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

 描述(申请人提供)：G蛋白偶联受体(GPCRs)是一个重要的膜蛋白超家族，由于它们定位于细胞表面，使它们很容易与小分子药物相互作用，因此成为所有商用药物的靶标。尽管对新药的资助始终如一，但在2013年FDA批准的24种新药中，只有4种针对GPCR超家族。这一低百分比可能部分是由于经常伴随治疗而来的大量副作用，部分原因是缺乏GPCRs的结构数据，以及对GPCR齐聚的功能后果普遍缺乏了解。越来越明显的是，GPCRs在膜上相互结合，形成(同或异)低聚物复合体，这些低聚体拓宽了细胞信号的范围。更好地了解推动寡聚的因素可能是靶向治疗的关键，首先了解低聚的后果，然后调节受体-受体的联系，例如通过设计以低聚体为靶点的基于结构的药物。为了追求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-功能关系中的知识差距，需要一些技术和方法创新，以及确定可行和有效的GPCR模型，以解决关于齐聚作用的功能影响、脂膜环境和水化的基本问题。我们识别了两个7TM受体，细菌蛋白视紫红质(PR)和全长人腺苷A2AGPCR，这两个受体是开发和测试所建议的工具以确定其在洗涤剂和脂膜中的低聚状态和结构的优秀系统。重要的是，PR和A2A受体都被证明在天然的脂质或细胞膜环境中都可以寡聚，这使得检验关于它们的结构-动力学-功能关系的关键假说具有非常重要的意义。这项拟议工作的创新之处在于选择了独特的生物物理工具，其中许多工具是由PI和合作者开发的。它们包括基于Gd3+自旋的标记的电子顺磁共振方法可以在2-6 nm范围内灵敏地捕获多个距离，Overhauser动态核极化可以直接绘制膜和蛋白质表面的水化动力学，以及有效的酵母表达协议来合成mg数量的A2A受体。这些独特的工具的结合使我们能够提出广泛的重要问题，例如：(1)7TM PR和A2A在脂膜中的低聚状态是什么？(2)脂膜的组成、动力学和水化作用是否调节齐聚？(3)观察到的PR和A2A的齐聚功能是什么？这些研究的重点是阐明天然脂膜环境中的7TM齐聚物的结构，与洗涤剂复合体的结构进行明确的比较，以及基于动力学的读出和评估蛋白质功能的方法，这对GPCR研究是新颖的，也是至关重要的。

 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.