforms (Nicoll et al., 1990 ), was attempted in order to clarify the structural scaffold for the conformational transition of CBD1 induced by Ca2+ binding and the structural difference from CBD2 at the atomic level.
The construct NCX1CBD1 corresponds to Val371–Glu508 of NCX1 plus an eight-residue N-terminal His tag that facilitated purification using Ni-chelate chromatography. The expression plasmid pHisNCX1CBD1 coding for NCX1CBD1 was constructed as follows. Two oligonucleotide primers were synthesized with NheI and BamHI restriction sites to facilitate cloning in frame into the His-tag fusion-protein expression vector pET-19a. PCR was performed with the canine NCX1 plasmid as a template for 30 cycles (367 K for 15 s, 330 K for 30 s and 341 K for 30 s). The amplified DNA was digested by NheI and BamHI and the resulting fragment (411 bp) was inserted into the corresponding sites of pET19a. Escherichia coli BL21 (DE3) cells harbouring pHisNCX1CBD1 were induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at the mid-exponential growth phase (OD600 = 0.5) and incubated for a further 3 h at 310 K. Cells were harvested by centrifugation, washed and resuspended in 50 mM Tris–HCl pH 7.5 containing 150 mM NaCl. The cells were disrupted by sonication and the lysate was centrifuged at 15 000 rev min−1 for 60 min. The supernatant fraction was loaded onto a Chelating Sepharose Fast Flow column equilibrated with 50 mM Tris–HCl pH 7.5 containing 150 mM NaCl and 10 mM imidazole. After washing the column with 50 mM Tris–HCl pH 7.5 containing 150 mM NaCl and 60 mM imidazole, His-tag fusion protein (HisNCX1CBD1) was eluted with 50 mM Tris–HCl pH 7.5 containing 150 mM NaCl and 300 mM imidazole. The eluted fraction containing HisNCX1CBD1 was dialysed overnight into 20 mM HEPES–NaOH pH 7.5 and was applied onto a MonoQ 5/50 GL column (GE Healthcare). HisNCX1CBD1 was eluted with a linear gradient of NaCl from 0 to 0.5 M in 20 mM HEPES–NaOH pH 7.5 and dialysed overnight into 5 mM Tris–HCl pH 7.5. The sample was concentrated to approximately 13 mg ml−1 using Centricon centrifugal concentrators (Millipore) and EDTA was added to a final concentration of 10 mM. The purity of the final preparation was estimated to be greater than 95% by SDS–PAGE. The size distribution of the protein molecule in solution was shown to be monodisperse by dynamic light-scattering measurements.
Initial crystallization screening took place using the sitting-drop vapour-diffusion method at 293 K and crystallization kits from Hampton Research. Crystallization drops consisting of 1 µl protein solution (13 mg ml−1 in 5 mM Tris–HCl, 10 mM EDTA pH 7.5) and 1 µl reservoir solution were equilibrated against 100 µl reservoir solution. Small hexagonal rod-shaped crystals were obtained using a reservoir condition of 30%(w/v) PEG 4000, 0.2 M ammonium sulfate and 0.1 M citrate buffer pH 5.0. To improve the quality of the crystals, we applied the micro-stirring technique. We shook the sitting-drop vapour-diffusion crystallization plates with a rotating shaker using a speed of 50 rev min−1. Details of this technique have been described elsewhere (Adachi, Matsumura et al., 2004 ; Adachi, Takano et al., 2004 ; Adachi et al., 2005 ). After screening the crystallization conditions and using the micro-stirring technique, diffraction-quality crystals were obtained using 22%(w/v) PEG 4000, 0.2 M ammonium sulfate and 0.1 M citrate buffer pH 5.0. Crystals grew within a few weeks to dimensions of approximately 0.2 × 0.1 × 0.1 mm (Fig. 1 ).
Native data were collected at 100 K using Cu Kα radiation from an FR-E rotating-anode X-ray generator (Rigaku Corp.) equipped with CMF optics (Osmic Inc.) and an R-AXIS VII detector (the crystal-to-detector distance was 135 mm). 90° of data were collected as 180 diffraction images using a 0.5° oscillation angle and an exposure time of 60 s (Fig. 2 ). The data were processed using the program CrystalClear (Rigaku MSC; Pflugrath, 1999 ).
Recombinant CBD1 was prepared in E. coli using biotechnology. Our initial crystallization trials with Ca2+-free CBD1, which were performed in a manner similar to that used to obtain the Ca2+-bound form, failed. However, the results of dynamic light-scattering experiments suggested a high likelihood of crystallization as the size distribution of the CBD1 molecules in the sample solution was narrow and monomodal. Thus, rescreening with various screening kits was continued and the micro-stirring method (Adachi et al., 2005 ) resulted in well shaped hexagonal plate-shaped crystals of Ca2+-free CBD1 at 293 K (Fig. 2 ). 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°. The V M value, the crystal volume per unit of protein molecular weight, was calculated to be 2.25 Å3 Da−1, indicating the presence of one molecule in an asymmetric unit, and a solvent content of about 55% was indicated from the V S value of 45.57% (Matthews, 1968 ); these V M and V S values are within the range usually observed for protein crystals.
A total of 25 923 reflections in the resolution range 26.72–3.00 Å were collected with 92.8% completeness and an R merge of 10.8%. We are attempting phase determination by molecular-replacement methods using the coordinates of Ca2+-bound CBD1 [PDB codes 2dpk (crystal structure; Nicoll et al., 2006 ) and 2fws (NMR structure; Hilge et al., 2006 )] and Ca2+-free/Ca2+-bound CBD2 (crystal structure; Besserer et al., 2007 ) as a search model. Structure determination of CBD1 using heavy-atom derivatives is also in progress. The X-ray structure analysis is promising for the elucidation of the structural scaffold for the Ca2+-dependent structural transformation, which is an essential component of the Ca2+-signalling pathway.