The 2.1 Å Resolution Structure of Cyanopindolol-Bound β1-Adrenoceptor Identifies an Intramembrane Na+ Ion that Stabilises the Ligand-Free Receptor

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.β1肾上腺素能受体（β1-adrenoceptor, β1AR）是一类可被内源性激动剂肾上腺素（adrenaline）与去甲肾上腺素（noradrenaline）激活的G蛋白偶联受体（G protein-coupled receptor, GPCR）。本研究解析了结合弱部分激动剂氰吡洛尔（cyanopindolol）的超热稳定型β1AR突变体的晶体结构，分辨率达2.1埃（Å）。研究人员采用脂质立方相（lipidic cubic phase）培养体系获得了高质量晶体（尺寸为100 μm的片状晶体），未借助常规用于GPCR结晶的胞内环3区T4溶菌酶（T4 lysozyme）或BRIL融合蛋白（BRIL fusion）辅助策略。本研究在膜内区域鉴定出一个与Asp872.50、Ser1283.39及3个水分子配位的钠离子，该钠离子属于跨膜螺旋1、2、3、6与7之间空腔内的更大范围水分子网络的组成部分。值得注意的是，尽管β1AR与腺苷A2A受体（adenosine A2A receptor）的全部α碳原子（Cα atoms）整体均方根偏差（root mean square deviation, rmsd）为2.4 Å，跨膜区氨基酸同一性仅为23%，但二者的水分子网络与钠离子却高度保守（rmsd仅0.3 Å）。钠离子不会对激动剂结合以及纳米抗体Nb80与β1AR的结合产生影响，但在无钠离子存在的条件下，受体的稳定性会降低7.5℃。对参与该钠离子或水分子网络配位的氨基酸侧链进行突变，可使β1AR的稳定性降低5至10℃。本研究数据表明，膜内钠离子与相关联的水分子网络可稳定β1AR的无配体状态，但仍允许受体形成激活态——该激活态伴随激动剂结合时钠离子结合口袋的塌陷。