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小叶主要含有磷脂酰胆碱和鞘磷脂，而磷脂酰乙醇胺和磷脂酰丝氨酸在内小叶中占主导地位。除了调节膜的结构外，脂质不对称性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，还没有直接的方法来创建不对称的脂质囊泡，这是不可或缺的系统，在体外研究蛋白质-脂质相互作用。在所提出的项目中，我们利用一种新的测定方法的基础上通过甲基-β-环糊精（MbetaCD）的脂质转移，以产生含有人G蛋白偶联受体（GPCR）的不对称蛋白脂质体。我们系统地改变脂蛋白体的外层单层的脂质成分，使其类似于真核细胞膜。因此，我们分析了配体结合和GPCR寡聚化的影响。此外，我们探讨了受体如何影响不同类型的脂质分配到脂质双层的外小叶的倾向。作为模型蛋白，我们研究了趋化因子受体CXCR 1，它是免疫和炎症反应的主要介质。CXCR 1选择性结合白细胞介素-8（IL-8），这被认为直接涉及脂质-水界面，因为受体N-末端在结合IL-8时从膜解离。为了阐明IL-8-CXCR 1结合的分子决定因素，我们应用等温滴定量热法（ITC）获得不同类型的不对称脂蛋白体中结合反应的热力学曲线。这些特征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、焓和熵。值得注意的是，在脂质双层环境中，目前不存在任何配体-GPCR相互作用的热力学曲线。为了解决膜的不对称性如何影响CXCR 1寡聚化，我们监测的超分子组织配体结合CXCR 1的帮助下，电子顺磁共振（EPR）光谱。为此，我们获得脉冲EPR光谱使用双电子-电子共振（DEER）来测量自旋标记的IL-8结合到CXCR 1的前体的自旋间距。此外，我们分析了CXCR 1如何影响不同类型的脂质的倾向，利用ITC为基础的脂质交换试验分配到双层膜的外叶。从ITC和ESR光谱，我们推断不同的脂质微环境如何影响超分子受体组装，这对活细胞中功能性纳米结构域的形成具有深远的影响。

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.