Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Graduate Program in Molecular Biophysics and Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Nat Chem Biol. 2020 Oct;16(10):1096-1104. doi: 10.1038/s41589-020-0567-0. Epub 2020 Jul 6.
Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with β-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.
B 类 G 蛋白偶联受体的肽配体通过两步结合过程发挥作用，但将其细胞外结合与细胞内受体-抑制蛋白相互作用联系起来的基本机制尚不完全清楚。我们使用 NMR、与质谱交联、信号实验和甲状旁腺激素 (PTH) 1 型受体 (PTHR) 的计算方法，表明 PTH C 端部分的初始结合在第二个结合事件发生之前限制了灵活的 PTH N 端信号表位的构象。在第二步中，一个“热点”PTH 残基 His9 插入到 PTHR 跨膜域中，变构地与受体-抑制蛋白偶联。PTHR 细胞内环 3 的构象变化允许与β-抑制蛋白的指环进行有利的相互作用。这些结果揭示了 PTHR-抑制蛋白复合物形成的结构决定因素，并表明两步结合机制通过配体和受体之间的协同波动进行，这种波动扩展到其他 B 类 G 蛋白偶联受体。
