Decoding the structure of GPR151 via NELiS
Abstract
Structure determination of orphan G protein–coupled receptors (GPCRs) is hindered by lack of known ligands and their inherent instability during purification. Conventional thermostability screening requires ligands or purified protein, limiting its utility for orphan GPCRs. Here, we present the Nb6-Enabled Ligand-Free Stabilization Platform (NELiS)—a ligand- and purification-independent method for identifying stabilizing mutations. Applied to GPR151, an orphan GPCR enriched in habenula and implicated in neuropsychiatric disorders, NELiS identified four mutations that significantly improved thermostability and expression, allowing receptor purification. Using the stabilized and purified receptor, we further found a high-affinity, GPR151-specific nanobody that facilitated structural determination. Structural analysis revealed unconventional activation-resistant features across canonical motifs and an autoinhibitory N-terminal region occupying the orthosteric pocket. Functional studies confirmed a unique activation mechanism and the critical role of the N terminus in receptor maturation and trafficking. These results establish NELiS as a generalizable tool for structural and functional investigation of orphan GPCRs.
Article Details
Journal Info
Proceedings of the National Academy of Sciences
National Academy of Sciences
Authors (11)
Yumeng Wang
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences
Luyu Fan
Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences
Qianqian Song
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences
Yaning Li
Jiayu Jin
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences
Qiaoyu Zhao
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences
Weijie Gu
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences
Xiangyi Shi
Shanghai Nanoport, Thermofisher Scientific
Dianfan Li
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences
Yao Cong
Sheng Wang