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High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor

Vadim Cherezov et al.

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The largest family of integral membrane proteins coded by the human genome comprises G protein–coupled receptors (GPCRs), with almost 1000 members (1, 2). These receptors communicate signals across cell membranes in response to an astonishing variety of extracellular stimuli—light, proteins, peptides, small molecules, hormones, and ions. Once activated, GPCRs trigger a cascade of responses inside the cell, primarily through interactions with their G protein partners, three-subunit regulators that are switched on and off by binding guanosine triphosphate (GTP) (thus accounting for their name). In addition, these receptors have been found to activate other, G protein–independent, signaling pathways. Their combined effects yield an amazingly diverse network of signals that must be exquisitely coordinated to ensure proper cellular function (3, 4).

Although drugs that act on GPCRs command more than 50% of the current market for human therapeutics, with annual revenues in excess of $40 billion, these drugs interact with only a fraction of the available receptors. Because of the importance of this protein family, there is an ongoing search for new drugs that act on GPCRs and that combine potent efficacy with high specificity. Of particular interest are the class A adrenergic receptors that respond to the hormones adrenaline and noradrenaline. These are the targets of current cardiac and asthma drugs that often have undesirable side effects. In addition, improved asthma drugs are needed in developing countries where the population and pollution levels are rapidly rising, along with the incidence of asthma. Structures of GPCRs can guide the development of more specific drugs and can be combined with traditional chemical screening methods to improve and accelerate drug discovery.

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Structure of the human β2-adrenergic receptor (blue) embedded in a lipid membrane and bound to a diffusible ligand (green), with cholesterol and palmitic acid (orange) between the two receptor molecules.

For protein structures to effectively guide drug design, it is critically important to maximize the available detail, as low-resolution structures can be ambiguous at best and misleading at worst. However, it remains a formidable challenge to obtain high-resolution structural data for membrane proteins. To accomplish this, we engineered the β2-adrenergic receptor to include lysozyme in place of one of the intracellular loops, which reduced conformational heterogeneity and facilitated crystal nucleation [see (5)]. Crystals were grown in a cholesterol-doped lipidic cubic phase that stabilized the receptor in a more natural membrane-like environment. We used a robot to set up more than 15,000 trials to optimize crystal growth in the extremely viscous lipidic cubic mesophase. We then evaluated the micrometer-size transparent crystals with a 10-μm x-ray beam. Our resulting 2.4 Å crystal structure of the human β2-adrenergic receptor successfully provides high-resolution detail.

The crystal structure of this important human membrane receptor reveals the details of its interactions with a diffusible ligand (the partial inverse agonist carazolol). In examining the structure, one can begin to appreciate the amazing structural plasticity of the GPCRs and how this allows them to recognize such a wide range of ligands critical for function within the human body. The ligand-binding site of the β2-adrenergic receptor is located in a position similar to that of the covalently bound ligand of rhodopsin, the light-absorbing, G protein–coupled receptor responsible for human vision. Key differences from rhodopsin are also observed, particularly in several of the kinked transmembrane helices and in the second extracellular loop, which in the β2-adrenergic receptor contains an unusual pair of disulfide bonds and an extra helix. This loop and the absence of structure in the N-terminal region of the receptor may be important for ligand binding.

Although this structure of a GPCR that recognizes a diffusible ligand furthers understanding of signal transduction and should facilitate the design of new drugs with fewer side effects, the structure alone cannot fully explain how ligand binding on the outside surface of a cell triggers internal signaling pathways. This will require characterization of how the receptor changes its conformation as it is activated. Follow-up structures or receptors bound to other ligands will be required to understand the different conformational states and how they transduce signals. It is possible that the active state will only be understood when a structure is obtained for a GPCR–G protein signaling complex with an agonist bound to the receptor. In addition, structural and complementary biophysical techniques (e.g., nuclear magnetic resonance) will help to resolve other key biological questions, including the effects of homodimerization or heterodimerization of the receptor, the nature of class B and C GPCR structures, and elucidation of cholesterol’s role in GPCR function.

Summary References

  1. S. Takeda, S. Kadowaki, T. Haga, H. Takaesu, S. Mitaku, FEBS Lett.520, 97 (2002).
  2. R. Fredriksson, M. C. Lagerstrom, L. G. Lundin, H. B. Schioth, Mol. Pharmacol.63, 1256 (2003).
  3. K. L. Pierce, R. T. Premont, R. J. Lefkowitz, Nat. Rev. Mol. Cell Biol.3, 639 (2002).
  4. R. J. Lefkowitz, S. K. Shenoy, Science308, 512 (2005).
  5. D. M. Rosenbaum et al., Science318, 1266 (2007); published online 25 October 2007 (10.1126/science.1150609).

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