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. 2014 Mar 24;9(3):e92727.
doi: 10.1371/journal.pone.0092727. eCollection 2014.

The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor

Jennifer L Miller-Gallacher  1 , Rony Nehmé  1 , Tony Warne  1 , Patricia C Edwards  1 , Gebhard F X Schertler  1 , Andrew G W Leslie  1 , Christopher G Tate  1
Affiliations

The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor

Jennifer L Miller-Gallacher et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: JLMG was the recipient of a Commonwealth Scholarship. Research in CGT's laboratory on G protein-coupled receptors is funded by the Medical Research Council (MRC U105197215), the Wellcome Trust and the BBSRC (BB/G003653/1). Research on GPCRs in CGT's and GFXS' laboratory is funded by Heptares Therapeutics; both CGT and GFXS are on the Scientific Advisory Board of Hepatares and are shareholders. In addition, CGT is a consultant for Heptares. Heptares and the other funding bodies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Structure of the intramembrane Na+ ion binding site.
(A) Cartoon of β1AR (grey) depicting the positions of ordered water molecules (red spheres), Na+ ions (purple spheres), lipids (green sticks) and cyanopindolol (stick representation: carbon, yellow; nitrogen, blue; oxygen, red). The extracellular surface is at the top of the figure with the N-terminus (N) and C-terminus (C) labelled appropriately. (B) Detail of the Na+ binding site with portions of H2 and H3 removed for clarity: red spheres, water molecules; purple sphere, Na+ ion; green sticks, amino acid side chains; yellow sticks, cyanopindolol; red dashed lines, polar contacts, as defined by PyMOL. (C) Diagrammatic representation of the hydrogen bond network. Hydrogen bonds are assigned as displayed in COOT with a maximum distance of 3.5 Å. Where more than four interactions are shown, those with more favourable distances and geometry have been selected, with the exception of waters coordinating the Na+ ion, where up to 5 interactions are shown. Only the last two digits of the water numbers are shown.
Figure 2
Figure 2. Conservation of the intramembrane Na+ ion and associated water network in β1AR and A2AR.
An alignment between cyanopindolol-bound β1AR (PDB code 4BVN) and ZM241385-bound A2AR (PDB code 4EIY) was performed and the positions of waters (small spheres), Na+ ions (large spheres) and ligands (sticks) compared superposed on the surface of representation of β1AR (grey): purple, β1AR; cyan, A2AR. The last two digits for the number of each water molecule in β1AR is shown in the bottom inset. The alignment was performed based on the polar side chains in the solvent channel that are conserved between β1AR and A2AR (β1AR/A2AR: N339/2847.49, N59/241.50, D87/522.50, S128/913.39, N335/2807.45, Ser336/2817.46, W303/2466.48) with an overall RMSD of 0.30 for 30 atoms.
Figure 3
Figure 3. The effect of Na+ ion concentration on the binding of agonist to β1AR.
(A) Competition binding assays were performed on β1AR in insect cell membranes using 3H-DHA and the agonist isoprenaline (Fig. S4) in the presence of either NaCl or choline chloride (0 mM, 150 mM, 1 M). IC50 values were converted to Ki values using the Cheng-Prusoff equation using values of 4 nM for the KD of 3H-DHA (0 mM, 150 mM or 1 M NaCl) and 10 nM for the concentration of 3H-DHA in the assay. Values (±SEM) were determined from 4 independent experiments performed in duplicate. (B) The activation of β1AR (black circles) and β1AR-D87A2.50 (green squares) by isoprenaline was monitored by measuring intracellular concentrations of cAMP in the cell lines 293-β1AR and 293-β1AR-D87A2.50. Expression levels of β1AR and β1AR-D87A2.50 were identical in both cell lines (Fig. S7). The EC50 for isoprenaline activation was 0.12±0.03 nM and 8.1±3.4 nM for β1AR and β1AR-D87A2.50, respectively. Results are the mean ± SEM for 4 independent experiments.
Figure 4
Figure 4. Thermostability of β1AR mutants.
(A) The thermostability of dodecylmaltoside-solubilised β1AR was determined in either 150 mM NaCl (red squares) or 150 mM choline chloride (blue circles) giving apparent Tms (±SEM) of 26.6±0.3°C (n = 4) and 19.1±0.6°C respectively (n = 2). (B) The thermostability of detergent-solubilised β1AR mutants in the presence of 150 mM NaCl was determined (n = 2) and compared to the thermostability of wild-type β1AR. Mutations were made of all the residues lining the Na+ ion pocket and associated water network, but the mutations D121A, W303F and Y333A did not express any functional receptor as defined by 3H-DHA binding (Fig. S8).
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