Lipid interactions of the human G-protein-coupled receptor chemokine receptor CXCR1 in asymmetric vesicles

大多数生物膜是高度不对称的。例如，哺乳动物细胞膜的外层小叶主要含有磷脂酰胆碱和鞘磷脂，而内层小叶主要含有磷脂酰乙醇胺和磷脂酰丝氨酸。除了调节膜的结构外，脂质不对称还影响膜蛋白的取向、纳米结构域的形成、蛋白质与膜的结合以及许多其他重要过程。然而，直到最近，还没有一种简单的方法来创造不对称的脂泡，这是体外研究蛋白质-脂质相互作用不可或缺的系统。在拟议的项目中，我们利用一种基于甲基-β-环糊精(MbetaCD)脂质转移的新方法来制备包含人G蛋白偶联受体(GPCR)的不对称蛋白脂质体。我们系统地改变蛋白脂质体外层的脂质成分，使其类似于真核细胞的细胞膜。因此，我们分析了这些因素对配体结合和GPCR寡聚的影响。此外，我们还探讨了受体如何影响不同类型的脂类进入脂类双层外层小叶的倾向。作为一个模型蛋白，我们研究了趋化因子受体CXCR1，它是免疫和炎症反应的主要介质。CXCR1选择性地与白细胞介素8(IL-8)结合，这被认为是直接涉及脂质-水界面，因为受体N端在与IL-8结合时从膜上解离。为了进一步阐明IL-8-CXCR1结合的分子决定因素，我们应用等温滴定量热法(ITC)获得了不同类型不对称蛋白脂质体中结合反应的热力学曲线。这样的图谱为药物发现研究提供了关键参数，包括结合亲和力、化学计量、热焓和熵。值得注意的是，在脂质-双层环境中，目前还不存在任何配体-GPCR相互作用的热力学曲线。为了解决膜不对称性如何影响CXCR1齐聚，我们利用电子顺磁共振(EPR)光谱监测了配体结合的CXCR1的超分子组织。为此，我们使用双电子-电子共振(DER)获得了脉冲EPR谱，以测量与CXCR1结合的自旋标记的IL-8原型的自旋间距离。此外，我们利用基于ITC的脂类交换实验分析了CXCR1如何影响不同类型脂类分配到双层膜外层小叶的倾向。根据ITC和ESR波谱，我们推断了不同的脂质微环境如何影响超分子受体组装，这对活细胞中功能纳米结构域的形成具有深远的意义。

Most biological membranes are highly asymmetric. For example, the outer leaflets of mammalian cellular membranes contain mainly phosphatidylcholine and sphingomyelin, whereas phosphatidylethanolamine and phosphatidylserine predominate in the inner leaflet. In addition to modulating the structure of membranes, lipid asymmetry affects the orientation of membrane proteins, the formation of nanodomains, the binding of proteins to the membrane, and many other vital processes. However, until recently, no straightforward approach has been available to create asymmetric lipid vesicles, which are indispensable systems for in vitro studies of protein-lipid interactions. In the proposed project, we utilize a novel assay based on lipid transfer by methyl-beta-cyclodextrin (MbetaCD) to generate asymmetric proteoliposomes containing a human G-protein-coupled receptor (GPCR). We systematically alter the lipid components of the outer monolayers of proteoliposomes such as to resemble that of eukaryotic cellular membranes. Thereby, we analyze the influence on ligand binding and GPCR oligomerization. In addition, we explore how the receptor influences the propensity of different types of lipids to partition into the outer leaflet of lipid bilayers. As a model protein, we study the chemokine receptor CXCR1, a major mediator of immune and inflammatory responses. CXCR1 selectively binds interleukin-8 (IL-8), which is suggested to directly involve the lipid-water interface, as the receptor N-terminus dissociates from the membrane upon binding of IL-8. To shed more light on the molecular determinants of IL-8-CXCR1 binding, we apply isothermal titration calorimetry (ITC) to obtain thermodynamic profiles of the binding reaction in different types of asymmetric proteoliposomes. Such profiles provide key parameters for drug-discovery studies including binding affinity, stoichiometry, enthalpy, and entropy. Notably, no thermodynamic profiles currently exist for any ligand-GPCR interaction in a lipid-bilayer environment. To address how membrane asymmetry affects CXCR1 oligomerization, we monitor the supramolecular organization of ligand-bound CXCR1 with the help of electron paramagnetic resonance (EPR) spectroscopy. To this end, we acquire pulsed EPR spectra using double electron-electron resonance (DEER) to measure interspin distances of spin-labeled IL-8 protomers bound to CXCR1. Moreover, we analyze how CXCR1 influences the propensity of different types of lipids to partition into the outer leaflet of bilayer membranes utilizing an ITC-based lipid-exchange assay. From both ITC and ESR spectroscopy, we deduce how different lipid micro-environments affect supramolecular receptor assemblies, which has far-reaching implications for the formation of functional nanodomains in living cells.