Structural Elucidation of GPCRs Using Engineered Protein Fusions

描述（由申请人提供）：需要G蛋白偶联受体（GPCR）的高分辨率三维结构，以了解这类主要跨膜信号蛋白的配体结合和激活机制。此外，阐明GPCR结构的能力将对新药的设计产生重大影响，因为这些受体是各种人类疾病的关键治疗靶点。我们已经取得了显着的进展，在分离衍射质量晶体的β 2肾上腺素能受体（β 2 AR），家庭A GPCR，使用的策略，其中折叠，结晶结构域整合到第三个细胞内环。该提案的第一个具体目标是解决这种工程化Beta2AR融合蛋白的高分辨率结构，使用我们所掌握的晶体优化和数据收集的所有策略：筛选添加剂和冷冻保护剂，利用先进的微焦点X射线光束线，如果需要，我们的构建体将进一步修改。该提议的第二个具体目的是将从融合蛋白策略中获得的知识应用于野生型Beta2AR的结构阐明。这将通过β 2AR和抗体片段之间的复合物的形成和结晶来实现。未来的目标将是在其他GPCR上测试受体融合晶体学的策略。在Beta2AR背景下成功使用的相同结构域将被克隆到几种不同的治疗相关受体中，这些受体与化学上不同的配体阵列结合。这些工程受体的表达，药理学和稳定性将进行评估，未来的目标是建立结晶试验。敷设总结：我们获得GPCR晶体结构的长期目标与公共卫生具有广泛的相关性，因为靶向这些受体的药物用于治疗心血管疾病、肺部疾病、炎症、糖尿病、肥胖症和精神疾病。如果我们能够理解GPCR激活中配体亲和力和特异性的分子基础，我们将获得一个重要的新工具，用于设计更有效的治疗方法，副作用更少。

DESCRIPTION (provided by applicant): The high-resolution three-dimensional structures of G protein coupled receptors (GPCRs) are required in order to understand the mechanisms of ligand binding and activation in this predominant class of transmembrane signaling proteins. In addition, the ability to elucidate the structures of GPCRs would make a significant impact in the design of new drugs, since these receptors are key therapeutic targets for a variety of human diseases. We have made significant progress in the isolation of diffraction-quality crystals for the Beta2 adrenergic receptor (Beta2AR), a Family A GPCR, using a strategy in which a folded, crystallizable domain is integrated into the third intracellular loop. The first specific aim of this proposal is to solve the high-resolution structure of this engineered Beta2AR fusion protein, using all of the strategies for crystal optimization and data collection at our disposal: additives and cryo-protectants will be screened, an advanced microfocus X-ray beamline will be utilized, and if necessary our construct will be further modified. The second specific aim of this proposal is to apply knowledge gained from the fusion protein strategy towards the structural elucidation of the wild-type Beta2AR. This will be accomplished through the formation and crystallization of complexes between the Beta2AR and antibody fragments. A future aim will be to test the strategy of receptor fusion crystallography on other GPCRs. The same domain that was used successfully in the context of the Beta2AR will be cloned into several different therapeutically relevant receptors, which bind to a chemically diverse array of ligands. The expression, pharmacology, and stability of these engineered receptors will be assessed, with the future goal of setting up crystallization trials. Lay summary: Our long-term objective of obtaining crystal structures for GPCRs has broad relevance for public health, in that drugs targeting these receptors are used in the treatment of cardiovascular disease, pulmonary disease, inflammation, diabetes, obesity, and psychiatric disorders. If we can understand the molecular basis for ligand affinity and specificity in GPCR activation, we will gain an important new tool in the design of more effective therapeutics with fewer side-effects.