Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N- terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R modulators. The HDX studies presented here for two GLP-1R peptide ligands indicates that the antagonist exendin- 4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

胰腺β细胞中胰高血糖素样肽-1受体（glucagon-like peptide-1 receptor, GLP-1R）的激活可增强胰岛素产生，是当前2型糖尿病（type 2 diabetes mellitus, T2DM）治疗的重要靶标。与其他B类G蛋白偶联受体（class B G protein-coupled receptors, GPCRs）类似，GLP-1R具备N端细胞外配体结合结构域。对肽类激动剂进行N端截短改造，可获得能够结合该细胞外结构域、但无法激活全长受体的拮抗剂。本研究的核心目标是借助氢氘交换（hydrogen/deuterium exchange, HDX）技术，解析肽配体与GLP-1R细胞外结构域（nGLP-1R）的酰胺氢键网络如何因结合相互作用发生改变，并以此技术平台验证潜在GLP-1R调节剂的直接结合事件。本文针对两种GLP-1R肽配体开展的HDX研究结果显示：与激动剂艾塞那肽（exendin-4）相比，拮抗剂艾塞那肽[9-39]（exendin-4[9-39]）在非离子型去污剂存在的环境中稳定性显著下降。此外，HDX技术可检测到艾塞那肽与艾塞那肽[9-39]的N端螺旋[Val19至Lys27]区域在与nGLP-1R结合后，其氢键网络发生稳定化。本研究还证实，配体结合可使nGLP-1R的氢键网络产生稳定化效应，并验证了潜在GLP-1R小分子配体与受体细胞外结构域的直接结合事件。