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      Structure of the human M 2 muscarinic acetylcholine receptor bound to an antagonist

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          The parasympathetic limb of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G protein coupled receptors (GPCRs) that mediate the response to acetylcholine released from parasympathetic nerves. 15 Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M 2 muscarinic acetylcholine receptor (M 2 receptor) is essential for the physiologic control of cardiovascular function through activation of G protein-coupled inwardly-rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of antagonist-bound M 2 receptor, the first human acetylcholine receptor to be characterized structurally. The antagonist QNB binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all 5 muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The M 2 receptor structure provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

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          Most cited references 34

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          Crystal structure of rhodopsin: A G protein-coupled receptor.

          Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.
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            Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.

            Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein-coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.
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              GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.

              The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

                Author and article information

                29 December 2011
                25 January 2012
                23 August 2012
                : 482
                : 7386
                : 547-551
                [1 ]Department of Life Science, Faculty of Science, Gakushuin University, Mejiro 1-5-1, Tokyo 171-8588, Japan
                [2 ]Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 CampusDrive, Stanford, California 94305, USA
                [3 ]Department of Medical Chemistry and Cell Biology, Kyoto University Faculty of Medicine, Konoe-cho, Yoshdida, Sakyo-Ku, Kyoto 606-8501, Japan
                [4 ]Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Konoe-cho, Yoshida, Sakyo-Ku, Kyoto 606-8501, Japan
                [5 ]Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
                [6 ]Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, California 94305, USA
                Author notes
                Correspondence and requests for materials should be addressed to B.K.K. ( kobilka@ ), T.H. ( tatsuya.haga@ ), or T.K. ( t-coba@ )

                These authors contributed equally to this work.


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                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R37 NS028471-21 || NS



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