The ICL3 Region and Activation

Up until recently, the many X-ray structures of rhodopsin and the ligand-mediated GPCRs had not revealed very much direct information on GPCR activation. In contrast, recent publications [31, 49-52] now provide tangible details of the GPCR activation mechanism. Two X-ray structures of invertebrate squid rhodopsin, which couples to Gq, were published [49, 50] in May and June of 2008, and demonstrate noteworthy features in the intracellular region of helices 5 and 6, and their ICL3 tether, which is 12 residues longer than bovine rhodopsin's ICL3. On the intracellular side, H5 and H6 are longer and rigid, relative to their bovine rhodopsin counterparts, and extend far from

Figure 16.3 View near the ICL3 of bovine rhodopsin (brown) and of bovine opsin (yellow) bound to Ga-CT (pink with its surface).

the core. In one X-ray structure [50], H5 and H6 comprise one boundary of a binding region for an octylglucoside in close proximity. The authors speculated that the region around the extended helices and the ICL3 tether may be involved in binding the G protein.

Within the next few months, two publications [31, 51] on bovine opsin X-ray structures, both of which are in the active conformation, appeared in July and September. The second structure provides direct support of the active state in that it is complexed with an 11-amino acid peptide, Ga-CT, derived from the C-terminus of the transducin Gat protein (ILENLKDCGLF, Gat [(340-350) K341L]) (see Fig. 16.3).

Relative to the earlier bovine rhodopsin structures with which it shares overall topological similarity, the opsin X-ray structure demonstrates changes mainly in the intracellular region. For example, there is a short helical turn in ICL1. More importantly, because the N-terminal region of opsin's ICL3 adopts an a- helical structure, the intracellular side of H5 is elongated by 1.5-2.5 helical turns relative to the rhodopsin X-ray structures (see Fig. 16.3). Also, H5 is tilted toward the other helices of the 7TM core. As expected from earlier experimental evidence [53-55], the intracellular side of H6 is tilted by 6-7 A away from the 7TM helical bundle, such that H5 and H6 are almost parallel and protrude from the intracellular region.

The highly conserved E(D)R135Y and NPxxY306(x)s,6F motifs help effect the stabilization of H5 and H6 in their intracellular positions within opsin. In rhodopsin, Arg135 of the E(D)RY motif within H3 interacts with Glu247 of H6. However, in opsin, this salt bridge is broken, H6 moves away from H3 so that H6 is further from the center of the helical core but approaches H5, H5

approaches H3 in a direction toward the helical bundle's center, and H5 and H6 are stabilized in their rearranged positions by interactions involving (1) Lys231 of H5 and Glu247 of H6, and (2) Tyr223 of H5 and Arg135 of H3. In opsin, Tyr306 of the NPxxY306(x)5j6F motif within H7 blocks the return of H6 to its position in rhodopsin by this residue's rotation into the helical core.

Stabilized by Tyr223, Arg135 is also involved in the binding of the Ga-CT peptide through hydrogen-bond formation involving the backbone carbonyl oxygen atom of Cys347 of Ga - CT. Ga- CT occupies a crevice bounded by Arg135, lipophilic residues of H5 and H6, and a hydrogen-bonding network involving H7 and H8. Conformational changes in the H7-H8 kink, which moves away from the helical core, and the rearrangement of H5 and H6 are required for Ga - CT binding - 50] , Detailed structural analysis in the ICL3 region would benefit from comparisons with the ligand-mediated X-ray structures. However, the full, native ICL3 loop is not contained in any of the ligand-mediated X - ray structures.

Relative to its position in the inactive state in rhodopsin, Trp265 of the "loggle switch" is shifted in opsin but does not display the expected rota-meric change; however, the lack of the rotameric change may be due to an empty binding pocket. In the Ga - CT- free opsin X - ray structure, electron density was not observed for Lys296, which is covalently bound to retinal in rhodopsin. However, electron density is observed in the opsin/Ga-CT complex, demonstrating that the binding of Ga-CT affects the chromophore-binding site. The authors suggest [51] that this observation supports the idea that the chromophore-binding site and the Ga-CT binding cleft are coupled during activation.

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