Petersen Jacob E, Pavlovskyi Artem, Madsen Jesper J, Schwartz Thue W, Frimurer Thomas M, Olsen Ole H
Section for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.
Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA; Center for Global Health and Infectious Diseases Research, Global and Planetary Health, College of Public Health, University of South Florida, Tampa, Florida, USA.
J Biol Chem. 2024 Dec;300(12):107948. doi: 10.1016/j.jbc.2024.107948. Epub 2024 Oct 30.
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 G and G-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-S297) and the position of the tryptophan residue within the canonical "toggle switch" motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F264) and a tyrosine in NK2R (NK2R-Y266), 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 Pro-Ile-Phe (PIF). This rotation establishes a tight hydrogen bond interaction from the tryptophan indole to the conserved serine (NK1R-S297) and a mainchain carbonyl (NK1R-A294) 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-S297 is consequently identified as a central hub for the activation of NKRs.
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1受体（NK1R）和神经激肽2受体（NK2R），这两种受体是G蛋白偶联受体，可传递G和G介导的下游信号。我们研究了受体正构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C末端甲硫氨酸紧密相互作用的残基。我们确定了一个保守的丝氨酸（NK1R-S297）以及典型的“切换开关”基序（TM6的CWxP）中色氨酸残基的位置，该位置与NK1R中的苯丙氨酸（NK1R-F264）和NK2R中的酪氨酸（NK2R-Y266）相邻，从而为神经肽C末端形成了不同的微环境。由于配体与切换开关之间的紧密相互作用，突变这些残基会导致NK1R和NK2R的活性发生显著变化。对活性和非活性NKR结构的结构分析表明，激活后切换开关残基的侧链旋转仅有微小变化。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，切换开关色氨酸吲哚基团和微开关基序Pro-Ile-Phe（PIF）的侧链发生了主要的协同运动。这种旋转建立了从色氨酸吲哚到保守丝氨酸（NK1R-S297）和TM7扭结中的主链羰基（NK1R-A294）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蛋白结合区域。因此，NK1R-S297被确定为NKR激活的中心枢纽。
