Department of Pharmacology and Chemical Biology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, China; College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, China.
J Biol Chem. 2021 Jan-Jun;296:100655. doi: 10.1016/j.jbc.2021.100655. Epub 2021 Apr 23.
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.
高度保守的氨基酸通常被预期在蛋白质超家族中具有相似的功能，包括 P2X 离子通道，其由细胞外 ATP 门控。然而，当相邻氨基酸不保守时，这些功能是否以及如何保守就变得不那么清楚了。在这里，我们研究了一个这样的例子，集中在高度保守的残基 P2X4 上，E118（大鼠 P2X4 编号，rP2X4），与人类神经性疼痛相关的 P2X 亚型。当我们比较 P2X4 的晶体结构与其他 P2X 亚型的晶体结构时，包括 P2X3、P2X7 和 AmP2X，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、双突变循环分析和分子建模来揭示 E118 与“喙”区域的氨基酸形成特定的接触，这有助于 ATP 与 rP2X4 结合。这些接触在其他亚型中不存在，因为喙区域的序列变异，导致这个保守残基与 ATP 识别和/或 P2X 受体的通道门控解耦。我们的研究提供了一个例子，即一个保守残基在功能蛋白中具有特定的作用，这是由相邻的非保守残基实现的。E118-喙区域接触所确立的独特作用为 P2X4 的亚型特异性抑制剂的开发提供了蓝图。
