Crystallization and structure determination of the angiotensin II type 1 receptor

描述（由申请人提供）：该R21提案的目标是获得血管紧张素II型1受体（AT1R）的高分辨率晶体结构。AT1R是a类G蛋白偶联受体（GPCR）超家族的成员，在心血管功能调控中发挥重要作用。作用于AT1R的药物目前用于治疗高血压和心力衰竭。AT1aR有三种不同类型的药物：(1)经典拮抗剂，血管紧张素受体阻滞剂（ARBs），稳定受体的非活性构象；(2)内源性激动剂血管紧张素II，稳定受体的活性构象，能够通过G蛋白和？-逮捕，(3)高度具体的？-抑制素偏向激动剂，稳定受体的构象，能够通过？没有任何可检测到的G蛋白活化。这样的吗? ?与传统的激动剂或拮抗剂不同，抑制素偏倚激动剂具有独特的药理和治疗特性，例如，它们降低血压（如拮抗剂），但增加心脏功能（如激动剂）。人们对这些不同类型的配体调节受体功能的结构基础知之甚少。我们建议通过R21机制开始对AT1aR的结构生物学进行详细的研究。我们的目标是证明我们可以获得高亲和力拮抗剂结合的AT1R的衍射质量晶体和高分辨率结构。拟议的工作是高风险和高影响的。如果成功，R21提案的结果将使我们能够获得资金，对不同构象状态下的AT1aR进行更彻底的结构表征。本提案的长期目标是确定三种不同构象的AT1aR的高分辨率晶体结构；ARB稳定的非活性构象；血管紧张素II稳定的经典活性构象；而主动构象仅能通过2个抑制因子发出信号，由a ？-逮捕偏颇激动剂。这些结构将有助于开发更安全、更有效的心力衰竭和高血压治疗方法。具体目的包括：1)生成AT1aR- t4溶菌酶融合蛋白，并调节这两个蛋白之间的连接物，优化AT1aR功能表达和稳定性。2)建立AT1aR的表达和纯化条件，用于晶体学实验。3)结晶并测定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. PUBLIC HEALTH RELEVANCE: Hypertension and heart failure represent major causes of morbidity and mortality in the United States. The renin-angiotensin system plays a central role in regulating cardiovascular function, and the angiotensin receptor is an important therapeutic target for cardiovascular disease. Successful completion of the aims of this proposal will facilitate the development of more selective and effective drugs for heart failure and hypertension.