104
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Activation and allosteric modulation of a muscarinic acetylcholine receptor

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β 2 adrenergic receptor (β 2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M 2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β 2AR and rhodopsin. We also report the structure of the M 2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.

          Related collections

          Most cited references43

          • Record: found
          • Abstract: found
          • Article: not found

          The dynamic process of β(2)-adrenergic receptor activation.

          G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins. Copyright © 2013 Elsevier Inc. All rights reserved.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Crystallizing membrane proteins using lipidic mesophases.

            A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Its most recent successes are the human-engineered beta(2)-adrenergic and adenosine A(2A) G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase and for setting up crystallizations in manual mode. Methods for harvesting microcrystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about 1 h.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Crystal structure of opsin in its G-protein-interacting conformation.

              Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.
                Bookmark

                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                2 April 2014
                20 November 2013
                5 December 2013
                05 June 2014
                : 504
                : 7478
                : 101-106
                Affiliations
                [1 ]Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305 USA
                [2 ]Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305 USA
                [3 ]Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA
                [4 ]Department of Chemistry and Pharmacy, Friedrich Alexander University, Schuhstrasse 19, 91052 Erlangen, Germany
                [5 ]Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Structural Biology Research Centre, VIB, Pleinlaan 2, B-1050 Brussels, Belgium
                [6 ]Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia
                [7 ]Neuroscience, Eli Lilly & Co., Indianapolis, IN 46285
                Author notes
                Correspondence and requests for materials should be addressed to B.K.K. ( kobilka@ 123456stanford.edu )
                [*]

                These authors contributed equally to this work.

                Article
                NIHMS529325
                10.1038/nature12735
                4020789
                24256733
                07d6e1e1-afe3-4c32-8f84-74a64b13b5fa

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Categories
                Article

                Uncategorized
                Uncategorized

                Comments

                Comment on this article