The Three-dimensional Structure of Bovine Rhodopsin Determined by Electron Cryomicroscopy

Abstract

G-protein-coupled receptors are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways, which activate cellular processes. Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light. Rhodopsin is the most extensively studied G-protein-coupled receptor, and knowledge about its structure serves as a template for other related receptors. We have gained detailed structural knowledge from the crystal structure (1), which was solved by x-ray crystallography in 2000 using three-dimensional crystals. Here we report a three-dimensional density map of bovine rhodopsin determined by electron cryomicroscopy of two-dimensional crystals with p22 1 2 1 symmetry. The usage of relatively small and disordered crystals made the process of structure determination challenging. Special attention was paid to the extraction of amplitudes and phases, since usable raw data were limited to a maximum tilt of 45°. In the refinement process, an improved unbending procedure was applied. This led to a final resolution of 5.5 Å in the membrane plane and ϳ13 Å perpendicular to it, making our electron density map the most accurate map of a G-protein-coupled receptor currently available by electron microscopy. Most important is the information we gain about the center of the membrane plane and the orientation of the molecule relative to the bilayer. This information cannot be retrieved from the three-dimensional crystals. In our electron density map, all seven transmembrane helices were identified, and their arrangement is in agreement with the arrangement known from the crystal structure (1). In the retinal binding pocket, a density peak adjacent to helix 3 suggests the position of the ␤-ionine ring of the chromophore, and in its vicinity several of the bigger amino acids can be identified. G-protein-coupled receptors (GPCRs)1 are a large group of integral membrane proteins that provide molecular links between extracellular signals and intracellular processes (2-7). Many neurotransmitters, hormones, and drugs produce their intracellular signaling through the mediation of G-protein-coupled receptors. The binding of a hormone or neurotransmitter causes a change in the structure of the receptor, which then activates a G-protein. Different members of the receptor family respond to different ligands, and the binding site for these ligands is in the membrane-embedded part of the protein (8, 9).One of the most widely studied GPCRs is rhodopsin (1, 10 -18). Rhodopsin, located in the retina disc membrane of the eye, is responsible for the visualization of dim light. It is composed of the protein opsin (ϳ40 kDa) covalently linked to 11-cis-retinal through Lys 296 of helix 7 (19). The chromophore 11-cis-retinal isomerizes upon light activation to its all-trans conformation, which changes the arrangement of the transmembrane helices (20). This triggers the signal transduction c...

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…Similar dimeric arrangements were found in previous structure determinations from two dimensional and three dimensional rhodopsin crystals [68,114] and rhodopsin models [115]. Based on atomic force microscopic studies [116], a model with TM4 and TM5 as interface of a physiological rhodopsin dimer was proposed [116,117,118].…”

Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay

“…The present study identified TMD III, TMD VI, and TMD VII as part of the ligand binding pocket, which is also the case for bioamine GPCRs (39 -41) and rhodopsin (42)(43)(44). This is further proof of a highly conserved feature among this very large family of receptors.…”

Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family

“…Molecular model of the residues proposed to be part of the structural basis for the high constitutive activity in the ghrelin receptor. A, molecular model of the ghrelin receptor built over the inactive structure of rhodopsin (60,61). The seven helical bundle is displayed without the loops as viewed from the extra-cellular side.…”