A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1

作者：Xiaoyan Wang, Dongsheng Liu, Ling Shen, Fahui Li, Yongze Li, Lingyun Yang, Tiandan Xu, Houchao Tao, Deqiang Yao, Lijie Wu, Kunio Hirata, Laura Bohn, Alexandros Makriyannis, Xiaohong Liu, Tian Hua, Zhi‐Jie Liu, Jiangyun Wang · 发表于：Journal of the American Chemical Society · 年份：2021 · DOI：10.1021/jacs.1c06847 · 被引用次数：86 · 研究领域：Pancreatic function and diabetes、Cannabis and Cannabinoid Research、Receptor Mechanisms and Signaling

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy ( 19 F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3′-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19 F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569’s distinct behavior. We demonstrate that our site-specific 19 F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.