Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site
Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site
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DOI：
10.1073/pnas.1212294109
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发表时间：
2012-11-06
影响因子：
11.1
通讯作者：
Fujiyoshi, Yoshinori
中科院分区：
文献类型：
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作者：
Abe, Kazuhiro;Tani, Kazutoshi;Fujiyoshi, Yoshinori
胃H+，K+-ATPase负责胃酸分泌。ATP驱动的H+进入胃是通过交换等量的K+有效地完成的，导致管腔pH接近1。由于可用于ATP水解的自由能有限，因此随着管腔pH的降低，转运的阳离子的化学计量比被认为是随着管腔pH的降低而变化的，从2H(+)/2K(+)到1H(+)/1K(+)，尽管这一假设的直接证据仍然很难找到。在这里，我们使用磷酸类似物氟化铝(Alf)和一个K+同系物(Rb+)，显示了脱磷过渡态H+，K+-ATPase的8角分辨结构，(Rb+)E2类似于Alf，这与前面不含Rb+的E2P状态不同。类似于Alf的(Rb+)E2跨膜阳离子结合部位的强密度很可能代表单一结合的Rb+离子，这明显不同于类似于Alf结构的无Rb+的E2AlF或结合K+的(K+)E2。放射性Rb-86(+)结合的测量表明，结合的化学计量因pH而异，与中性pH相比，在酸性结晶条件下，Rb+的结合量大约有一半。这些数据为H+，K+-ATPase的1H(+)/1K(+)/1ATP转运模式提供了结构和生化证据，从热力学角度讲，这是产生10(6)倍质子梯度的先决条件。结合在(Rb+)E2中观察到的类似于Alf结构的β亚基N端和P结构域之间释放的稳定E2P的相互作用，我们提出了H+，K+-ATPase的改进的载体运输模型，该模型必须在胃腔的高酸性状态下占优势。
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-angstrom resolution structure of H+, K+-ATPase in the transition state of dephosphorylation, (Rb+)E2 similar to AlF, which is distinct from the preceding Rb+-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb+) E2 similar to AlF highly likely represents a single bound Rb+ ion, which is clearly different from the Rb+-free E2AlF or K+-bound (K+) E2 similar to AlF structures. Measurement of radioactive Rb-86(+) 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 beta subunit's N terminus and the P domain observed in the (Rb+) E2 similar to 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.