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.

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol–Bound β1-Adrenergic Receptor

“…6). The receptor is in the inactive state and is virtually identical to the structure of the cyanopindolol-bound receptor (PDB code 4BVN; root-mean-square deviation (RMSD) 0.19 Å over 2062 atoms) (Miller-Gallacher et al, 2014). The major difference in the ligand-binding pocket between b 1 AR bound to either cyanopindolol or 7-methylcyanopindolol is that the hydroxyl group of Ser215 5.46 is displaced 0.8 Å from its position when cyanopindolol is bound in a direction away from the center of the receptor.…”

“…45, 29.25, 45.63, 50.17, 69.38, 71.22, 101.62, 104.73, 111.47, 114.42, 115.24, 117.72, 126.70, 138.40, and 151.32. Expression, Purification, and Crystallization of b 1 AR The b44-TS construct was used, which contained additional thermostabilizing mutations, I129 3.40 V, E130 3.41 W, and Y343 7.53 L (Miller and Tate, 2011), to the previously published turkey (Meleagris gallopavo) b 1 AR construct b44-m23 (Warne et al, 2009. The construct used here is identical to that used for the structure determination of b 1 AR at 2.1-Å resolution (Miller-Gallacher et al, 2014), except that it contains the E130 3.41 W mutation to improve the amount of functional receptor expressed and Asp322 is identical to the wild-type receptor instead of being mutated to Lys to form a salt bridge in the extracellular region. None of these mutations affect the structure of the binding pocket of the receptor.…”

“…Images were processed with MOSFLM (Leslie, 2006) and AIMLESS (Evans, 2006(Evans, , 2011. The structure was solved by molecular replacement with PHASER (McCoy et al, 2007) using the b 1 AR-LCP structure with cyanopindolol bound (Miller-Gallacher et al, 2014) as a starting model. Refinement, rebuilding, and validation were carried out with REFMAC5 (Murshudov et al, 1997(Murshudov et al, , 2011, COOT (Emsley et al, 2010), and MOLPROBITY (Davis et al, 2007;Chen et al, 2010).…”

“…In vivo, the receptors are activated by both adrenaline and noradrenaline, with important clinical roles in the modulation of cardiac output (b 1 AR) and bronchodilatation (b 2 AR). Recent successes in the structure determination of both b 1 AR (Warne et al, 2008Moukhametzianov et al, 2011;Christopher et al, 2013;Miller-Gallacher et al, 2014) and b 2 AR Wacker et al, 2010;Rosenbaum et al, 2011;Rasmussen et al, 2011a,b) have led to a molecular understanding of receptor activation (Lebon et al, 2012). Binding of a full agonist to the receptors results in a contraction of the ligandbinding pocket by 1.0 Å for b 1 AR and 1.2 Å for b 2 AR and a rotamer change of Ser 5.46 so that it forms a hydrogen bond with the para-hydroxyl group of the catecholamine moiety of the agonist.…”

“…Structures of b 1 AR bound to full agonists , partial agonists , weak partial agonists (Warne et al, 2008;Moukhametzianov et al, 2011;Miller-Gallacher et al, 2014), and biased agonists have been determined. All these structures are in an inactive state, so the structures with agonists represent the encounter complex between the ligand and receptor before the receptor becomes activated.…”