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.
Highlights
There are three b-adrenoceptors encoded by the human genome, b1AR, b2AR and b3AR, which are all members of the G protein-coupled receptor (GPCR) superfamily [1,2]
The high thermostability of b36-m23 allowed its structure to be determined from crystals grown in octylthioglucoside [11] or Hega-10 [10], with complete data sets being collected from single crystals, but it formed only small crystals in lipidic cubic phase (LCP)
The overall dimensions of the ligand binding pocket are identical between the structures of b1AR determined in LCP compared to OTG, with the distance between the Cas of Ser2115.42 and Asn3297.39, which changes when either an agonist or antagonist is bound [36], being identical (16.0 A ) to within experimental error
Summary
There are three b-adrenoceptors (bARs) encoded by the human genome, b1AR, b2AR and b3AR, which are all members of the G protein-coupled receptor (GPCR) superfamily [1,2]. The development of novel engineering strategies for GPCRs [3] has allowed the structures of both b1AR and b2AR to be determined bound to a variety of agonists, partial agonists and inverse agonists [4,5,6,7,8,9,10,11,12]. Binding of a full agonist causes a contraction of the ligand binding pocket by ,1 Aand the rotamer conformational changes of Ser2155.46 and Ser2125.43, which combine to weaken the helixhelix interactions between H3-H4-H5 [10]. Binding of partial agonists stabilises the contraction of the ligand binding pocket and the rotamer conformation change of Ser2125.43, but not a rotamer change of Ser2155.46 (Ref [10]). Many questions still remain about both the dynamics of these processes and about the structures themselves, which may be answered through higher resolution structures of Number of crystals
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