Equilibrium between Metarhodopsin-I and Metarhodopsin-II Is Dependent on the Conformation of the Third Cytoplasmic Loop

Abstract

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln 238 and Glu 239 , in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.Rhodopsin is a G-protein-coupled receptor (GPCR) 2 characterized by seven transmembrane helices (H1 through H7) (1). It is an efficient photoreceptor, with the chromophoric retinal cofactor undergoing photoisomerization from 11-cis to all-trans upon activation. The retinal isomerization propagates conformational changes through the lightexcited rhodopsin protein to a meta-stable active state, metarhodopsin-II, which is in equilibrium with the inactive metarhodopsin-I precursor (see Fig. 1A). Metarhodopsin-II binds to the heterotrimeric G-protein transducin (Gt) and triggers GDP release followed by GTP uptake (2, 3).A large fraction of existing drugs and drugs under development target GPCRs (4). The rhodopsin system is considered a prototypical model for the GPCR superfamily, and recent work has produced new insights on the biochemistry of rhodopsin (1, 2). However, many questions remain unanswered, particularly with respect to the mechanism of receptor and G-protein activation.Metarhodopsin-II is characterized by its specific ability to bind and activate multiple copies of the heterotrimeric G-protein transducin (Gt ␣␤␥ ) in rapid s...

Lipids Alter Rhodopsin Function via Ligand-like and Solvent-like Interactions

“…Our simulations suggest that the hydrogenbonding ability of PE headgroups can favor the formation of secondary structure in rhodopsin's ICL3. These findings illustrate the structural plasticity of this region and help reconcile the different ICL3 conformations observed in existing dark-state crystal structures (72,113,114). Our dark-state trajectories were initialized from the tetragonal P4 1 crystal structure solved by Okada et al (72) (PDB: 1U19), in which ICL3 is entirely unstructured.…”

The Dependence of Retinal Degeneration Caused by the Rhodopsin P23H Mutation on Light Exposure and Vitamin A Deprivation

“…Pharmacological chaperones for P23H rhodopsin that are not chromophore analogues are also possible, as evidenced by the observations that monoclonal antibodies can stabilize metarhodopsin-1, 45 and disulfide bonds can stabilize rhodopsin against denaturation. 46 However, the necessary increase in stability may be difficult to achieve, as previous measurements indicate that binding of 11-cis retinal increases the activation energy and ⌬H for unfolding of wild-type opsin because of thermal denaturation by 30% to 60%.…”

Crystal structure of a photoactivated deprotonated intermediate of rhodopsin

“…It has been suggested (18) that upon formation of Meta II, a change in the conformation of Tyr-268 (6.51) (induced by the chromophore) could stabilize the Glu-181 anion and modify its water-modulated interaction with Glu-113 (4.70), the residue that interacts with the protonated Schiff base nitrogen (19). The reorganization of loop C-III has been inferred recently from monoclonal antibody studies (20). Residues 175 and 176 are also different between the two structures, possibly because of the flexibility of the adjacent Gly-174 residue.…”