Molecular Structure of Rhodopsin

The extracellular surface domain of Rho is comprised of the amino-terminal tail (NT) and three interhelical loops (E1, E2, and E3) (Fig. 1) [6]. There is significant secondary structure in the extracellular domain and several intra- and inter-domain interactions. The E2 loop is extremely interesting in that it is folded deeply into the core of the membrane-embedded region of Rho. In addition to contacts with the chromophore (11-ds-retinol), E2 forms extensive contacts with other extracellular regions. The P3 and P4 strands, which arise from E2, run anti-parallel. The P4 strand is situated more deeply within the membrane-embedded region of Rho than the P3 strand. The P4 strand is adjacent to the chromophore and forms the extracellular boundary, or roof, of the ligand-binding pocket. A disulfide bond between Cys-110 and Cys-187, which forms the extracellular end of H3, is highly conserved among all class A GPCRs.

More than one-half of the 348 amino acid residues in Rho make up the seven transmembrane segments (H1 to H7) included in the membrane-embedded domain. The crystal structure of this domain is remarkable for a number of kinks and distortions of the individual transmembrane segments, which are otherwise generally a-helical in secondary structure. Many of these distortions from idealized secondary structure were not accounted for in molecular graphics models of Rho based on projection density maps obtained from cryoelectron microscopy [7]. H7 is the most highly distorted of the seven transmembrane helical segments. There are kinks at two Pro residues, Pro-291 and Pro-303. In addition, the helix is irregular around the region of residue Lys-296, which is the chromophore attachment site. Pro-303 is a part of the highly conserved Asn/Pro/ X/X/Tyr motif (Asn-302/Pro-303/Val-304/Ile-305/Tyr-306 in Rho).

The membrane-embedded domain of Rho is also characterized by the presence of several intramolecular interactions that may be important in stabilizing the ground state structure of the receptor. One of the hallmarks of the molecular physiology of Rho is that it is essentially silent biochemically in the dark. The bound chromophore serves as a potent pharmacological inverse agonist to minimize activity. The Rho structure reveals numerous potentially stabilizing intramolecular interactions, some mediated by the chro-mophore and others arising mainly from interhelical interactions that do not involve the chromophore-binding pocket directly. For example, a complex H-bond network appears to link H6 and H7. The key interaction here is between Met-257 and Asn-302. The precise functional importance of the highly conserved Asn/Pro/X/X/Tyr motif (Asn-302/Pro-303/Val-304/Ile-305/Tyr-306 in Rho) is unclear. However, one key structural role is to mediate several interhelical interactions. The side chains of Asn-302 and Tyr-306 project toward the center of the helical bundle. The hydroxyl group of Tyr-306 is close to Asn-73 (cytoplasmic border of H2), which is also highly conserved. A key structural water molecule may facilitate an H-bond interaction between Asn-302 and Asp-83 (H2). A recent mutagenesis study of the human platelet-activating factor receptor showed that replacement of amino acids at the positions equivalent to Asp-78 and Asn-302 in Rho with residues that could not H bond prevented agonist-dependent receptor internalization and G-protein activation [8].

The 11-ds-retinol chromophore is a derivative of vitamin A!, with a total of 20 carbon atoms (Fig. 2). The binding site of the chromophore lies within the membrane-embedded

Figure 2 Photoisomerization of the 11-cis-retinylidene chromophore (RET) to its 11-trans form is the only light-dependent event in vertebrate vision. The RET chromophore is a derivative of vitamin A1 with a total of 20 carbon atoms. The structure of the chromophore in rhodopsin appears to be 6s-cis 11-cis 12s-trans 15-anti-retinylidene protonated Schiff base. The planar surfaces are meant to depict the twists about the C-6-C-7 and C-12-C-13 bonds. Photoisomerization in Rho occurs on an ultrafast time scale, with photorhodopsin as the photoproduct formed on a femtosecond time scale [23]. The photolyzed pigment then proceeds through a number of well-characterized spectral intermediates. As the protein gradually relaxes around 11-trans RET, protein-chromophore interactions change and distinct Xmax values are observed. Important photochemical properties of Rho in the rod cell disc membrane include a very high quantum efficiency (~ 0.67 for Rho versus ~ 0.20 for RET in solution) and an extremely low rate of thermal isomerization.

Figure 2 Photoisomerization of the 11-cis-retinylidene chromophore (RET) to its 11-trans form is the only light-dependent event in vertebrate vision. The RET chromophore is a derivative of vitamin A1 with a total of 20 carbon atoms. The structure of the chromophore in rhodopsin appears to be 6s-cis 11-cis 12s-trans 15-anti-retinylidene protonated Schiff base. The planar surfaces are meant to depict the twists about the C-6-C-7 and C-12-C-13 bonds. Photoisomerization in Rho occurs on an ultrafast time scale, with photorhodopsin as the photoproduct formed on a femtosecond time scale [23]. The photolyzed pigment then proceeds through a number of well-characterized spectral intermediates. As the protein gradually relaxes around 11-trans RET, protein-chromophore interactions change and distinct Xmax values are observed. Important photochemical properties of Rho in the rod cell disc membrane include a very high quantum efficiency (~ 0.67 for Rho versus ~ 0.20 for RET in solution) and an extremely low rate of thermal isomerization.

Figure 3 The RET chromophore-binding pocket of bovine Rho. The RET chromophore-binding pocket is shown from slightly above the plane of the membrane bilayer looking between transmembrane segments H1 and H7. Several amino acid residues are labeled, including the Schiff base counterion Glu-113. At least three residues appear to interact with the C-19 methyl group of the chromophore: Ser-118, Ile-189, and Tyr-268. The C-19 methyl group might provide a key ligand anchor that couples chromophore isomerization to protein conformational changes. Some additional key amino acid residues are labeled, including the Cys-187, which forms a highly conserved disulfide bond with Cys-110.

Figure 3 The RET chromophore-binding pocket of bovine Rho. The RET chromophore-binding pocket is shown from slightly above the plane of the membrane bilayer looking between transmembrane segments H1 and H7. Several amino acid residues are labeled, including the Schiff base counterion Glu-113. At least three residues appear to interact with the C-19 methyl group of the chromophore: Ser-118, Ile-189, and Tyr-268. The C-19 methyl group might provide a key ligand anchor that couples chromophore isomerization to protein conformational changes. Some additional key amino acid residues are labeled, including the Cys-187, which forms a highly conserved disulfide bond with Cys-110.

domain of the receptor (Fig. 3). All seven transmembrane segments and part of the extracellular domain contribute interactions with the bound chromophore. The chromophore is located closer to the extracellular side of the transmembrane domain of the receptor than to the cytoplasmic side. Glu-113 serves as the counterion for the Schiff base attraction of the chromophore to Lys-296. In all, at least 16 amino acid residues are within 4.5 A of the chromophore: Glu-113, Ala-117, Thr-118, Gly-121, Glu-122, Glu-181, Ser-186, Tyr-191, Met-207, His-211, Phe-212, Phe-261, Trp-265, Tyr-268, Ala-269, and Ala-292. The most striking feature of the binding pocket is the presence of many polar or polarizable groups to coordinate an essentially hydrophobic ligand.

The cytoplasmic domain of Rho is comprised of three cytoplasmic loops and the carboxyl-terminal tail: C1, C2, C3, and CT. Loops C1 and C2 are resolved in the crystal structure, but only residues 226 to 235 and 240 to 246 are resolved in C3. CT is divided into two structural domains. C4 extends from the cytoplasmic end of H7 at Ile-307 to Gly-324, just beyond two vicinal Cys residues (Cys-322 and Cys-323), which are posttranslationally palmitoylated. The remainder of CT extends from Lys-325 to the carboxyl terminus of Rho at Ala-348. The crystal structure does not resolve residues 328 to 333 in CT.

A number of cytoplasmic proteins are known to interact exclusively with the active state of the receptor (R*). Because the crystal structure depicts the inactive Rho structure that does not interact significantly with cytoplasmic proteins, the structure can provide only indirect information about the relevant R* state. Perhaps the most extensively studied receptor-G-protein interaction is that of bovine Rho with Gt. Detailed biochemical and biophysical analysis of the R*-Gt interaction has been aided by mutagenesis of the cytoplasmic domain of bovine Rho. Numerous Rho mutants defective in the ability to activate Gt have been identified. Several of these mutant receptors were studied by flash photolysis [9], light-scattering [10], or proton-uptake assays [11]. The key overall result of these studies is that C2, C3, and H8 are involved in the R*-Gt interaction.

H8 is a cationic amphipathic helix that may bind a phos-pholipid molecule, especially a negatively charged phospho-lipid such as phosphatidylserine. In fact, spectroscopic evidence has been reported to show an interaction between Rho and a lipid molecule that is altered in the transition of Rho to metarhodopsin II, the spectrally defined form of R* [12]. H8 points away from the center of Rho, and the area of the membrane surface covered by the entire cytoplasmic surface domain appears to be roughly large enough to accommodate Gt in a one-to-one complex.

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