Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Biomolecular NMR and Center for Integrated Protein Science Munich at Department Chemie, Technical University of Munich, Lichtenbergstraße 4, 85747, Garching, Germany.
Nat Commun. 2020 Feb 3;11(1):669. doi: 10.1038/s41467-020-14526-3.
G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the β-adrenergic receptor to agonist stimulation and coupling to a G-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed.
G 蛋白偶联受体（GPCRs）是一种变构信号蛋白，可将细胞外刺激传递穿过细胞膜。使用 F NMR 和定点标记，我们研究了β-肾上腺素能受体跨膜螺旋 6 和 7 的细胞质区域对激动剂刺激和与 G 蛋白模拟纳米体的偶联的反应。激动剂结合表明受体在两种非活性状态和预激活形式之间处于平衡状态，随着配体效能的增加，预激活形式的比例越来越大。纳米体偶联导致完全活跃的三元受体复合物的存在，其数量与激动剂效能直接相关，与部分激动剂一致。虽然对于不同的激动剂，活性状态三元复合物中螺旋 6 的环境处于明确定义的构象中，显示出很少的构象灵活性，但螺旋 7 上高度保守的 NPxxY 模体的环境仍然是动态的，采用多种激动剂特异性构象，这意味着该区域在受体功能中具有进一步的作用。还观察到一种非活性纳米体偶联的三元受体形式。
