Substance P and neurokinin A are closely related neuropeptides belonging to the tachykinin family. Their receptors are neurokinin one receptor (NK1R) and neurokinin two receptor (NK2R), G protein–coupled receptors that transmit Gs and Gq-mediated downstream signaling. We investigate the importance of sequence differences at the bottom of the receptor orthosteric site for activity and selectivity, focusing on residues that closely interact with the C-terminal methionine of the peptide ligands. We identify a conserved serine (NK1R-S2977.45) and the position of the tryptophan residue within the canonical “toggle switch” motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F2646.51) and a tyrosine in NK2R (NK2R-Y2666.51), giving rise to distinct microenvironments for the neuropeptide C terminals. Mutating these residues results in dramatic activity changes in both NK1R and NK2R due to a close interaction between the ligand and toggle switch. Structural analysis of active and inactive NKR structures suggests only a minor change in sidechain rotation of toggle switch residues upon activation. However, extensive molecular dynamics simulations of receptor:neuropeptide:G protein complexes indicate that a major, concerted motion happens in the toggle switch tryptophan indole group and the sidechains of the microswitch motif PIF. This rotation establishes a tight hydrogen bond interaction from the tryptophan indole to the conserved serine (NK1R-S2977.45) and a mainchain carbonyl (NK1R-A2947.41) in the kink of TM7. This interaction facilitates communication with the NPxxY microswitch motif of TM7, resulting in stabilization of the G protein–binding region. NK1R-S2977.45 is consequently identified as a central hub for the activation of NKRs.

中文翻译：

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速激肽 NK1 和 NK2 受体中神经肽介导的激活机制的分子决定因素


P 物质和神经激肽 A 是属于速激肽家族的密切相关的神经肽。它们的受体是神经激肽一受体 （NK1R） 和神经激肽二受体 （NK2R），它们是 G 蛋白偶联受体，可传递 Gs 和 Gq 介导的下游信号传导。我们研究了受体正位点底部的序列差异对活性和选择性的重要性，重点关注与肽配体的 C 末端蛋氨酸紧密相互作用的残基。我们鉴定了一个保守的丝氨酸 （NK1R-S2977.45） 和色氨酸残基在经典“拨动开关”基序中的位置，TM6 的 CWxP，毗邻 NK1R 中的苯丙氨酸 （NK1R-F2646.51） 和 NK2R 中的酪氨酸 （NK2R-Y2666.51），从而为神经肽 C 末端产生不同的微环境。由于配体和拨动开关之间的密切相互作用，这些残基突变会导致 NK1R 和 NK2R 的活性发生显著变化。活性和非活性 NKR 结构的结构分析表明，激活后拨动开关残基的侧链旋转仅发生微小变化。然而，受体：神经肽：G 蛋白复合物的广泛分子动力学模拟表明，拨动开关色氨酸吲哚基团和微动开关基序 PIF 的侧链中发生了重大的协同运动。这种旋转在 TM7 的扭结中建立了从色氨酸吲哚到保守丝氨酸 （NK1R-S2977.45） 和主链羰基 （NK1R-A2947.41） 的紧密氢键相互作用。这种相互作用促进了与 TM7 的 NPxxY 微开关基序的通讯，从而稳定了 G 蛋白结合区。NK1R-S2977.45 因此被确定为 NKR 激活的中心枢纽。