Cholesterol modulates the dimer interface of the β₂-adrenergic receptor via cholesterol occupancy sites

Abstract

The β2-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β2-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β2-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.

Figure 1

Schematic representation of the β₂-adrenergic receptor. (a) Top view of the receptor with individual helices marked. (b) Starting structure of the two monomers of the β₂-adrenergic receptor in the POPC bilayer. The two receptors are shown in shades of blue corresponding to panel a; lipid molecules are shown in gray, the phosphate bead of lipid is in orange, and the surrounding water molecules are in blue. (c) Time course of dimerization of the β₂-adrenergic receptor in POPC bilayers with increasing cholesterol concentration. The minimum distance between two receptors (defined as the distance between the closest beads from two individual receptors, as shown in the figure) during the course of the simulation is plotted for receptor association in POPC bilayers alone (black); and in the presence of 9% (red), 30% (green) and 50% (blue) cholesterol concentration. A representative simulation from each of the four systems is plotted. See Materials and Methods for other details. To see this figure in color, go online.

Figure 2

Schematic representation depicting the modulation of transmembrane helices of the β₂-adrenergic receptor involved at the dimer interface with increasing cholesterol concentration. (a and e) POPC bilayer. (b–d and f–h) POPC bilayers containing 9% (b and f), 30% (c and g), and 50% (d and h) cholesterol. Representative top and side views of the transmembrane helices are shown for clarity. The transmembrane helices that comprise the dimer interface are colored and labeled. The remaining helices are colored gray. See Materials and Methods for other details. To see this figure in color, go online.

Figure 3

Contact maps depicting the helix-helix interactions between the two receptors. (a) POPC bilayer. (b–d) POPC bilayers containing 9% (b), 30% (c), and 50% (d) cholesterol. The values were calculated as an average over all simulations and normalized by the time of occurrence and simulation length. A cutoff distance of 0.5 nm was used to determine the contact residues. To see this figure in color, go online.

Figure 4

Cholesterol occupancy at the β₂-adrenergic receptor (maximum occupancy time of cholesterol, i.e., the maximum time a given cholesterol molecule was continuously bound to each of the transmembrane helices). (a–f) The values shown are normalized and averaged for three simulations at increasing cholesterol concentrations: 9% (a and b), 30% (c and d), and 50% (e and f). The simulations were divided into two regimes: the monomer regime (a, c, and e) and the dimer regime (b, d, and f). A maximum occupancy time of one implies that a given cholesterol molecule was present at the given site throughout the entire simulation time, and a value of zero implies it was always absent from that site. The error bars represent the Standard Deviation (SD) between the simulations. See Materials and Methods for further details. To see this figure in color, go online.

Figure 5

Cholesterol-binding sites on transmembrane helix IV of the β₂-adrenergic receptor. (a) The CCM site observed in the crystal structure (30). (b) The cholesterol-binding site identified in the coarse-grained simulations that directly corresponds to the CCM site. (c–f) A high dynamics was observed for the cholesterol and additional conformations of the cholesterol near the site. The backbone of transmembrane helix IV is shown in blue, and the side chains of the amino acid residues R151, I154, and W158 are shown in gray. The bound cholesterol molecule is shown in magenta and the polar bead representing the -OH group is depicted in blue. For clarity, the surrounding receptors, lipid, cholesterol, and water molecules are not shown. See Materials and Methods for further details. To see this figure in color, go online.

Figure 6

Comparison of the dimer interfaces of the β₁-adrenergic receptor and the β₂-adrenergic receptor. (a and b) Crystal structure of the β₁-adrenergic receptor (83). (c and d) Dimer structures of the b2-adrenergic receptor obtained from coarse-grained simulations at 0% and 50% cholesterol concentration, respectively. The transmembrane helices that comprise the dimer interface are colored and labeled. The remaining helices are colored gray. To see this figure in color, go online.