A dynamic view of GPCR-G protein complexes: insight into partial agonism and G protein selectivity

人体是由许多细胞组成的，这些细胞大量地协同工作以维持生命。每个细胞周围都有一层脂膜，大多数化学物质都不能穿透它。然而，为了正常地协同工作，各个细胞需要相互交流，并对外部需求做出反应或调整。为了帮助做到这一点，许多传感器蛋白，即所谓的G蛋白偶联受体(GPCRs)嵌入到脂肪细胞的包膜中。在人类中有超过850种不同类型的GPCRs，它们的任务是感知细胞外部广泛配体分子(激动剂)的存在，并通过脂膜传递这一信息。GPCRs擅长这一点，因为每种受体都专门结合一组特定的配体。配基结合改变了受体的形状，进而允许其他蛋白质，即所谓的转导蛋白，与细胞膜内侧的受体部分结合。将转导器与gpr偶联，然后激活广泛的细胞信号过程，这些过程指示细胞做出反应并采取必要的行动。净效应是细胞外部存在的一种特定类型的配体已经传递到细胞内部。由于其作为细胞传感器的核心作用，GPCRs调节着广泛的生理过程，因此对人类的健康和疾病功能至关重要。不足为奇的是，很大比例的处方药针对的是GPCR。GPCR的一个决定性特征是它们的流动性。它们可以很容易地改变构象和激动剂结合影响的毫不费力，这与这些受体的内在功能密切相关。同样，GPCRs与其转导G蛋白以动态方式相互作用，但目前对这些GPCRG蛋白复合体如何相互作用知之甚少。大多数结构研究都依赖于揭示静态络合物中GPCRs的方法，在静态络合物中，结合伙伴通常被设计成稳定的形式。然而，这忽略了这样一个事实，即结合伙伴保持固有的移动性，并且可以采用一系列不同的构象。因此，我们建议使用一种名为核磁共振的方法进行研究，这种方法可以在保持这些蛋白质自然流动性的同时对其进行研究。我们将更详细地研究GPCRs是如何与它们的转导蛋白相互作用的，特别是异源三聚体G蛋白家族。在保持这些蛋白质的动态性质的同时，获得对这些相互作用的分子细节的结构洞察，是提高我们对这些受体如何工作的理解的关键。通过我们对GPCRb1AR的研究，我们将了解GPCRG蛋白相互作用的分子决定因素。我们将发现复合体的动态性质如何与结合伙伴的性质有关，以及它们的构象如何随着结合的激动剂的类型和相互作用的G蛋白的类型而变化。将我们从核磁共振中观察到的分子与这些复合体的生物物理性质相关联，如结合伙伴的亲和力、复合体的形成速度以及核苷酸交换速度如何受到影响，将使我们了解这些构象差异如何影响信号功能。在增加我们对这些受体-转导复合体以及GPCRs和G蛋白如何动态相互作用的一般知识的同时，我们的工作也将告诉我们两个主要的悬而未决的问题，这两个问题对这些蛋白质的功能至关重要：为什么某些配体与受体结合时会导致温和的、低于最大反应的反应？为什么特定的gpr与G蛋白家族中选定的一个成员相互作用，而不是另一个？这两个问题都对细胞信号有直接的影响，我们的研究将提供关键分子决定因素的动态视角。

The human body is made of many cells, which work together in large numbers to sustain life. Each cell is surrounded by a lipid membrane that is impenetrable to most chemicals. However, in order to work properly together, individual cells need to communicate with each other and respond or adjust to external needs. To help with that, a host of sensor proteins, so-called G protein-coupled receptors (GPCRs) are embedded in the lipid cell envelope. There are over 850 different kinds of GPCRs in humans and their task is to sense the presence of a wide range of ligand molecules (agonists) on the outside of the cell and to communicate that information across the lipid membrane. GPCRs excel at this, as each kind of receptor is specialised to bind a particular set of ligands. Ligand binding changes the shape of the receptor, which in turn allows other proteins, so-called transducers, to bind to that part of the receptor on the inside of the cell membrane. Coupling of the transducer to the GPCR then activates a wide range of cellular signaling processes, which instruct the cell to respond and take necessary actions. The net effect is that the presence of a particular type of ligand on the outside of a cell has been communicated to the cell interior. Owing to their central role as sensors of the cell, GPCRs regulate a wide range of physiological processes and hence are central to human function in health and disease. Not surprisingly, a large proportion of prescription drugs target GPCRs.A defining feature of GPCRs is their mobility. They can easily change conformation and the effortlessness with which agonist-binding influences this strongly relates to the intrinsic function of these receptors. Similarly, GPCRs interact with their transducer G proteins in a dynamic fashion but how these GPCR-G protein complexes interact is currently poorly understood. Most structural investigations have relied on methods that reveal the GPCRs within static complexes, where the binding partners have usually been engineered into a stabilised form. This overlooks the fact, however, that the binding partners remain inherently mobile and can adopt a range of different conformations. We propose therefore to conduct studies using a method called NMR, which allows investigation of these proteins while retaining their natural mobility. We will investigate in greater detail how GPCRs interact with their transducers, in particular the family of heterotrimeric G proteins. Obtaining structural insight into the molecular details of these interactions, while preserving the dynamic nature of these proteins, is key for improving our understanding of how these receptors work. Through our investigations with the GPCR b1AR we will learn the molecular determinants of GPCR interactions with G proteins. We will discover how the dynamic nature of the complexes relate to the properties of the binding partners and how their conformations vary with the type of agonist bound and the type of G protein that interacts. Correlating our molecular observations from NMR with biophysical properties of these complexes, such as affinity of the binding partners, how rapidly the complexes are formed and how the rate of nucleotide exchange is affected, will inform us on how these conformational differences affect signaling function. While increasing our general knowledge of these receptor-transducer complexes and how GPCRs and G proteins dynamically interact, our work also will inform us on two major unresolved questions that are key for the function of these proteins: Why do certain ligands when bound to a receptor result in a moderated, less than maximal response? Why does a particular GPCR interact with a selected member of the G protein family preferably over another one? Both questions have direct implications for cellular signaling and our investigations will provide a dynamic perspective of the key molecular determinants.