Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers

Abstract

The G protein-coupled receptor (GPCR) rhodopsin self-assembles into supramolecular structures in native bilayers, but the structural determinants of receptor oligomerization are not known. We carried out multiple self-assembly coarse-grained molecular dynamics (CGMD) simulations of model membranes containing up to 64 molecules of the visual receptor rhodopsin over time scales reaching 100 μs. The simulations show strong preferential interaction modes between receptors. Two primary modes of receptor-receptor interactions are consistent with umbrella sampling/potential of mean force (PMF) calculations as a function of the distance between a pair of receptors. The preferential interfaces, involving helices (H) 1/8, 4/5 and 5, present no energy barrier to forming a very stable receptor dimer. Most notably, the PMFs show that the preferred rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1/H2 and amphipathic H8 at the extracellular and cytoplasmic surfaces, respectively. This dimer orientation is in line with earlier electron microscopy, X-ray, and cross-linking experiments of rhodopsin and other GPCRs. Less stable interfaces, involving H4 and H6, have a free energy barrier for desolvation (delipidation) of the interfaces and appear to be designed to stabilize "lubricated" (i.e., lipid-coated) dimers. The overall CGMD strategy used here is general and can be applied to study the homo- and heterodimerization of GPCRs and other transmembrane proteins. Systematic extension of the work will deepen our understanding of the forces involved in the membrane organization of integral membrane proteins.

Figure 1

Receptor organization upon self-assembly in ten independent simulations of 16 receptors (a) and 64 receptors (c). The protein-to-lipid ratio is 1/100. The white area and the orange protein correspond to the unit cell of the simulation box. Black spheres were placed on Cys316 in H8 to ease the visualization of the receptor orientation. (b) A schematic defines the potential sites of interactions of a single rhodopsin as viewed from the cytoplasmic surface. (d) Graphical definition of φ₁ and φ₃ used in subsequent analysis.

Figure 2

Conformational analysis of the arrangement of receptor dimers formed in self-assembly simulations, and projections of the dimer conformations onto (φ₁, φ₃) angle space. (a) The dimer conformations are represented by dots colored according to the buried ASA (high a_b, bound dimer; a_b=0, unbound dimer). The use of a_b as a third dimension and a viewing from the side of the higher a_b emphasizes the (φ₁, φ₃) subspace primarily explored by the receptor dimers when bound. (b) Subsets of dimer conformations used in the clustering analysis are color-coded according to the cluster to which they belong. The six most populated clusters are highlighted. The blue dots lacking a black rim correspond to dimers from any of the other clusters (7 to 301). (c) Structures representative for the ten most populated clusters of the receptor dimers. These ten clusters are organized into three groups based on the similarity of their relative orientation. (The representative structure of the cluster number 286 is also shown.) When relevant, a grey disk underlines the most populated cluster of the group. For each group the population (%) of the most prominent cluster is given together with the sum (Σ) over the entire group.

Figure 3

Potentials of mean force (PMFs) of receptor interfaces. (a) Molecular system used to generate the PMFs: two receptors (H8 is orange) embedded in a membrane bilayer (grey) solvated by water (blue). The lipid and water molecules are only partially shown to feature the receptors. The dimensions of the simulation box are given in nm. The protein-to-lipid ratio was 1/328. (b) PMFs are expressed as a function of the interfacial distance (d’). The set of restrains used to control the distance (d) and the relative orientation (φ₁, φ₃) of the receptors in shown in the inset. (c) Illustration of the interfaces; a_b is the protein burial (nm²).

Figure 4

Dimer interaction interface. (a) Interfacial lipids in site2–site2 (weak) dimer. (b) Interacting-residue network in the site1–site1 (strong) dimer. The palmitoyl chains (palm) are shown in cyan stick, an orange dashed line encircles the residues from H1 and H2 involved in the contacts, and in the middle of the bilayer a few interacting residues are shown within an ochre dashed circle. Side chains shown in green were found to be relevant for the dimer in the X-ray packing data ^,. Side chains shown in yellow were revealed from the CGMD data: Trp35, Leu99, Ser38, Leu46 and potentially Tyr43 through an interaction with Phe45’.

Figure 5

Model of the rows-of-dimers organization of rhodopsin molecules after a 16-μs* CGMD simulation. The starting conformation was built according to the cell dimensions determined from AFM images of rhodopsin in disc membranes prepared from mouse retinas^, (Supporting Information Fig. S3). The lipid molecules are shown by cyan dots placed at the location of the phosphate groups. Rhodopsins are shown in deep red using cylinders for the helices and grey tubes for the backbone trace. The large orange spheres are centered on Thr242 to show the H6 protrusion. The monoclinic unit cell (γ =85°) is outlined by a black box; the view is from the cytoplasmic surface. Note that the protein-to-lipid ratio is with 1/36 about twice as large as in bovine with 1/70 (see Methods for details).