A Specific Cholesterol Binding Site Is Established by the 2.8 Å Structure of the Human β2-Adrenergic Receptor

Abstract

The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.

Coarse-grained modeling of allosteric regulation in protein receptors

“…2F), our model identifies residues 196-200 and 295-305 which are located near residues 203, 204, 207, 293, 308, and 312, which in turn are known to be involved in ligand binding (41,42,49). Residues 136-140 are in close spatial proximity to the DRY motif (residues 130-132), and residues 226-230 are located near the assumed β-arrestin binding site (40).…”

“…Residues 136-140 are in close spatial proximity to the DRY motif (residues 130-132), and residues 226-230 are located near the assumed β-arrestin binding site (40). Interestingly, the allosteric coupling map also identifies residues 62-66, 148-152, and 335-340, which are located near residues 70, 147-151, 154, 158, and 341 which may be involved in cholesterol binding (49). Residues 95-99 and 178-183, which form solvated loops, may interact with allosteric modulators.…”

“…This suggestion needs to be examined further, because the higher flexibility of the protein termini [the "tip effect" (50)] might influence the allosteric analysis to some extent. Experiments indicate that the protein termini may indeed engage in interactions with signaling and scaffold proteins (42) or lipids (49). As such, the role of the protein termini in signal transduction requires additional investigation.…”

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

“…For example, cholesterol modulates agonist binding to oxytocin receptors (Gimpl and Fahrenholz, 2002), 5-HT receptors (Pucadyil and Chattopadhyay, 2004) and μ-opioid receptors (Qiu et al, 2011), whereas other GPCRs are less influenced (Oates and Watts, 2011). Stability studies have demonstrated the binding of cholesterol molecules to a conserved motif located between helices 1-4 (Hanson et al, 2008). Recently, relevant phospholipids were found to affect GPCR function.…”

Identification of two distinct inactive conformations of the β 2 -adrenergic receptor reconciles structural and biochemical observations

“…T he recent crystal structures of the ␤ 2 -adrenergic receptor (␤ 2 AR) represented a significant advance in the study of G protein-coupled receptors (GPCRs) (1)(2)(3)(4), which constitute the largest class of both human membrane proteins and drug targets (5). ␤ 2 AR, an important target for cardiac and asthma drugs, has long served as the prototypical ligand-binding GPCR and has been extensively characterized in experimental work over several decades (6).…”