Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland.
Biomolecular NMR Spectroscopy Platform, ETH Zürich, 8093 Zürich, Switzerland.
Molecules. 2020 Dec 17;25(24):5984. doi: 10.3390/molecules25245984.
In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[CH] labelled thermostabilized β adrenergic receptor from turkey in association with a variety of different effectors, including mini-G and nanobody 60 (Nb60), which have not been previously studied in complex with β adrenergic receptor (βAR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tβAR stabilized by the native-like mini-G protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.
在这项工作中，我们研究了与各种不同效应器（包括先前未通过 NMR 研究与β肾上腺素能受体（βAR）复合的 mini-G 和纳米抗体 60（Nb60））结合的来自火鸡的稳定化β肾上腺素能受体的甲硫氨酸ε-[CH]标记的甲基核磁共振（NMR）谱。与 Nb60 结合的 pindolol 和 Nb60 诱导受体的高度相似的非活性状态，非常类似于静止状态构象集合。我们表明，在结合 mini-Gs 或纳米抗体 80（Nb80）后，整个受体发生了巨大的变构变化。由天然样 mini-G 蛋白稳定的 tβAR 构象与目前使用的替代 Nb80 诱导的构象高度相似。有趣的是，在这两种情况下都存在残留动力学，而在静止状态下未观察到残留动力学。最后，我们再现了一种药物相关的情况，其中拮抗剂以竞争性方式消除了受体与 mini-G 蛋白的相互作用，验证了我们制剂的功能完整性。因此，所呈现的系统非常适合在体外再现 G 蛋白偶联受体（GPCR）的激活循环的各个步骤，并为未来更具天然样系统的功能和药理学特性提供了基础。
