Department of Molecular and Cell Biology (R.C.A., S.D., A.B.D., R.S., R.J.E.) and Leicester Institute of Structural and Chemical Biology (R.S.), University of Leicester, Leicester, United Kingdom; and Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq (S.D.).
Department of Molecular and Cell Biology (R.C.A., S.D., A.B.D., R.S., R.J.E.) and Leicester Institute of Structural and Chemical Biology (R.S.), University of Leicester, Leicester, United Kingdom; and Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq (S.D.)
Mol Pharmacol. 2018 May;93(5):553-562. doi: 10.1124/mol.117.111021. Epub 2018 Mar 13.
P2X7 receptor (P2X7R) activation requires ∼100-fold higher concentrations of ATP than other P2X receptor (P2XR) subtypes. Such high levels are found during cellular stress, and P2X7Rs consequently contribute to a range of pathophysiological conditions. We have used chimeric and mutant P2X7Rs, coupled with molecular modeling, to produce a validated model of the binding mode of the subtype-selective antagonist A438079 at an intersubunit allosteric site. Within the allosteric site large effects on antagonist action were found for point mutants of residues F88A, D92A, F95A, and F103A that were conserved or similar between sensitive/insensitive P2XR subtypes, suggesting that these side-chain interactions were not solely responsible for high-affinity antagonist binding. Antagonist sensitivity was increased with mutations that remove the bulk of side chains around the center of the binding pocket, suggesting that the dimensions of the pocket make a significant contribution to selectivity. Chimeric receptors swapping the left flipper (around the orthosteric site) reduced both ATP and antagonist sensitivity. Point mutations within this region highlighted the contribution of a P2X7R-specific aspartic acid residue (D280) that modeling suggests forms a salt bridge with the lower body region of the receptor. The D280A mutant removing this charge increased ATP potency 15-fold providing a new insight into the low ATP sensitivity of the P2X7R. The ortho- and allosteric binding sites form either side of the -strand Y291-E301 adjacent to the left flipper. This structural linking may explain the contribution of the left flipper to both agonist and antagonist action.
P2X7 受体 (P2X7R) 的激活需要比其他 P2X 受体 (P2XR) 亚型高约 100 倍的 ATP 浓度。这种高水平存在于细胞应激期间，因此 P2X7R 有助于多种病理生理状况。我们使用嵌合体和突变体 P2X7R，并结合分子建模，产生了一种经过验证的亚型选择性拮抗剂 A438079 在亚基间变构位点结合模式的模型。在变构位点中，对于在敏感/不敏感 P2XR 亚型之间保守或相似的残基 F88A、D92A、F95A 和 F103A 的点突变，发现对拮抗剂作用有很大影响，这表明这些侧链相互作用并不是高亲和力拮抗剂结合的唯一原因。与去除结合口袋中心周围大部分侧链的突变体相比，拮抗剂敏感性增加，这表明口袋的尺寸对选择性有重要贡献。交换左翼（位于正位点周围）的嵌合受体降低了 ATP 和拮抗剂的敏感性。该区域内的点突变突出了 P2X7R 特异性天冬氨酸残基 (D280) 的贡献，建模表明该残基与受体的下半身区域形成盐桥。去除该电荷的 D280A 突变体使 ATP 效力增加了 15 倍，为 P2X7R 的低 ATP 敏感性提供了新的见解。正位和变构结合位点形成于邻近左翼的β-链 Y291-E301 的两侧。这种结构连接可能解释了左翼对激动剂和拮抗剂作用的贡献。
