ジャーナル: Proc Natl Acad Sci U S A / 年: 2012 タイトル: Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site. 著者: Kazuhiro Abe / Kazutoshi Tani / Thomas Friedrich / Yoshinori Fujiyoshi / 要旨: Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a ...Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K(+) congener (Rb(+)), the 8-Å resolution structure of H(+),K(+)-ATPase in the transition state of dephosphorylation, (Rb(+))E2~AlF, which is distinct from the preceding Rb(+)-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb(+))E2~AlF highly likely represents a single bound Rb(+) ion, which is clearly different from the Rb(+)-free E2AlF or K(+)-bound (K(+))E2~AlF structures. Measurement of radioactive (86)Rb(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb(+) is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H(+),K(+)-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb(+))E2~AlF structure, we propose a refined vectorial transport model of H(+),K(+)-ATPase, which must prevail against the highly acidic state of the gastric lumen.
履歴
登録
2012年10月13日
登録サイト: PDBE / 処理サイト: PDBE
改定 1.0
2012年11月7日
Provider: repository / タイプ: Initial release
改定 1.1
2013年1月16日
Group: Database references / Derived calculations / Other
モード: BRIGHT FIELD / 倍率(公称値): 40000 X / 最大 デフォーカス(公称値): 3480 nm / 最小 デフォーカス(公称値): 830 nm
撮影
フィルム・検出器のモデル: KODAK SO-163 FILM
回折
平均測定温度: 4 K
検出器
日付: 2010年3月23日
放射波長
相対比: 1
反射
解像度: 8→129 Å / Num. obs: 39197 / % possible obs: 73.2 %
-
解析
ソフトウェア
名称
バージョン
分類
MRC
モデル構築
SITUS
精密化
MRC
SUITE
データスケーリング
MRC
位相決定
3次元再構成
解像度: 8 Å / 対称性のタイプ: 2D CRYSTAL
精密化
解像度: 8→129 Å / Num. reflection obs: 4166 / σ(F): 0 詳細: ALL REGIONS WERE MODELED STEREOCHEMICALLY. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2219. (DEPOSITION ID: 11197).