Structural analysis of 2D crystals of gastric H+,K+-ATPase in different states of the transport cycle
Structural analysis of 2D crystals of gastric H+,K+-ATPase in different states of the transport cycle
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DOI：
10.1016/j.jsb.2007.12.005
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发表时间：
2008-05-01
影响因子：
3
通讯作者：
Fujiyoshi, Yoshinori
中科院分区：
文献类型：
--
作者：
Nishizawa, Tomohiro;Abe, Kazuhiro;Fujiyoshi, Yoshinori
H+，K+-ATP酶利用ATP泵送质子穿过胃膜。我们使用电子晶体学和限制性胰蛋白酶蛋白水解来研究H+，K+-ATP酶的构象变化。在低pH值下，不加核苷酸（E-1态），以及在氟铝酸盐和ADP存在下（模拟E1P.ADP态），用洗涤剂增溶的H+，K+-ATP酶获得了有序的二维晶体。在这两种状态下，用H+，K+-ATP酶的冷冻水合二维晶体获得的投影图看起来非常相似，表明在从E-1到E1P.ADP状态的过渡期间只有很小的构象变化。这一结果与相关ATP酶SERCA的X射线晶体结构不同，后者揭示了E-1和E1P.ADP状态下的显著不同构象。为了进一步表征H+，K+-ATP酶在其转运周期中的构象变化，我们用胰蛋白酶进行了有限的蛋白水解。所有检测的H+，K+-ATP酶的状态，包括存在于2D晶体中的E-1和E1P.ADP状态，在消化模式中显示出特征性差异。虽然有限的蛋白水解实验的结果表明，H+，K+-ATP酶在运输周期的不同阶段采用不同的构象，投影图表明，在H+，K+-ATP酶的结构重排比在相关的SERCA ATP酶中观察到的要小得多。(c)2007年爱思唯尔公司All rights reserved.
The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E-1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1P.ADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E-1 to the E1P.ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E-1 and E1P.ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E-1 and E1P.ADP states present in the 2D crystals, showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase. (c) 2007 Elsevier Inc. All rights reserved.