Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.
Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.
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DOI：
10.1074/jbc.m114.584623
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发表时间：
2014-10-31
期刊：
影响因子：
--
通讯作者：
Fujiyoshi Y
中科院分区：
文献类型：
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作者：
Abe K;Tani K;Fujiyoshi Y
背景：胃H+，K+- atp酶质子泵实现胃酸分泌。结果：新确定的（SCH）E2·MgF结构为（SCH）E2·BeF和（Rb+）E2·AlF光闭态的混合构象。结论：通过对e2p相关结构的比较，发现A-M2连接体对酶的构象变化起着重要作用。意义：A-M2连接体介导的构象变化在H+、K+-ATPase和Ca2+-ATPase以及其他p型atpase之间是保守的。胃H+，K+- atp酶是一种atp驱动的质子泵，负责胃酸化，是抗溃疡药物的分子靶点。在这里，我们展示了它在E2P模拟状态下的低温电子显微镜（EM）结构，与氟化镁（MgF）结合，以及它的K+竞争拮抗剂SCH28080，通过二维晶体的电子晶体学在7 Å分辨率下确定。与其他e2p相关的低温电镜结构进行系统比较，发现（SCH）E2·MgF态的分子构象有显著区别。虽然（SCH）E2·MgF态的A结构域的方位位置与E2·AlF（氟化铝）态相似，跨膜腔门是关闭的，但根据对SCH28080结合的E2·BeF（氟化铍）态结构的观察，（SCH）E2·MgF态的跨膜螺旋排列显示出由结合的SCH28080在其腔内施加的光开构象。因此，（SCH）E2·MgF状态的分子构象代表了一种混合的整体结构，其中其细胞质和管腔的一半似乎由与酶各自部分结合的磷酸盐类似物和拮抗剂独立调节。分子构象的比较表明，连接A结构域和跨膜螺旋2的连接子区域（A- m2连接子）介导了光门控的调控。在H+，K+- atp酶中观察到的管腔门控的机制原理与在肌浆网Ca2+- atp酶和其他p型atp酶中观察到的机制原理一致，并且很可能在p型atp酶家族中普遍保守。
Background: The gastric H+,K+-ATPase proton pump achieves gastric acid secretion. Results: A newly determined (SCH)E2·MgF structure represents a hybrid conformation of (SCH)E2·BeF and luminal-closed (Rb+)E2·AlF states. Conclusion: Comparison of E2P-related structures revealed that the A-M2 linker importantly contributes to conformational changes of the enzyme. Significance: A-M2 linker-mediated conformational change is conserved between H+,K+-ATPase and Ca2+-ATPase and perhaps other P-type ATPases. Gastric H+,K+-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K+-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H+,K+-ATPase is consistent with that observed in sarcoplasmic reticulum Ca2+-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.