B2AR Laid Bare

β2AR at 2.4 Å resolution Credit: Courtesy of Raymond Stevens With roughly 1,000 different members, G-protein-coupled receptors (GPCRs) are the largest family of proteins coded by the human genome. Involved in sensing a panoply of physiological and environmental signals, including hormones, neurotransmitters, odorants, tastes and light, GPCRs are the targets of a pharmacopeia of drugs, from beta blockers to antihistamines. Until recently, however, high-resolution crystal structures were known for only the light receptor rhodopsin. In 2007, Brian Kobilka, a biochemist at Stanford University, and Raymond Stevens, a chemist at Scripps Research Institute, cracked the structure of β2-adrenergic receptor (β2AR), a receptor of adrenaline involved in cardiovascular and pulmonary function. After slogging away at the membrane molecule for close to two decades, Kobilka and Stevens had "a parallel and complementary series of technological breakthroughs," recalls Stevens. Kobilka and colleagues stabilized β2AR by tacking on an antibody fragment. In parallel, he also replaced one of the receptor's flexible intracellular loops with a small, stable protein called the T4 lysozyme, and teamed up with Stevens to apply newly-developed biophysical techniques and lipid-based crystallography methods to solve the structure at 2.4 Å resolution. "We didn't know which [method] was going to work, so we tried both," says Kobilka. "And they ended up both working at about the same time." In two Science papers 1,2 - this month's Hot Papers - and one other paper in Nature, 3 researchers showed that β2AR and rhodopsin have significantly different extracellular loop configurations, which explained differences in ligand binding between rhodopsin and all other non-light sensing GPCRs. The three-dimensional β2AR structure should pave the way for structure-based drug discovery, says Michel Bouvier of the University of Montreal. "We can probably now start designing compounds that will have the exact characteristics we want for a drug." A streak of structures The β2AR structure was not the last stop for GPCR research, notes Ad IJzerman, from the Leiden/Amsterdam Center for Drug Research. "We've got two down and 998 to go," he says. Since the Hot Papers were published, researchers have worked out the architecture of several other GPCRs, including the closely related β1AR, a heart rate and blood pressure regulator. Structural biologist Gebhard Schertler of the UK Medical Research Council's Laboratory of Molecular Biology in Cambridge successfully crystallized β1AR last June after using site-directed mutagenesis to make the receptor more thermostable. 4 The β1AR and β2AR structures were remarkably similar, with identical amino acid residues in their primary ligand-binding sites. But Schertler found subtle differences in the funnel-like entrance to the binding pocket, which he hopes can be exploited to design better drugs. Currently, inhalers that stimulate β2AR to ease lung tension during asthma attacks also activate β1AR, which causes the heart to beat faster if the dose is not controlled carefully, he notes. "If you want to work out selectivity between drugs then you want to know the differences between very similar receptors," Schertler says. Rhodopsin and both adrenergic receptors were crystallized in complex with inactivating ligands. But to fully understand GPCR dynamics, structures of receptors in complex with their G-protein activators are needed, says Kobilka. Last summer, Oliver Ernst, a biophysicist at the Charité University Hospital in Berlin, obtained two structures of opsin, a protein component of rhodopsin: one ligand-free, and the other in complex with a small, synthetic peptide derived from a G-protein sub-unit that stabilized opsin in an active state. 5 Both structures can now be used as templates for modeling active GPCRs, which should make computational drug screening easier, says Ernst. Minding the binding Stevens, together with IJzerman, worked out the next GPCR structure late last year. Using the same T4 lysozyme replacement technique that worked for the β2AR, Stevens and his colleagues obtained a close-up picture of the A2A adenosine receptor, a receptor that plays a role in a host of physiological processes and is blocked by caffeine. 6 The A2A adenosine receptor's binding pocket was distinct from that of the rhodopsin and the adrenergic receptors, indicating that each GPCR may have a unique ligand binding location and orientation. The GPCR structures rolled out over the past two years were "really the climax of decades of work," says IJzerman. "At the moment," adds Schertler, "we are really getting a molecular picture of [GPCR] receptors, and we see the beginnings of a truly molecular pharmacology." For example, Bouvier and his graduate student Martin Audet modeled the binding of nine β2AR ligands - including agonists, inverse agonists, and neutral antagonists for different pathways - and showed that the way the ligands docked in the receptor's crystal structure correlated with their distinct activation roles. 7 But there's plenty of work remaining before structure-based drug discovery can be fully realized, says Kobilka. "Crystallography only gives us a snapshot of a single conformational state. To really understand how these proteins work, we'll need other information about their dynamics," which can be gleaned from other biophysical techniques, such as nuclear magnetic resonance spectroscopy, and by crystallizing receptors in their active states. "It's just the beginning," he says. Data derived from the Science Watch/Hot Papers database and the Web of Science (Thomson ISI) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. V. Cherezov et al., "High-resolution crystal structure of an engineered human b2-adrenergic G protein-coupled receptor," Science, 318:1258-65, 2007. (Cited in 177 papers) D.M. Rosenbaum et al., "GPCR engineering yields high-resolution structural insights into b2-adrenergic receptor function," Science, 318:1266-73, 2007. (Cited in 90 papers) References 1. V. Cherezov et al., "High-resolution crystal structure of an engineered human b2-adrenergic G protein-coupled receptor," Science, 318:1258-65, 2007. (cited in 177 papers) 2. D.M. Rosenbaum et al., "GPCR engineering yields high-resolution structural insights into b2-adrenergic receptor function," Science, 318:1266-73, 2007. (cited in 90 papers) 3. S.G. Rasmussen et al., "Crystal structure of the b2 adrenergic G-protein-coupled receptor," Nature, 450:383-7, 2007. 4. T. Warne et al., "Structure of a b1-adrenergic G-protein-coupled receptor," Nature, 24:486-91, 2008. 5. P. Scheerer et al., "Crystal structure of opsin in its G-protein-interacting conformation," Nature, 455:497-502, 2008. 6. V.-P. Jaakola et al., "The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist," Science, 322:1211-7, 2008. 7. M. Audet and M. Bouvier, "Insights into signaling from the b2-adrenergic receptor structure," Nat Chem Biol, 4:397-403, 2008.

B2AR Laid Bare

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Elie Dolgin

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February 2009

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