Zhao Wen-Shan, Sun Meng-Yang, Sun Liang-Fei, Liu Yan, Yang Yang, Huang Li-Dong, Fan Ying-Zhe, Cheng Xiao-Yang, Cao Peng, Hu You-Min, Li Lingyong, Tian Yun, Wang Rui, Yu Ye
From the School of Life Sciences and Key Laboratory of Preclinical Study for New Drugs of Gansu Province School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China, the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
the Institute of Medical Sciences and Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
J Biol Chem. 2016 Apr 8;291(15):7990-8003. doi: 10.1074/jbc.M115.711127. Epub 2016 Feb 10.
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.
在结构解析前的时代，人们对关键残基/基序在P2X受体通道功能中的作用已有了显著认识。近期P2X受体的结构解析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P2X4编号）在整个P2X家族中高度保守，且与人P2X受体的功能丧失多态性有关。先前的研究表明它们参与直接的ATP结合。然而，P2X的晶体结构显示这两个残基形成了一个亚基间盐桥，位于远离ATP结合位点的位置。因此，有必要重新评估这个盐桥在P2X受体中的作用。在此，我们提出这个结构元件在蛋白质稳定性和通道门控中起关键作用，而非直接参与ATP相互作用和通道组装。通过结合诱变、电荷交换和二硫键交联，我们揭示了这个盐桥对正常P2X4通道功能的严格要求。这个盐桥可能有助于稳定与该盐桥和α2螺旋结构耦合的β2,3-折叠的弯曲构象。强烈扭结的β2,3对于P2X4受体中头部结构域、背鳍结构域、右鳍状结构域和环β7,8之间的结构域-结构域相互作用至关重要。与β2,3-折叠向α2螺旋的弯曲角度相反或沿其方向进行二硫键交联，分别导致P2X4受体功能丧失和功能增强。进一步在β2,3-折叠之间的连接子中插入具有大侧链的氨基酸或α2螺旋的构象变化，干扰β2,3的扭结构象，导致P2X4受体功能丧失。所有这些发现为理解Asp-85和Arg-309之间的盐桥及其结构耦合的β2,3-折叠对P2X受体功能的贡献提供了新的见解。
