Probing the Receptor Dimer Interfaces of G-Protein Coupled Receptor in Model Membranes

Abstract

How the components of the G protein-coupled receptor (GPCR) “signalosome” assemble and function in the membrane bilayer is not known. Using a multidisciplinary approach combining computational and experiment work that leverages recent structural data, we present a model for the supramolecular structure of the rod cell disc membrane phototransduction machinery. We first show a comparison of multiple rhodopsin dimer interfaces in a model membrane using a coarse-grained molecular dynamics simulation approach accumulating more than a millisecond of simulation time. To characterize the preferred binding interface of a pair of rhodopsins, we determined the potentials of mean force as a function of the distance between two membrane-embedded receptors. The interfaces probed include helix 4 (H4), H4/H5, H5, H6 and H1/H8. The results show that the most stable rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1 and H2 at the extracellular side and amphipathic H8 at the cytoplasmic one. The existence of the H1/H8 dimer was unambiguously corroborated by crosslinking experiments in which we identified CYS316 in H8 as the site of a chemical crosslink between two rhodopsins in native ROS disc membrane using proteolysis, CNBr cleavage, and high-resolution liquid-chromatography mass-spectroscopy (LC/MS). We then show how secondary interaction surfaces appear to stabilize extended “lubricated” rows of these dimers as seen earlier in atomic force microscopy studies. The synthesis of the new rhodopsin dimer orientation with the structures of the R∗/Gt complex in two different orientations and 3-D densities of the complex obtained earlier by electron-microscopy argues for novel alternatives for the supramolecular organization of ROS membrane. The model we propose suggests the possibility for an efficient one-dimensional mechanism for Gt to search for active receptor (R∗) even under low light conditions.