Rhodopsin is the only GPCR that utilizes photochemistry to generate an activating ligand in situ, by converting the chromophore from its inverse agonistic form into its agonistic form. Tethering the ligand to the protein via a Lys side chain serves two purposes: (1) the inverse agonist 11-cw-retinal stabilizes the inactive receptor conformation and (2) permanent occupancy of the binding pocket ensures maximal sensitivity to light and a fast response necessary for phototransduction.
The 2.8 A crystal structure of rhodopsin, the first high-resolution structure of a GPCR with a bound ligand, has been solved and refined [22,23]. The predicted seven transmembrane helices and the covalent linkage between 11 -ds-retinal and opsin, via Lys296 in helix VII, was confirmed. Rhodopsin shows an ellipsoidal shape (view parallel to the disk membrane plane, Figures 3 and 4) with a seven transmembrane helix region, and an extracellular and intracellular region each consisting of three interhelical loops and a terminal tail (NH2 or COOH, respectively). Approximately equal amounts of the protein mass are distributed to the two solvent-exposed regions (Figures 2 and 3); however, the degree of association of the polypeptide segments is different in both regions. Only a few interactions are seen in the cytoplasmic region, whereas the extracellular parts associate significantly with each other, making the second extracellular loop (E-II) fit tightly within a limited space inside the bundle of helices (Figure 4). A twisted P-hairpin is formed by part of this loop, creating a plug upon which the retinal lies. The P-strand of loop E-II is connected to helix III by a highly conserved disulfide bridge among GPCRs between Cys187 (loop E-II) and Cys110 (helix III). Another feature of the extracellular region is its post-translational modification: N-glycosylation at Asn2 and Asn15.
The seven transmembrane helices of rhodopsin contain a mixture of a- and 310-helices, and vary in length from 19 to 34 residues, in the degree of bending, kinking and twisting, and also in the tilt angles with respect to the expected membrane surface (for details see [22,23]). A significant feature found in the crystal structure is a strong distortion by one of the most conserved residues among GPCRs, Pro267 in helix VI, causing the largest helix bend (36°) seen in the structure , This is of special importance, as movement of the cytoplasm-facing part of helix VI is involved in formation of the active receptor conformation [24,25], and mutations at this position exert long-range effects on the structure of the third cytoplasmic loop . H-V and H-III are not significantly bent, although Pro and Gly residues are present. In contrast, a deviation of helix II from an ideal helix by 30° is due to the flexibility in the Gly-Gly sequence in the middle of this helix. Helix VII shows a considerable distortion and elongation in the region around the retinal attachment site Lys296 and contains two prolines, Pro291 and Pro303. The latter is part of the highly conserved NPxxY motif (Figure 2) found in many GPCRs. This motif, whose function is still unknown, might be involved in the formation of a structural domain that keeps the receptor in the inactive conformation. The structure revealed an additional, non-transmembrane helix. The short cytoplasmic helix VIII, adjacent to helix VII, runs parallel to the cytoplasmic surface and is terminated by palmitoylated cysteines (Cys322 and Cys323), fixing the helix to the membrane (Figures 2 and 4). This cytoplasmic helix is part of the binding site for the
Figure 2. Two-dimensional model of bovine rhodopsin. Half of the polypeptide chain of the apoprotein is embedded in the disk membrane and forms seven transmembrane helices (represented by blue cylinders). A cytoplasmic amphiphatic helix is terminated by palmitoylated Cys322 and Cys323 (violet filled circles) and is represented as a purple cylinder. Visual pigments consist of the opsin apoprotein and the chromophore 11-cw-retinal, vitamin A aldehyde, which is attached via a protonated Schiff base linkage to the e-amino group of a Lys side chain in helix VII (Lys296 is represented by a black circle). The counter-ion of the protonated Schiff base is Glu113 and is shown as a red circle. Photoisomerization of the chromophore to all-irans-retinal leads to activating conformational changes concomitant with exposure of binding sites for Gt at the cytoplasmic side of the receptor. The cytoplasmic surface consists of loops connecting successive helices and the C-terminus (shown as yellow filled circles with a red periphery). Loops C-II, C-III, and the cytoplasmic helix, are involved in interaction with Gt. The regulatory proteins RK and arrestin also bind to the cytoplasmic side although with different loop preferences (see text). Preferred phosphorylation sites for RK at rhodopsin's C-terminus are represented by brown filled circles. A highly conserved disulfide bridge stabilizes the inactive receptor conformation bridge (Cys residues involved are represented by yellow filled circles). Asn2 and Asn15 at the N-terminus carry carbohydrate chains.
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