A conserved residue in the P2X4 receptor has a nonconserved function in ATP recognition.
A conserved residue in the P2X4 receptor has a nonconserved function in ATP recognition.
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DOI：
10.1016/j.jbc.2021.100655
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Chen PF;Ma XF;Sun LF;Tian Y;Fan YZ;Li P;Xiao Z;Zhu MX;Guo CR;Li C;Yu Y;Wang J
通常预计高度保守的氨基酸在蛋白质超家族中具有相似的功能，包括由细胞外ATP门控的P2 X离子通道的功能。然而，当邻近的氨基酸不保守时，这些功能是否以及如何保守变得不太清楚。在这里，我们研究了一个这样的情况下，集中在高度保守的残基P2 X4，E118（大鼠P2 X4编号，rP 2X 4），P2 X亚型与人类神经性疼痛。当我们将P2 X4的晶体结构与其他P2 X亚型（包括P2 X3、P2 X7和AmP 2X）的晶体结构进行比较时，我们观察到E118的侧链取向略有改变。我们使用蛋白质嵌合体，双突变体循环分析和分子建模来揭示E118与“喙”区域中的氨基酸形成特异性接触，这有助于ATP与rP 2X 4结合。这些接触不存在于其他亚型中，因为喙区的序列变异，导致该保守残基与ATP识别和/或P2 X受体的通道门控解耦。我们的研究提供了一个例子，保守残基与功能蛋白质的特定作用，使相邻的非保守残基。由E118-喙区域接触建立的独特作用为P2 X4亚型特异性抑制剂的开发提供了蓝图。
Highly conserved amino acids are generally anticipated to have similar functions across a protein superfamily, including that of the P2X ion channels, which are gated by extracellular ATP. However, whether and how these functions are conserved becomes less clear when neighboring amino acids are not conserved. Here, we investigate one such case, focused on the highly conserved residue from P2X4, E118 (rat P2X4 numbering, rP2X4), a P2X subtype associated with human neuropathic pain. When we compared the crystal structures of P2X4 with those of other P2X subtypes, including P2X3, P2X7, and AmP2X, we observed a slightly altered side-chain orientation of E118. We used protein chimeras, double-mutant cycle analysis, and molecular modeling to reveal that E118 forms specific contacts with amino acids in the “beak” region, which facilitates ATP binding to rP2X4. These contacts are not present in other subtypes because of sequence variance in the beak region, resulting in decoupling of this conserved residue from ATP recognition and/or channel gating of P2X receptors. Our study provides an example of a conserved residue with a specific role in functional proteins enabled by adjacent nonconserved residues. The unique role established by the E118-beak region contact provides a blueprint for the development of subtype-specific inhibitors of P2X4.
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