Structural analysis and molecular modeling of high affinity binding and activation of β1-adrenergic receptor

β1-肾上腺素受体高亲和力结合和激活的结构分析和分子建模

基本信息

批准号：
11672210
负责人：
KUROSE Hitoshi
金额：
$ 2.24万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

(-)-RO363 is one of the β_1-selective agonists, and the affinity of (-)-RO363 for β_1AR is about 100-fold and 3000-fold higher than for β_2AR and β_3AR, respectively. Therefore, (-)-RO363 can be used as a tool to examine the binding site of β_1AR and a role in receptor activation by β_1-selective agonists. We made chimeric β_1/β_2ARs and Ala-substituted β_1ARs, and found several key amino acids responsible for β_1-selective binding of (-)-RO363 (Leu^<110> and Thr^<117> in the second transmembrane domain (TMD 2), and Phe^<359> in TMD 7). To analyze a role of these amino acids in the activation step, we made several mutants of a constitutively active (CA-) β_1AR with Ala substitution of the key amino acids responsible for β_1-selective binding. The degree of decrease in the affinity of CA-β_1AR for (-)-RO363 was essentially the same as that of wild type β_1AR when mutated at Leu^<110> and Thr^<117>. However, the affinity was decreased in Ala-substituted mutant of Phe^<359> compared to that of wild type β_1AR.Therefore, we concluded that Phe^<359> may be required for a conformational change of β_1AR induced by binding of (-)-RO363, and Leu^<110> and Thr^<117> may be necessary for the initial binding of (-)-RO363 with high affinity and Phe^<359> interacts with the N-substituent of (-)-RO363. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of β_1AR form a binding pocket ; the methoxy phenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu^<110>, Thr^<117>, and Phe^<359>, and Leu^<110> and Phe^<359> interact with the phenyl ring of (-)-RO363, whereas Thr^<117> forms hydrogen bond with the methoxy group of (-)-RO363. These results can be also used for better drug design.

（ - ） - RO363是β_1选择性激动剂之一，β_1AR的（ - ） - RO363的亲和力分别比β_2AR和β_3AR高约100倍和3000倍。因此，（ - ） - RO363可以用作检查β_1AR的结合位点的工具，并用β_1选择性激动剂在受体激活中的作用。我们制作了嵌合β_1/β_2AR和ALA取代的β_1ars，发现了几个关键氨基酸，负责（ - ） - ro363（LEU^<110>和THR^<117>）的β_1选择性结合，在第二个Transmmbrane域中（TMD 2），以及Phe^<359> in tmd 7> in tmd 7>为了分析这些氨基酸在激活步骤中的作用，我们用ALA取代了负责β_1选择性结合的关键氨基酸的几个突变体（CA-）β_1AR。当在Leu^<110>和Thr^<117>突变时，Ca-β_1AR对（ - ） - RO363的亲和力的降低与野生型β_1AR的降低程度与野生型β_1AR的降低程度相同。然而，与野生型β_1AR相比，Phe^<359>的ALA取代突变体中的亲和力降低了。因此，我们得出的结论可能需要pHE^<359>可能需要β_1AR的构象变化来引起（通过（ - ） - RO363和LEU^<110>^<117>的结合而引起的β_1AR的构象变化 - 具有高亲和力和phe^<359>与（ - ） - ro363的n-近感相互作用。基于这些结果，我们为（ - ） - RO363构建了一个三维模型。该模型表明β_1AR的TMD 2和TMD 7形成一个结合口袋。（ - ） - ro363的N-拟合的方法氧苯基似乎位于Leu^<110>，Thr^<117>和Phe^<359>和Leu^<110>和Phe^<110>和Phe^<110>和Phe^<359>与（ - RO363的苯基环）相互作用的空腔中。（ - ） - RO363。这些结果也可以用于更好的药物设计。