Role of lipid membrane and hydration on the oligomerization and function of PR and A2A

 描述（由申请人提供）：G蛋白偶联受体（GPCR）是一种重要的膜蛋白超家族，由于其定位于细胞表面，使其易于与小分子药物相互作用，因此已成为近40%市售药物的靶标。尽管对新药的持续资助，但2013年FDA批准的24种新药中只有4种针对GPCR超家族。这种低百分比可能部分是由于经常伴随治疗的多种副作用，其部分是由于缺乏GPCR的结构数据以及对GPCR寡聚化的功能后果的普遍理解不足而引起的。越来越明显的是，GPCR在膜中彼此缔合以形成（同源或异源）寡聚复合物，并且这些寡聚物拓宽了细胞信号传导的范围。更好地了解驱动寡聚化的因素可能是靶向治疗的关键，首先了解寡聚化的后果，然后调节受体-受体结合，例如通过设计基于结构的药物来靶向寡聚体群体。为了实现设计和合理化针对特定GPCR寡聚体的治疗方法的雄心勃勃的目标，必须首先针对结构-动力学-功能关系中的知识空白，需要一些技术和方法创新，以及确定可行和有效的GPCR模型，以解决有关寡聚化，脂质膜环境和水合作用的功能影响的基本问题。我们确定了两个7 TM受体，细菌变形视紫红质（PR）和全长的人腺苷A2 A GPCR，作为优秀的系统开发和测试所提出的工具，以确定其低聚状态和结构的洗涤剂和脂质膜。至关重要的是，PR和A2 A受体已被证明在天然脂质或细胞膜环境中寡聚化，这使得测试关于其结构-动力学-功能关系的关键假设非常重要。拟议工作的创新在于选择独特的生物物理工具，其中许多是由PI和合作者开发的。它们包括Gd 3+自旋标记的电子顺磁共振方法，以灵敏地捕获2-6 nm范围内的多个距离，Overhauser动态核极化，以直接绘制膜和蛋白质表面水合动力学，以及用于合成mg量A2 A受体的有效酵母表达方案。这些独特工具的组合使我们能够提出广泛的重要问题，例如：（1）脂质膜中7 TM PR和A2 A的寡聚状态是什么？(2)脂质膜的组成、动力学和水合作用调节寡聚化吗？(3)观察到的PR和A2 A寡聚化的功能作用是什么？所提出的研究的重点是阐明天然脂质膜环境中的7 TM寡聚体结构，与去污剂复合物中获得的结构进行明确的比较，以及基于动力学的方法来阅读和评估蛋白质功能，这对于GPCR研究是新颖的和至关重要的。

 DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are an important superfamily of membrane proteins that have been a target of nearly 40% of all commercially available pharmaceuticals due to their localization at the cell surface, making them easily accessible to interact with small molecule drugs. Despite consistent funding for new drugs, only 4 of the 24 new drugs approved by the FDA in 2013 targeted the GPCR superfamily. This low percentage is likely due, in part, to a multitude of side effects that often accompany treatment, which arise in part from a lack of structural data for GPCRs and a generally poor understanding of functional consequences of GPCR oligomerization. It has become increasingly evident that GPCRs associate with each other in membranes to form (homo- or hetero-) oligomeric complexes and that these oligomers broaden the range of cell signaling. A better understanding of the factors that drive oligomerization would potentially be key for targeted therapies to, first, understand the consequence of, and then, to modulate receptor-receptor association, e.g. by designing structure-based drugs to target an oligomer population. To pursue such ambitious goals of designing and rationalizing therapeutics that target a specific GPCR oligomer, knowledge gaps in structure-dynamics-function relationships must first be targeted, requiring a number of technological and methodological innovations, as well as the identification of viable and effective GPCR models to address basic questions regarding the functional impact of oligomerization, the lipid membrane environment, and hydration. We identify two 7TM receptors, the bacterial proteorhodopsin (PR) and the full-length human adenosine A2A GPCR that serve as excellent systems to develop and test the proposed tools to determine their oligomeric state and structure in detergent and lipid membranes. Crucially, both the PR and A2A receptors have been shown to oligomerize in native lipid or cell membrane environments, making it highly significant to test key hypotheses on their structure-dynamics-function relationships. The innovation of the proposed work lies in the choice of unique biophysical tools, many of which were developed by the PI and collaborators. They include electron paramagnetic resonance methods of Gd3+ spin-based labels to sensitively capture multiple distances in the 2-6 nm regime, Overhauser dynamic nuclear polarization to directly map out membrane and protein surface hydration dynamics, and effective Yeast expression protocols for synthesizing mg quantities of A2A receptors. The combination of these unique tools permits us to cast broadly important questions, such as: (1) What is the oligomeric state of the 7TM PR and A2A in lipid membranes? (2) Do lipid membrane composition, dynamics and hydration tune oligomerization? (3) What is the functional role of oligomerization observed for PR and A2A? The emphasis of the proposed studies on elucidating 7TM oligomer structure in native lipid membrane environment, explicit comparison to structures obtained in detergent complexes and the dynamics-based approach to reading out and evaluating protein function is novel and critically important for GPCR studies.