Stabilization of XRay Structures

Due to its high abundance in bovine retina [ 19] and the limitations in the purification and crystallization of other GPCR proteins [20], bovine rhodopsin, until recently, was the only GPCR for which a three-dimensional (3D) structure was determined. Creating challenges to crystallization, other GPCR proteins are found in low concentrations, are conformationally flexible, and are unstable in detergent solutions. Since the bovine rhodopsin X-ray determinations, the first GPCR structures published were those of the p2-adrenoreceptor (Protein Data Bank [PDB] accession IDs 2R4R, 2R4S, and 2RH1) [21-23] complexed with the picomolar-affinity inverse agonist carazolol (2RH1). These p2-adrenoreceptor X-ray structures, which are also the first published ligand-

mediated GPCR X-ray structures, were solved by two different approaches, both of which utilized a companion protein to stabilize the flexible intracellular loop 3 (ICL3). The authors used a number of other techniques, including ligand-affinity chromatography and embedding in a lipid cubic phase, to obtain crystallizable protein [24-26].

In the first p2-adrenoreceptor X-ray determinations [21], which resulted in two 3.4-A resolution structures with a poorly resolved active site, a p2AR-Fab5 complex helped stabilize the two almost identical proteins and facilitate crystallization. In an attempt to improve the packing of the extracellular domains and thereby improve the electron density, the authors inserted a TEV (tobacco etch virus) cleavage site after the 24th amino acid of the N-terminus; however, this effort did not lead to a significant crystallographic result. In the second approach [22, 23] , the authors replaced the flexible ICL3 loop, which may contribute to unrestricted helical movements, with residues from T4 lyso-zyme (T4L), and the fusion helped stabilize and crystallize the protein. T4L adds a polar surface that is important for forming crystal lattice contacts and restricting helical motions. To simulate a membrane-like environment and thus help stabilize the protein, the authors grew the crystals in a cholesterol-doped lipidic cubic phase. These innovative efforts were rewarded by a 2.4-A resolution structure of p2AR-T4L complexed with the partial inverse agonist carazolol. Because the authors observed only minor differences when they compared the p2AR-T4L X-ray structure with the lower-resolution wild-type p2AR-Fab5 X-ray complex, they concluded that the p2AR-T4L X-ray structure, except for ICL3, is probably sufficiently similar in conformation to that of the native protein. A second high- resolution p2AR X - ray structure was reported [27] , this time in complex with the partial inverse agonist timolol. Splicing of T4L into the ICL3 region was again successful, without significant changes to the overall structure (relative to that observed in the first p2AR-T4L X- r ay determination). Consistent with the introduction of a different ligand, only small conformational changes in the binding site were observed. The crystal packing is different in the two high-resolution p2AR structures, in that the p2AR/timolol complex contains two cholesterol binding sites that are not involved in crystal packing. The cholesterol binding sites may be involved in cholesterol-mediated thermal stabilization, allosteric modulation of the high - affinity agonist binding state, and receptor trafficking (refer to Reference 27 and references therein).

An X-ray structure of a protein that is closely related to p2AR, the turkey p1 receptor complexed with the antagonist cyanopindolol, was published recently I 28] , Employing a strategy to identify a combination of mutations in p1AR that result in thermal stability, the authors found a set of six point mutations that sufficiently stabilized the complex without the introduction of a companion (Fab5 or T4L) protein. It should be noted that residues in ICL3 and the C-terminus were also deleted. The mutated protein was stable in many detergents used for crystallization and remained in an antagonist conformation even when the ligand was no longer present. Interestingly, the high - resolution p1AR and p2AR X-ray structures are very similar, indicating an absence of significant artifacts introduced by the use of a companion protein in the two approaches used for the p2AR structures. The p1AR and p2AR ligand-binding cavities are very similar, showing differences of the scale expected for different complexed ligands, with small conformational variations in the binding-site residues. The source of p1-versus-p2 selectivity is not obvious, because the amino acid residues in the vicinity of the ligand differ by very little.

The reported ligand-mediated GPCR X-ray structures described above are the monoaminergic amine systems of p1AR and p2AR. A recently published X-ray structure is now available for a more distantly related Class A GPCR, the A2a adenosine receptor in complex with the high-affinity antagonist ZM241385 [29] [ The issue of thermal instability of this GPCR was addressed mainly by the T4L fusion approach, in which the ICL3 segment of Leu209 to Ala221 was replaced with T4L. Also, to improve the likelihood of crystallization, the C-terminus of the receptor, from Ala317 to Ser412, was deleted. The ZM241385 antagonist binding in this complex is markedly different from the binding of ligands in the p1AR and p2AR structures (vide infra).

Motivated by an earlier approach developed [30] for the selective extraction of rhodopsin, Park et al. [31] selectively extracted bovine opsin from rod cell disc membranes, thereby obtaining crystals of opsin in its native state at 2.9-Â resolution, without further purification steps and without protein structural modifications.

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