The Overall Topology of the 7TM Region

Electron microscopy experiments [32] with a resolution of 7 Â first showed that the transmembrane region of bacteriorhodopsin was, to a large extent, comprised of a-helices that are approximately perpendicular to the plane of the membrane. Later electron microscopy and electron diffraction efforts [33] with a resolution of about 6.5 Â demonstrated that bacteriorhodopsin was comprised of seven rod-shaped features (i.e., a-helices) with a possible linking of adjacent rods by polypeptide chains, which were later recognized to be alternating intracellular and extracellular loops. Electron cryomicroscopy experiments [34] showed that bovine rhodopsin structure also adopts a 7TM a - helical configuration. However, unlike the nearly parallel a - helical arrangement in the bacteriorhodopsin structure, only four a -helices in bovine rhodop-sin are nearly perpendicular to the plane of the membrane, with an unexpected tilting of one of the four helices; the other three helices are more highly tilted. Moreover, the transmembrane helices are arranged differently in bacteriorho-dopsin and bovine rhodopsin.

The determination of a low-resolution [35] and, later, a higher-resolution [ 36] bacteriorhodopsin X- ray structure motivated their use in drug design [6, 37] as templates to model GPCRs of interest. To refine the bacteriorhodop-sin- based models, information derived from site- directed mutagenesis data, affinity labeling, and structure-activity relationships have been utilized. Examples of the use of bacteriorhodopsin as a drug-design template have been reviewed [12,38-41].

The first X-ray diffraction structure of a GPCR, namely, bovine rhodopsin at 2.8-Â resolution, became available in 2000. Subsequently, as enumerated in Table 16.1 , an additional 17 (bovine and squid) (rhod)opsin X-ray structures were elucidated. Especially with respect to the transmembrane region, the 18 (rhod)opsin X-ray structures are very superimposable, and the covalently bound chromophore, retinal (when present), varies minimally in position.

In the development of homology models, the X-ray structures of bovine rhodopsin were expected to provide a significant advantage relative to the bacteriorhodopsin X-ray structures, because bacteriorhodopsin is not a GPCR and bovine rhodopsin has at least a marginal increase in homology to GPCRs of interest for drug discovery efforts. In addition, the a-helices of the X-ray structures of bovine rhodopsin and bacteriorhodopsin differ significantly [40] in their positions, orientations, and packing -39, 42, 43] , Moreover, a-helix kinks appear in bovine rhodopsin, but the a-helices are more regularly shaped in bacteriorhodopsin.

Despite low sequence identity and the presence of a different chromo-phore/ligand when comparing bovine rhodopsin to the human p2AR, the turkey p1AR, and the human A2a adenosine receptor, their overall topology is quite similar in the transmembrane region, as observed in X-ray structures (see Table 16.1). However, one notices changes in the tertiary structure and the positions of helices I, III, IV, V, and VI (e.g., when comparing the p 2 receptor and rhodopsin) [21-23].

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