title: Crystallization and preliminary X-ray crystallographic analysis of Ca2+-free primary Ca2+-sensor of Na+/Ca2+ exchanger.
abstract: 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%.
authors.affiliation: Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Mima
authors.forename: Chika
authors.initials: C
authors.lastname: Kawai
authors.forename: Kumsun
authors.initials: K
authors.lastname: Paku
authors.forename: Koji
authors.initials: K
authors.lastname: Tomoo
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Ishida
authors.forename: Shigeru
authors.initials: S
authors.lastname: Sugiyama
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kitatani
authors.forename: Hiroshi Y
authors.initials: HY
authors.lastname: Yoshikawa
authors.forename: Syou
authors.initials: S
authors.lastname: Maki
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Adachi
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Satoshi
authors.initials: S
authors.lastname: Murakami
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Inoue
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Mori
authors.forename: Satomi
authors.initials: S
authors.lastname: Kita
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwamoto
mesh_terms: 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
chemical_list: D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; D002118:Calcium