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.

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. Conservation of the intramembrane Na⁺ ion and associated water network in β₁AR and A_2AR.

An alignment between cyanopindolol-bound β₁AR (PDB code 4BVN) and ZM241385-bound A_2AR (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, A_2AR. 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 A_2AR (β₁AR/A_2AR: N339/284^7.49, N59/24^1.50, D87/52^2.50, S128/91^3.39, N335/280^7.45, Ser336/281^7.46, W303/246^6.48) with an overall RMSD of 0.30 for 30 atoms.

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 K_i values using the Cheng-Prusoff equation using values of 4 nM for the K_D 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-D87A^2.50 (green squares) by isoprenaline was monitored by measuring intracellular concentrations of cAMP in the cell lines 293-β₁AR and 293-β₁AR-D87A^2.50. Expression levels of β₁AR and β₁AR-D87A^2.50 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-D87A^2.50, respectively. Results are the mean ± SEM for 4 independent experiments.

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 T_ms (±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).