Temperature dependent photolabeling of the human angiotensin II type 1 receptor reveals insights into its conformational landscape and its activation mechanism
Temperature dependent photolabeling of the human angiotensin II type 1 receptor reveals insights into its conformational landscape and its activation mechanism
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DOI：
10.1016/j.bcp.2010.06.004
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发表时间：
2010-10-01
影响因子：
5.8
通讯作者：
Escher, Emanuel
中科院分区：
文献类型：
--
作者：
Arsenault, Jason;Cabana, Jerome;Escher, Emanuel
我们提出了一个光亲和标记研究的人血管紧张素II（AngII）1型受体（hAT（1））和一个组成型活性突变体（CAM）N111 G hAT（1）在多个温度下使用的对苯甲酰基-L-苯丙氨酸（Bpa）含有AngII类似物I-125-[Sar（1），Bpa（8）] AngII和甲硫氨酸邻近方法（MPA）。通过在hAT（1）及其CAM中通过诱变引入选择性地与Bpa反应的Met残基，我们能够鉴定受体-配体复合物中Bpa部分周围的残基的位置。在这里，我们通过控制和改变（从-20至50摄氏度）进行光标记的温度来改进这种表征。用CNBr消化hAT（1）Met突变体以及CAM双突变体光标记的受体，并通过放射性和光密度分析来定量片段化模式。许多重要的和显着的变化，在碎片模式中观察到的功能的光解温度和本构活化的背景下。配体-受体复合物随着温度的升高而变得越来越灵活，即Bpa部分可以更容易地标记越来越远的残基。这些片段化模式被转换成距离的限制，包括到一个模拟退火协议，以探索这些构象变化的程度。在组成性激活的背景下，第6跨膜结构域（TM 6）被发现表现出相对向外的运动，而TM 2和5被发现移动更接近配体结合位点。TM 3显示轻微移位。(C)2010年爱思唯尔公司All rights reserved.
We present a photoaffinity labeling study of the human Angiotensin II (AngII) type 1 receptor (hAT(1)) and a constitutively active mutant (CAM) N111G hAT(1) at multiple temperatures using a p-benzoyl-L-phenylalanine (Bpa) containing AngII analogue I-125-[Sar(1), Bpa(8)] AngII and the Methionine Proximity Approach (MPA). By introducing Met residues, which react selectively with Bpa, by mutagenesis in hAT(1) and its CAM, we were able to identify the position of residues that surround the Bpa moiety in the receptor-ligand complexes. Here we refined this characterization by controlling and varying (from -20 to 50 degrees C) the temperature at which the photolabeling was carried out The hAT(1) Met mutant, as well as CAM double mutant, photolabeled receptors were digested with CNBr and the fragmentation patterns were quantified by radioactive and densitometric analysis. Many important and significant changes in the fragmentation patterns were observed as function of both the temperature of photolysis and the context of constitutive activation. The ligand-receptor complex was increasingly flexible as temperature was increased, i.e. that the Bpa moiety could more easily label increasingly distant residues. These fragmentation patterns were converted into distance constraints that were included into a simulated annealing protocol in order to explore the extent of these conformational changes. In the context of constitutive activation, the 6th transmembrane domain (TM6) was found to exhibit a relative outward movement while TM2 and 5 were found to move closer to the ligand binding site. TM3 showed a slight displacement. (C) 2010 Elsevier Inc. All rights reserved.