Temperature-dependent variations of ligand-receptor contact points in hAT1
Temperature-dependent variations of ligand-receptor contact points in hAT1
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DOI：
10.1002/psc.875
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发表时间：
2007-09-01
影响因子：
2.1
通讯作者：
Escher, E.
中科院分区：
文献类型：
--
作者：
Arsenault, J.;Renaud, M. P. La Haye;Escher, E.
光亲和标记法经常用于研究蛋白质，包括肽能G蛋白偶联受体(GPCR)。为此，二苯甲酮光标记物已被广泛用于识别配体结合口袋中的许多接触残基。根据人血管紧张素11I型受体HAT(1)的光化学性质和甲硫氨酸优先掺入二苯甲酮的特性，采用迭代蛋氨酸诱变策略确定了HAT(1)的三维结合环境。这导致了配体结合受体结构的构建。本研究调查了温度对其中一些接触点的可达性的影响。在-15℃到37℃的温度范围内，用二苯甲酮配体(125)I-[Sar1，BPA(8)]AngII对HAT、受体和来自迭代突变研究的两个有代表性的Met突变体(H256M-HAT(1)和F293M-HAT(1))进行光标记，标记的受体被部分纯化并用溴化氰消化以确定接触点或片段。在整个温度范围内，HAT(1)和F293M-HAT(1)的第7跨膜结构域(TMD)的受体接触或标记没有变化。然而，观察到H256M-HAT(1)的配体-受体接触随温度的变化。在-15℃时，H256M标记与HAT(1)相同，表明相互作用是第7 TMD所特有的。在更高的温度和37℃下观察到显著的标记变化，标记几乎仅发生在第6个TMD的突变残基H256M-HAT(1)。同时对该靶蛋白不同区域的竞争标记表明，在生理温度下，配体-受体结构变得越来越不稳定，而在低温下，更紧密、低迁移率和低能量的构象占优势。版权所有(C)2007欧洲肽协会和John Wiley&Sons，Ltd.
Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin 11 type I receptor hAT(1) has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The,present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT, receptor and two representative Met mutants (H256M-hAT(1) and F293M-hAT(1)) from the iterative mutagenesis study were photolabelled with the benzophenoneligand (125) I-[Sar1, Bpa(8)]AngII at temperatures ranging from - 15 degrees C to 37 degrees C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT(1) and F293M-hAT(1) across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT(1) was observed. At -15 degrees C, H256M labelling was identical to that of hAT(1), indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37 degrees C labelling occurred almost exclusively at mutated residue H256M-hAT(1) in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.