Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR
Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR
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DOI：
10.1016/j.bpj.2014.10.011
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发表时间：
2014-11-18
影响因子：
3.4
通讯作者：
Milon, Alain
中科院分区：
文献类型：
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作者：
Gater, Deborah L.;Saurel, Olivier;Milon, Alain
胆固醇与G蛋白偶联受体（GPCRs）的结合及其在膜中活性的调节是了解其功能的基本问题。尽管在β（2）肾上腺素能受体（β（2）AR）和其他GPCR的高分辨率X射线结构中鉴定了胆固醇结合位点，但胆固醇对该受体的结合亲和力以及游离胆固醇和结合胆固醇之间的交换率仍然未知。在这项研究中，我们报告了β（2）AR中存在两类胆固醇结合位点。通过分析β（2）AR在胆固醇立方相（LCP）中的解折叠温度作为胆固醇浓度的函数，我们观察到具有亚nM亲和力常数的胆固醇的高亲和力协同结合。与此相反，饱和转移差（STD）NMR实验显示存在的第二类胆固醇结合位点，在快速交换的STD NMR时间尺度。作为胆固醇浓度的函数的STD信号的滴定提供了其解离常数的100 mM的下限。然而，这些结合位点对胆固醇和β（2）AR都是特异性的，如使用麦角固醇和对照膜蛋白（KpOmpA）的对照实验所示。我们假设这种特异性是由高亲和力结合的胆固醇分子介导的，并提出在高亲和力结合位点周围形成瞬时胆固醇簇。
Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the beta(2) adrenergic receptor (beta(2)AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in beta(2)AR. By analyzing the beta(2)AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and beta(2)AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.