The Binding Site

In X-ray structures of bovine or squid rhodopsin (see Table 16.1 and Fig. 16.1a,b), retinal is covalently bound and tightly enclosed in a mainly lipophilic binding pocket in the inactive state. In addition, the bovine rhodopsin binding site is blocked by the ECL2, which helps completely enclose retinal by folding into the receptor (see Fig. 16.1a,b). The topology of the rhodopsin binding site raises questions about whether rhodopsin X-ray structures can serve as reliable templates to model other GPCRs of interest. Are gross changes to the position, orientation, and kinking of a - helices necessary to accommodate ligands of different sizes, or are the requisite conformational changes (e.g., in rotational states of the residues) mainly localized to the binding site? Also, how would one expand rhodopsin's tight binding cavity to create a more reliable template from which to construct a homology model, whether for antagonists or agonists? For example, if one used an inactive-state bovine rhodopsin X-ray structure as a template, the construction of a reliable active-state homol-ogy model would be very difficult and may involve attempts to expand the binding site and rearrange the 7TM a-helices; these conformational changes are difficult to predict.

Figure 16.1 (a,b) Two orthogonal views of an overlay of the ECL2 region of PDB entries 1U19 (bovine rhodopsin in brown with retinal), 2RH1 (the human p2AR in pink with carazolol), and 3EML (the human A2a adenosine receptor in green with ZM241385); (c) the "toggle switch" region in the same proteins as in (a) and (b) viewed from inside the core toward the EC side. Note that the residues corresponding to Phe290 in the human p2AR are Ala in rhodopsin and His in A2a. All components of Figs. 16.1, 16.2, 16.3, and 16.5 were created in Maestro [94].

Figure 16.1 (a,b) Two orthogonal views of an overlay of the ECL2 region of PDB entries 1U19 (bovine rhodopsin in brown with retinal), 2RH1 (the human p2AR in pink with carazolol), and 3EML (the human A2a adenosine receptor in green with ZM241385); (c) the "toggle switch" region in the same proteins as in (a) and (b) viewed from inside the core toward the EC side. Note that the residues corresponding to Phe290 in the human p2AR are Ala in rhodopsin and His in A2a. All components of Figs. 16.1, 16.2, 16.3, and 16.5 were created in Maestro [94].

The binding sites in the X-ray structures of the human p2AR and the turkey p1AR are more open than in the rhodopsin X-ray structures, due to changes in the ECL2 architecture (see below) and a kinking of helix I toward the binding pocket in rhodopsin. Some of the transmembrane helices of the A.a adenosine receptor X- ray structure are shifted or kinked relative to either rhodopsin or the adrenergic receptors. The binding of the antagonist ZM241385 in the A2a adenosine receptor X-ray structure [29] is very different from that of retinal or the adrenergic receptor ligands (see Fig. 16.1a,b). Relative to the binding of retinal or the adrenergic receptor ligands, ZM241385 is positioned closer to the extracellular region and shifted toward helices VI and VII, providing very little overlap with the other X-ray ligands. In the A2 a adenosine receptor X-ray structure, the binding cavity is easily accessible to ligands. The observed differences in binding mode and ligand orientation in the A2a adenos-ine receptor X-ray structure may indicate that selectivity differences among other GPCRs may be due to receptor plasticity, rather than being derived from the variation of amino acids on a nearly conformationally invariant backbone. These binding-site differences suggest severe challenges in the use of X-ray structures as homology modeling templates for remotely related GPCRs (also see Reference 22). An excellent illustration of this concern are recently published studies [44] demonstrating that the binding mode of ZM241385 is not accurately predicted when X-ray structures of remotely related GPCRs, the p2AR and bovine rhodopsin, are used as templates for homology models of the A2a adenosine receptor. In the A2a homology model based on the highresolution carazolol p2 X-ray structure, the binding of ZM241385 is similar to that of carazolol in its respective X-ray structure. The bovine rhodopsin-based model produces a similar discrepancy.

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