A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of 2,3-Sheet Essential for Channel Function of P2X4 Receptors
A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of 2,3-Sheet Essential for Channel Function of P2X4 Receptors
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高度保守的盐桥可稳定对 P2X4 受体通道功能至关重要的 beta2,3-Sheet 的扭结构象。
DOI：
10.1074/jbc.m115.711127
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Ye Yu
中科院分区：
文献类型：
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作者：
Wen-Shan Zhao;Meng-Yang Sun;Liang-Fei Sun;Yan Liu;Yang Yang;Li-Dong Huang;Ying-Zhe Fan;Xiao-Yang Cheng;Peng Cao;You-Min Hu;Lingyong Li;Yun Tian;Rui Wang;Ye Yu
在前结构时代，对P2 X受体通道功能中关键残基/基序的理解已经取得了重大进展。最近的结构测定P2 X受体使我们能够重新评估这些残基/基序的作用。残基Arg-309和Asp-85（大鼠P2 X4编号）在整个P2 X家族中高度保守，并参与人P2 X受体的功能丧失多态性。以前的研究表明，它们参与了ATP的直接结合。然而，P2 X的晶体结构表明，这两个残基形成了一个位于远离ATP结合位点的亚基间盐桥。因此，有必要重新评估这种盐桥在P2 X受体中的作用。在这里，我们建议的关键作用，这种结构元素在蛋白质的稳定性和通道门控，而不是直接的ATP相互作用和通道组装。结合诱变，电荷交换，和二硫键交联，我们揭示了这个盐桥在正常的P2 X4通道功能的严格要求。该盐桥可能有助于稳定与该盐桥和α2-螺旋结构偶联的β 2，3-折叠的弯曲构象。强扭结的β 2，3是P2 X4受体中头域、背鳍域、右鳍域和环β 7，8之间的域-域相互作用所必需的。与β 2，3-折叠朝向α2-螺旋的弯曲角度相反或沿着方向的二硫键交联分别导致P2 X4受体的功能丧失和功能获得。将具有大侧链的氨基酸进一步插入β 2，3-折叠之间的接头或α2-螺旋的构象变化，干扰β 2，3的扭结构象，导致P2 X4受体功能丧失。这些发现为理解Asp-85和Arg-309之间的盐桥及其结构偶联的β 2，3-折叠对P2 X受体功能的贡献提供了新的见解。
Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt bridge in P2X receptors. Here, we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide cross-linking, we revealed the stringent requirement of this salt bridge in normal P2X4 channel function. This salt bridge may contribute to stabilizing the bending conformation of the β2,3-sheet that is structurally coupled with this salt bridge and the α2-helix. Strongly kinked β2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and loop β7,8 in P2X4 receptors. Disulfide cross-linking with directions opposing or along the bending angle of the β2,3-sheet toward the α2-helix led to loss-of-function and gain-of-function of P2X4 receptors, respectively. Further insertion of amino acids with bulky side chains into the linker between the β2,3-sheet or the conformational change of the α2-helix, interfering with the kinked conformation of β2,3, led to loss-of-function of P2X4 receptors. All these findings provided new insights in understanding the contribution of the salt bridge between Asp-85 and Arg-309 and its structurally coupled β2,3-sheet to the function of P2X receptors.