Design and preparation of the class B G protein-coupled receptors GLP-1R and GCGR for 19F-NMR studies in solution,The FEBS Journal

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Design and preparation of the class B G protein-coupled receptors GLP-1R and GCGR for 19F-NMR studies in solution
The FEBS Journal ( IF 5.5 ) Pub Date : 2020-12-23 , DOI: 10.1111/febs.15686
Huixia Wang _{1,

2,

3,

4} , Wanhui Hu ₁ , Dongsheng Liu ₁ , Kurt Wüthrich _{1,

5}

Affiliation

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 ¹⁹F-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]).

中文翻译：

用于溶液中 19F-NMR 研究的 BG 类蛋白偶联受体 GLP-1R 和 GCGR 的设计和制备

人胰高血糖素样肽 1 受体 (GLP-1R) 和胰高血糖素受体 (GCGR) 是 BG 类蛋白偶联受体 (GPCR)，它们分别通过与胰高血糖素样肽 1 (GLP- 1) 和胰高血糖素 (GCG)。这些多肽激素参与脂质和胆酸代谢的调节，因此在糖代谢和糖尿病的发病机制中起重要作用。，这引起了这些 GPCR 作为药物靶点的浓厚兴趣。因此，GLP-1R 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，因此它们的结构是众所周知的。在这里，我们介绍了在溶液中使用核磁共振 (NMR) 光谱来补充分子结构的基础，其中包含有关分子内动力学以及与生理配体和外在候选药物相互作用的热力学和动力学的信息。这包括生成新的近野生型 GLP-1R 和 GCGR 构建体，优化洗涤剂胶束和纳米圆盘中 NMR 研究的溶液条件，氟 19 NMR 探针的翻译后化学引入，以及序列- ^{19 的}具体任务贴在本地半胱氨酸上的 F 标签。添加负变构调节剂 (NAM) NNC0640 对于获得我们 NMR 实验所需的长期稳定性至关重要，我们报告了对 NNC0640 与 GLP-1R 跨膜结构域结合引起的变构效应的新见解。 GLP-1R[TMD])。

更新日期：2020-12-23

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