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The 2.1 A resolution structure of cyanopindolol-bound ?1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

ABSTRACT: The ?1-adrenoceptor (?1AR) 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 ?1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 ?m 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 ?1AR and the adenosine A2A receptor (rmsd of 0.3 Å), despite an overall rmsd of 2.4 Å for all C? atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to ?1AR 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 ?1AR by 5-10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of ?1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.

SUBMITTER: Miller-Gallacher JL

PROVIDER: S-EPMC3963952 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Miller-Gallacher Jennifer L JL Nehmé Rony R Warne Tony T Edwards Patricia C PC Schertler Gebhard F X GF Leslie Andrew G W AG Tate Christopher G CG

PloS one 20140324 3

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 ...[more]

PMID: 24663151

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Project description:Background Autoantibodies against the second extracellular loop of the β1-adrenoceptor (β1- AA ) act similarly to agonist of β1-adrenergic receptor, which plays an important role in the pathophysiological characteristics of ventricular remodeling. Recently, considerable lines of evidence have suggested that CTRP9 (C1q tumor necrosis factor-related protein 9) is a potent cardioprotective cardiokine and protects the heart from ventricular remodeling. The aim of this study was to determine the role of CTRP 9 in ventricular remodeling induced by β1- AA . Methods and Results Blood samples were collected from 131 patients with coronary heart disease and 131 healthy subjects. The serum levels of β1- AA and CTRP 9 were detected using ELISA . The results revealed that CTRP 9 levels in β1- AA -positive patients were lower than those in β1- AA -negative patients, and serum CTRP 9 concentrations were inversely correlated with β1- AA . β1- AA monoclonal antibodies (β1- AA mAbs) were administered in mice with and without rAAV 9- cTnT -Full Ctrp9- FLAG virus for 8 weeks. Reverse transcription-polymerase chain reaction/Western analysis showed that cardiomyocyte CTRP 9 expression was significantly reduced in β1- AA mAb-treated mice. Moreover, compared with the β1- AA mAb alone group, cardiac-specific CTRP 9 overexpression improved cardiac function, attenuated adverse remodeling, and ameliorated cardiomyocyte apoptosis and fibrosis. Mechanistic studies demonstrated that CTRP 9 overexpression decreased the levels of G-protein-coupled receptor kinase 2 and promoted the activation of AMP-dependent kinase pathway. However, cardiac-specific overexpression of CTRP 9 had no effect on the levels of cAMP and protein kinase A activity elevated by β1-AAmAb. Conclusions This study provides the first evidence that the long-term existence of β1- AA mAb suppresses cardiac CTRP 9 expression and exaggerates cardiac remodeling, suggesting that CTRP 9 may be a novel therapeutic target against pathologic remodeling in β1- AA -positive patients with coronary heart disease.

Dataset Information

The 2.1 A resolution structure of cyanopindolol-bound ?1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

Publications

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

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