title: The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.
abstract: The β1-adrenoceptor (β1AR) is a G protein-coupled receptor (GPCR) that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable β1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 μm plates) were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between β1AR and the adenosine A2A receptor (rmsd of 0.3 Å), despite an overall rmsd of 2.4 Å for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to β1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of β1AR by 5-10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of β1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Miller-Gallacher
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Rony
authors.initials: R
authors.lastname: Nehmé
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Tony
authors.initials: T
authors.lastname: Warne
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Edwards
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Gebhard F X
authors.initials: GF
authors.lastname: Schertler
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Andrew G W
authors.initials: AG
authors.lastname: Leslie
authors.affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Tate
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D010869:Pindolol / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability / Q000187:drug effects; D043705:Receptor, Adenosine A2A / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*; D013696:Temperature; D014422:Turkeys
chemical_list: D000319:Adrenergic beta-Antagonists; D043705:Receptor, Adenosine A2A; D018342:Receptors, Adrenergic, beta-1; C047975:cyanopindolol; D012964:Sodium; D010869:Pindolol