A

Figure 3. Charge distribution and dimensions of rhodopsin. The space-filling model shows an ellipsoidal shape of rhodopsin, 75 A long and perpendicular to the membrane, while the transmembrane domain is 41 A high. The length and width of the elliptic footprint on the plane at the middle of the membrane are roughly 45 and 37 A, respectively [23], The position of the chromophore is indicated by a plane. Negative charges are in red, positive charges are in blue.

C-terminus of the Gta-subunit and plays a role in the regulation of Gty binding [27].

Rhodopsin's C-terminal penta-peptide region (residues 344-348), GVAPA, is essential for the translocation of newly synthesized rhodopsin molecules from the inner to the outer segment of the rod photoreceptor cell [28]. In GPCRs, inactivation of the activated receptor is frequently achieved by phosphorylation of the C-terminal Ser and Thr residues and subsequent binding of arrestin. The major phosphorylation sites on rhodopsin are Ser338, Ser343 and Ser334 [29-31]. Although the structure of the C-terminus is poorly determined, H-bond interactions between this region and parts of the third cytoplasmic loop are conceivable. Photoactivation of rhodopsin would break these interactions, allowing binding of RK and phosphorylation of the hydroxyl groups [32],

The members of the rhodopsin subfamily, making up ~ 90% of all known GPCRs, share many key structural features (Figures 2 and 4), such as a disulfide bond between helix III and the extracellular region and a tripeptide sequence D(E)RY(W) located at the intracellular end of helix III. These

C-tail

Figure 4. Crystal structure of bovine rhodopsin. The seven transmembrane helices are labeled I to VII, cytoplasmic and the extracellular loops as C-I, -II, -III and C-tail, and E-I, -II, -III and N-tail. A short cytoplasmic helix (VIII), corresponding to Lys311-Cys322, is found between H-VII and the C-tail and runs parallel to the membrane surface. Two palmitoyl groups are attached to Cys322 and Cys323 and anchor the C-tail to the membrane. Carbohydrate chains are oriented toward the intradiskal (extracellular) face of rhodopsin, and are attached to Asn2 and Asn15.

Figure 4. Crystal structure of bovine rhodopsin. The seven transmembrane helices are labeled I to VII, cytoplasmic and the extracellular loops as C-I, -II, -III and C-tail, and E-I, -II, -III and N-tail. A short cytoplasmic helix (VIII), corresponding to Lys311-Cys322, is found between H-VII and the C-tail and runs parallel to the membrane surface. Two palmitoyl groups are attached to Cys322 and Cys323 and anchor the C-tail to the membrane. Carbohydrate chains are oriented toward the intradiskal (extracellular) face of rhodopsin, and are attached to Asn2 and Asn15.

residues are critical for proper protein folding and G-protein activation, respectively. There are also several other highly conserved residues with a frequency of occurrence >90% that define the rhodopsin family, such as an Asn-Asp pair located in helix I and helix II, respectively, Pro residues in helices V and VI, aromatic residues in helices IV and VI, and the NPxxY motif in helix VII. The crystal structure of bovine rhodopsin offers the most reliable model for these conserved features (Figures 2 and 4), for all members of this sub-family of GPCRs.

The area of rhodopsin projected into the membrane plane is <1500 A2. Inclusion of the cytoplasmic helix VIII elongates the cytoplasmic region, resulting in an area that is sufficiently large to dock a single trimeric Gt holoprotein on the surface. Receptor dimerization appears to be important for the function of GPCRs [33]. However, for rhodopsin, a lack of dimerization upon activation was concluded from the absence of light-induced changes of the diffusional speed within functional rods [34]. From spectroscopic measurements, monitoring the binding of Gt to R*, a 1:1 stoichiometry was determined [35] (see below); however, the dimer between two rhodopsin molecules was observed in the crystals of rhodopsin (Figure 5). These dimers are stabilized by hydrophobic interactions (Figure 5A) and by the dipoledipole interaction (Figure 5B). Such dimerization in detergent implies that, in vivo, there would be repulsion rather than association of two or more rhodopsin molecules, unless phospholipids play an important role in compensating for this repulsion.

3.3.2 The inactive ground state

Vertebrate rhodopsin, like other visual pigments, contains 11-m-retinal as a chromophore covalently bound to the e-amino group of a lysine side chain via a Schiff base linkage. By its spectral properties, the Schiff base is likely to be protonated, but a protonated Schiff base of 11-m-retinal formed from

Figure 5. Rhodopsin dimer in the asymmetric crystal unit. (A) The asymmetric unit of the rhodopsin crystals contains two rhodopsin molecules [22,23], Helices are shown as red rods, P-strands are shown as blue arrows. (B) The dimer of rhodopsins within the asymmetric unit is held together by hydrophobic interactions and by dipole interactions between monomers. Red and blue represent negative and positive electric fields of the protein dimer.

Figure 5. Rhodopsin dimer in the asymmetric crystal unit. (A) The asymmetric unit of the rhodopsin crystals contains two rhodopsin molecules [22,23], Helices are shown as red rods, P-strands are shown as blue arrows. (B) The dimer of rhodopsins within the asymmetric unit is held together by hydrophobic interactions and by dipole interactions between monomers. Red and blue represent negative and positive electric fields of the protein dimer.

n-butylamine in free methanol solution absorbs at 440 nm. Thus, it was recognized early that charged groups on opsin are required to tune the position of the spectral maximum of bound 11 -cz's-retinal (see [36]). The shift from 440 nm to that found in rhodopsin (500 nm) or color pigments is due to the interaction with the protein environment ("opsin shift") (see [36]). The environment of the chromophore is depicted in Figure 6. Important for spectral tuning is the position Glu181, a residue in the (3-hairpin plug structure brought into proximity of carbon C12 of the polyene chain of retinal due to the Cys110-Cys187 disulfide bridge. In red and green color pigments Glu181 is replaced by a

Figure 6. Chromophore-binding site of bovine rhodopsin. Schematic of the side chains surrounding the 11-c/s-retinylidene group (drawn in orange), viewed upside down from within the disk membrane. The side chains are drawn as ball and sticks and colored by elements. Retinal forms a protonated Schiff base with Lys296. Glu113 serves as counter-ion and forms a salt bridge with the protonated Schiff base. The P-ionone ring is mainly kept in place by residues Trp265, Phe261 (both from helix VI) and Glu122

(from helix III).

Figure 6. Chromophore-binding site of bovine rhodopsin. Schematic of the side chains surrounding the 11-c/s-retinylidene group (drawn in orange), viewed upside down from within the disk membrane. The side chains are drawn as ball and sticks and colored by elements. Retinal forms a protonated Schiff base with Lys296. Glu113 serves as counter-ion and forms a salt bridge with the protonated Schiff base. The P-ionone ring is mainly kept in place by residues Trp265, Phe261 (both from helix VI) and Glu122

(from helix III).

histidine residue which, in combination with a chloride anion, is likely to be involved in the spectral tuning of these pigments (see [74]). Position Glu122 was implicated in regulating the rate of decay of the active species and the rate of regeneration [37].

The crystal structure of ground state bovine rhodopsin with bound inactivating 11-cw-retinal represents a template for GPCRs in their least active conformation. Compared to light-activated rhodopsin, the basal activity of free opsin is less than 1/106 [38]. An important element in keeping the receptor in its inactive conformation is a salt bridge between Lys296, the retinal attachment site in helix VII and its counter-ion, Glu113 in helix III, which is neutralized in active forms of rhodopsin [39]. Removal of the charge at either position, Lys296 or Glu113, leads to increased basal activity of opsin, referred to as constitutive activity. Corresponding salt bridges between helix III and helix VII were shown to be critical for other GPCRs (see, for example, [40]). In rhodopsin, the salt bridge is formed between the protonated Schiff base nitrogen of Lys296 and Glu113. The counter-ion increases the Ka of the Schiff base by several orders of magnitude (reviewed in [41]) and prevents its spontaneous hydrolysis. Binding of 1 \-cis-retinal to opsin, however, reduces the basal activity of free opsin further by a factor of 104 [38], classifying this retinal isomer as an inverse agonist. This reaction is exothermic (AH = -11 kcal moF1, [42]) and is used to build up inactivating structural constraints seen in the ground state structure. This is reflected in the lowest crystallographic temperature factors of the structure in the region around the retinal Schiff base. The structure also revealed several H-bonded networks and hydrophobic interactions which connect neighboring helices, stabilizing the ground state (for details see [22,23,43]). Helix VII, which carries the retinal, shows most connections and interacts with all helices except helix IV and V. In contrast, helix VI is only connected to helix VII via hydrophobic interactions, which is what may allow light-induced helix movement, as mentioned earlier. Which of these interactions are caused by the presence of the 11-c/s-retinal and which structural constraints are broken or change upon light-activation of rhodopsin are largely unknown.

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