Crystallization and structure determination of the angiotensin II type 1 receptor

描述(申请人提供)：本R21提案的目标是获得血管紧张素II 1型受体(AT1R)的高分辨率晶体结构。AT1R是A、G类蛋白偶联受体超家族中的一员，在心血管功能调节中发挥重要作用。作用于AT1R的药物目前被用于治疗高血压和心力衰竭。AT1aR有三类不同的药物：(1)经典的拮抗剂，血管紧张素受体阻滞剂(ARB)，它将受体稳定在不活跃的构象中，(2)内源性激动剂血管紧张素II，它稳定受体的活跃构象，能够通过G蛋白和？-拦截素发出信号，以及(3)高度特异的？-arrestin偏向激动剂，它稳定受体的构象，能够仅通过？-拦截素发出信号，而不会检测到G蛋白的激活。这种偏向arrestin的激动剂具有独特的药理和治疗特性，不同于经典的激动剂或拮抗剂，例如，它们降低血压(如拮抗剂)，但提高心功能(如激动剂)。关于这些不同类型的配体调节受体功能的结构基础，人们知之甚少。我们建议通过R21机制开始详细研究AT1aR的结构生物学。我们这个提议的目标是证明我们可以获得衍射质量的晶体和高分辨结构的AT1R与高亲和力的拮抗剂结合。拟议的工作是高风险和高影响的。如果成功，这项R21提案的结果将使我们能够获得资金，以更彻底地表征不同构象状态下的AT1aR的结构。这项建议的长期目标是确定AT1aR在三种不同构象中的高分辨晶体结构；由ARB稳定的非活性构象；由血管紧张素II稳定的经典活性构象；以及能够仅通过由α-arrestin偏向激动剂稳定的2-阻滞剂发出信号的活性构象。这些结构将有助于开发更安全和更有效的治疗心力衰竭和高血压的方法。具体目标包括：1)构建AT1aR-T4溶菌酶融合蛋白，并调节这两个蛋白之间的连接子，以优化AT1aR的功能表达和稳定性。2)建立AT1aR的表达和纯化条件，用于结晶学实验。3)结晶并测定了AT1aR的X射线晶体结构。

DESCRIPTION (provided by applicant): The goal of this R21 proposal is to obtain a high-resolution crystal structure of the angiotensin II type 1 receptor (AT1R). The AT1R is a member of the Class A G protein coupled receptor (GPCR) superfamily that plays important roles in the regulation of cardiovascular function. Drugs acting on the AT1R are currently used in the treatment of hypertension and heart failure. There are three distinct classes of drugs for the AT1aR: (1) classical antagonists, angiotensin receptor blockers (ARBs) that stabilize the receptor in an inactive conformation, (2) the endogenous agonist angiotensin II that stabilizes an active conformation of the receptor capable of signaling through both the G proteins and the ? -arrestins, and (3) a highly specific ? -arrestin biased agonist that stabilizes a conformation of the receptor that is capable of signaling exclusively through ? -arrestins without any detectable activation of G proteins. Such ??arrestin biased agonists have unique pharmacological and therapeutic properties, distinct from classical agonists or antagonists, e.g. they lower blood pressure (like antagonists) but increase cardiac performance (like agonists). Little is known about the structural basis by which these different types of ligands regulate receptor function. We propose to begin a detailed investigation of the structural biology of the AT1aR through an R21 mechanism. Our goal for this proposal is to demonstrate that we can obtain diffraction quality crystals and a high-resolution structure of the AT1R bound to a high-affinity antagonist. The proposed work is high-risk and high-impact. If successful, the outcome of this R21 proposal will enable us to obtain funding for a more thorough structural characterization of the AT1aR in different conformational states. The long-term objective of this proposal is to determine the high-resolution crystal structures of the AT1aR in three different conformations; the inactive conformation stabilized by an ARB; the classical active conformation stabilized by angiotensin II; and an active conformation capable of signaling through only 2- arrestins stabilized by a ? -arrestin biased agonist. These structures will facilitate the development of safer and more effective therapeutics for heart failure and hypertension. Specific Aims include: 1) Generate an AT1aR-T4lysozyme fusion protein and adjust the linkers between these two proteins to optimize AT1aR functional expression and stability. 2) Establish conditions for expression and purification of AT1aR for crystallography trials. 3) Crystallize and determine the X-ray crystal structure of the AT1aR.