Determining the environment of the ligand binding pocket of the human angiotensin II type I (hAT1) receptor using the methionine proximity assay
Determining the environment of the ligand binding pocket of the human angiotensin II type I (hAT1) receptor using the methionine proximity assay
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DOI：
10.1074/jbc.m413653200
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发表时间：
2005-07-22
影响因子：
4.8
通讯作者：
Escher, E
中科院分区：
文献类型：
--
作者：
Clément, M;Martin, SS;Escher, E
肽激素血管紧张素 II (AngII) 以延伸构象与跨膜结构域内的 AT(1)（(a) 下条形血管紧张素 (t) 下条形 (1) 下条形）受体结合，其 C 端残基与 Phe-293/Asn-294 处的跨膜结构域 VII 相互作用。该结合口袋的分子环境仍有待阐明。二苯甲酮光标记与目标结构中的蛋氨酸残基的优先结合使我们能够设计一种称为蛋氨酸邻近测定的实验方法，该方法基于系统诱变和光标记来确定该结合袋的分子环境。纯化一系列用 I-125-[Sar(1), p'-苯甲酰基-L-Phe(8)] AngII 或用 I-125-[Sar(1), p''-甲氧基-对'-苯甲酰基-L-Phe(8)] AngII 光标记的 44 个跨膜结构域 III、VI 和 VII X -> Met 突变体，并用溴化氰消化。一些突变体产生的消化模式与野生型人类 AT(1) 观察到的消化模式不同，表明它们与 AngII 的 8 位有新的受体接触。以下残基形成该结合袋：跨膜结构域 (TMD) III 中的 L112M 和 Y113M； TMD VI 中的 F249M、W253M、H256M 和 T260M；以及 TMD VII 中的 F293M、N294M、N295M、C296M 和 L297M。这些接触的同源建模和整合使我们能够开发出与人类 AT(1) 相互作用的基于证据的分子模型，该模型与视紫红质-视网膜相互作用非常相似。
The peptide hormone angiotensin II (AngII) binds to the AT(1) ((a) under bar ngiotensin (t) under bar ype (1) under bar) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. A series of 44 transmembrane domain III, VI, and VII X -> Met mutants photolabeled either with I-125-[Sar(1), p'-benzoyl-L-Phe(8)] AngII or with I-125-[Sar(1), p ''-methoxy-p'-benzoyl-L-Phe(8)] AngII were purified and digested with cyanogen bromide. Several mutants produced digestion patterns different from that observed with wild type human AT(1), indicating that they had a new receptor contact with position 8 of AngII. The following residues form this binding pocket: L112M and Y113M in transmembrane domain (TMD) III; F249M, W253M, H256M, and T260M in TMD VI; and F293M, N294M, N295M, C296M, and L297M in TMD VII. Homology modeling and incorporation of these contacts allowed us to develop an evidence-based molecular model of interactions with human AT(1) that is very similar to the rhodopsin-retinal interaction.