Adrenaline - activated structure of β_2-adrenoceptor stabilized by an engineered nanobody

摘要

这项研究报告了与不同激动剂(即BI167107、hydroxybenzyl isoproterenol和内源性激动剂"肾上腺素")形成复合物的、完全具有活性的人β-2-肾上腺素受体(β_2AR)的三个结构。)β_2AR是一种"G-蛋白耦合受体"(GPCR),后者是很多临床药物作为作用目标的普遍存在的膜蛋白。我们对它们与其内源性激动剂相结合的分子过程及其激发效应蛋白的分子过程仍很不了解。尽管所研究的三种激动剂在化学性质上具有多样性，但这三种激动剂都能稳定受体中高度相似的活性态。微妙的结构差别让我们对一个GPCR是怎样被多种激动剂激发的(这是对药物开发极为重要的一个现象)有了认识。%G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β_2-adrenoceptor (β_2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β_2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β-2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ～80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β_2AR and related GPCRs.