Design and preparation of the class B G protein-coupled receptors GLP-1R and GCGR for 19F-NMR studies in solution
Design and preparation of the class B G protein-coupled receptors GLP-1R and GCGR for 19F-NMR studies in solution
复制标题
用于溶液中 19F-NMR 研究的 B 类 G 蛋白偶联受体 GLP-1R 和 GCGR 的设计和制备
DOI：
10.1111/febs.15686
复制
发表时间：
2021-01-09
期刊：
影响因子：
5.4
通讯作者：
Wuthrich, Kurt
中科院分区：
文献类型：
--
作者：
Wang, Huixia;Hu, Wanhui;Wuthrich, Kurt
人胰高血糖素样肽-1受体（GLP-1 R）和胰高血糖素受体（GCGR）是B类G蛋白偶联受体（GPCR），分别通过与胰高血糖素样肽-1（GLP-1）和胰高血糖素（GCG）相互作用而被激活。这些多肽激素参与脂质和胆酸代谢的调节，在糖代谢和糖尿病的发病机制中起着重要作用，因此这些GPCR作为药物靶点引起了人们的极大兴趣。因此，GLP-1 R和GCGR已通过X射线晶体学和冷冻电子显微镜（cryo-EM）进行了广泛研究，因此其结构众所周知。在这里，我们提出的基础，在解决方案中使用核磁共振（NMR）光谱，以补充分子结构与信息的分子内动力学和热力学和动力学的相互作用与生理配体和外源性候选药物。这包括GLP-1 R和GCGR的新型近野生型构建体的产生，洗涤剂胶束和纳米盘中NMR研究的溶液条件的优化，氟-19 NMR探针的翻译后化学引入，以及附着于固有半胱氨酸的F-19标记的序列特异性分配。添加负变构调节剂（NAM）NNC 0640对于获得我们的NMR实验所需的长期稳定性至关重要，我们报告了NNC 0640与GLP-1 R（GLP-1 R [TMD]）跨膜结构域结合产生的变构效应的新见解。
The human glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are class B G protein-coupled receptors (GPCRs) that are activated by interactions with, respectively, the glucagon-like peptide-1 (GLP-1) and glucagon (GCG). These polypeptide hormones are involved in the regulation of lipid and cholic acid metabolism, and thus play an important role in the pathogenesis of glucose metabolism and diabetes mellitus, which attracts keen interest of these GPCRs as drug targets. GLP-1R and GCGR have therefore been extensively investigated by X-ray crystallography and cryo-electron microscopy (cryo-EM), so that their structures are well known. Here, we present the groundwork for using nuclear magnetic resonance (NMR) spectroscopy in solution to complement the molecular architectures with information on intramolecular dynamics and on the thermodynamics and kinetics of interactions with physiological ligands and extrinsic drug candidates. This includes the generation of novel, near-wild-type constructs of GLP-1R and GCGR, optimization of the solution conditions for NMR studies in detergent micelles and in nanodiscs, post-translational chemical introduction of fluorine-19 NMR probes, and sequence-specific assignments of the F-19-labels attached to indigenous cysteines. Addition of the negative allosteric modulator (NAM) NNC0640 was critically important for obtaining the long-time stability needed for our NMR experiments, and we report on novel insights into the allosteric effects arising from binding of NNC0640 to the transmembrane domain of GLP-1R (GLP-1R[TMD]).