Crystallization and preliminary X-ray crystallographic analysis of Ca2+-free primary Ca2+-sensor of Na+/Ca2+ exchanger.
The plasma-membrane Na(+)/Ca(2+) exchanger (NCX) regulates intracellular Ca(2+) levels in cardiac myocytes. Two Ca(2+)-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. The binding of Ca(2+) to CBD1 results in conformational changes that stimulate exchange to exclude Ca(2+) ions, whereas CBD2 maintains the structure, suggesting that CBD1 is the primary Ca(2+)-sensor. In order to clarify the structural scaffold for the Ca(2+)-induced conformational transition of CBD1 at the atomic level, X-ray structural analysis of its Ca(2+)-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 P6(2)22 or P6(4)22, with unit-cell parameters a = b = 56.99, c = 153.86 A, beta = 120 degrees , and contained one molecule per asymmetric unit (V(M) = 2.25 A(3) 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 A using an R-AXIS detector and gave a data set with an overall R(merge) of 10.8% and a completeness of 92.8%.
Mima
Masashi
M
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan.
Kawai
Chika
C
Paku
Kumsun
K
Tomoo
Koji
K
Ishida
Toshimasa
T
Sugiyama
Shigeru
S
Matsumura
Hiroyoshi
H
Kitatani
Tomoya
T
Yoshikawa
Hiroshi Y
HY
Maki
Syou
S
Adachi
Hiroaki
H
Takano
Kazufumi
K
Murakami
Satoshi
S
Inoue
Tsuyoshi
T
Mori
Yusuke
Y
Kita
Satomi
S
Iwamoto
Takahiro
T
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; D002118:Calcium