Two Classes of Cholesterol Binding Sites for the β 2 AR Revealed by Thermostability and NMR

Abstract

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 ?2 adrenergic receptor (β2AR) 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 β2AR. By analyzing the β2AR 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 β2AR, 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.

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

“…These sites are in agreement with those found in the crystallographic structures of GPCRs (Cherezov et al, 2007; Hanson et al, 2008; Gimpl, 2016; Warne et al, 2011; Liu et al, 2012; Gater et al, 2014). …”

“…The occupancy of two cholesterol molecules at EC1 is in good agreement with the crystal structure of the adenosine receptor A 2A AR (Liu et al, 2012), while IC2 is so far unidentified among the experimentally determined structures (Gater et al, 2014). Notably, the cholesterol binding residues of the three interaction sites are conserved to a large degree among β 2 AR orthologues (Figure 2—figure supplement 2, Figure 2—figure supplement 3, Figure 2—figure supplement 4), indicating that these sites have conserved during the evolution of the receptor.…”

“…Based on X-ray crystal structures cholesterol has specific contacts with β 2 AR (Cherezov et al, 2007; Hanson et al, 2008), suggesting that β 2 AR has binding sites for cholesterol. Spectroscopic (Gater et al, 2014) and MD simulation (Cang et al, 2013; Prasanna et al, 2014; Lee et al, 2012) studies have reported direct interactions between cholesterol and GPCRs, including β 2 AR. Experimental data show that cholesterol binding to β 2 AR changes its structural properties (Hanson et al, 2008; Zocher et al, 2012).…”

Na+ and solute diffusion in aqueous channels of Myverol bicontinuous cubic phase: PGSE NMR and computer modelling

“…BCPs have attracted attention for use in a wide variety of areas, but by far the most intense focus has been on their use as drug delivery vehicles . The interest in BCPs as drug delivery vehicles is because of their large loading capacity for both hydrophilic and hydrophobic molecules .…”

“…[2] While minimal surfaces describe the general structure of BCPs, the bilayer itself is far from static, showing thermal oscillations in the form of standing waves about the corresponding nodal surface. [3][4][5][6] BCPs have attracted attention for use in a wide variety of areas, [7][8][9][10][11][12][13][14][15] but by far the most intense focus has been on their use as drug delivery vehicles. [16][17][18] The interest in BCPs as drug delivery vehicles is because of their large loading capacity for both hydrophilic and hydrophobic molecules.…”