The 2.1 Å Resolution Structure of Cyanopindolol-Bound β1-Adrenoceptor Identifies an Intramembrane Na+ Ion that Stabilises the Ligand-Free Receptor

The beta(1)-adrenoceptor (beta(1)AR) 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 beta(1)AR mutant bound to the weak partial agonist cyanopindolol to 2.1 angstrom resolution. High-quality crystals (100 mm 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 Asp87(2.50), Ser128(3.39) 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 beta(1)AR and the adenosine A(2A) receptor (rmsd of 0.3 angstrom), despite an overall rmsd of 2.4 angstrom for all C alpha atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to beta(1)AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5 degrees 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 beta(1)AR by 5-10 degrees C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of beta(1)AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.