Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Abstract

Rhodopsin is an extensively studied member of the G proteincoupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.Rhodopsin is the photoreceptor molecule present in vertebrate eyes, which senses light stimuli and initiates a signaling cascade mediated by the G protein. Rhodopsin is a member of the family-A G protein-coupled receptors (GPCRs), 3 and therefore it is presumed to have diverged from a diffusible ligand binding GPCR (1). However, in contrast with other GPCRs where diffusible ligands interact with their receptors strictly by noncovalent interactions, the binding of the retinal to opsin involves a covalent bond between the receptor and the ligand. Rhodopsin has the 11-cis-retinal chromophore covalently bound in its protein moiety acting as an inverse agonist in the dark, and light absorption causes the generation of the agonist all-trans-retinal through cis-trans photoisomerization of the chromophore (2).The covalent bond is formed between the aldehyde group of the retinal and the ⑀-amino group of lysine 296 4 located at the transmembrane helix VII of rhodopsin, through a protonated Schiff base linkage (3, 4). Formation of the protonated Schiff base is an important structural feature for photoreception, because the protonation of the Schiff base causes electron delocalization of the chromophore, resulting in a red shift of the absorption spectrum of rhodopsin, which allows it to absorb visible light (5). Moreover, the positive charge of Lys-296 and the negative charge of Glu-113 form an ionic pair, thus forming an intramolecular switch known as the opsin lock, which locks the receptor in an inactive state (6, 7). This ionic interaction is maintained in ...

Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II

“…3). Full activation of rhodopsin, however, requires an intact Schiff base bond to the receptor (42,43), which seems to require the rotation of the chromophore during activation (11). Interestingly, this rotation is not observed in bacteriorhodopsin, another well-studied retinal binding 7TM protein.…”

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation

“…Experimental data were fitted by a single exponential function, and the v light was estimated as previously described (41,42). The k d was measured by a fluorescence assay as previously described (42,43). The assay mixture consisted of 20 nM pigments (60 nM for zebrafish, tiger salamander, Mexican salamander and newt blue, Xenopus blue T47L, and tiger salamander blue M47T to increase signal to noise ratio), 5 μM GTPγS, 0.015% DDM, 50 mM Hepes (pH 6.5), 140 mM NaCl, 5.8 mM MgCl 2 , and 1 mM DTT.…”

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

“…In its basal resting state, it consists of a polypeptide chain, opsin, and its cognate chromophore, the vitamin A aldehyde derivative 11-cis-retinal, which acts as an inverse agonist preventing G-protein activation in the dark (3,11). The retinal chro-mophore is responsible for light absorption in the visual process (12), and, upon photon capture, it isomerizes to its all-trans configuration yielding the active Rh photointermediate metarhodopsin II (MetaII) capable of binding and activating the G-protein transducin (Gt) (13)(14)(15).…”