The three-dimensional structure of the complex of non visual repressor protein and GPCR was successfully resolved

Recently, Xu Huaqiang group, Yu Xuekui group and Congyao group of national protein science center of Shanghai Institute of Biochemistry and cell (Shanghai) made great progress in GPCR transmembrane signal transduction field For the first time, the frozen electron microscope structure of non visual repressed protein 2 and neurotensin receptor 1 was analyzed, The mechanism of signal integration of GPCR coupled with non visual repressor protein was discussed The research results were published in cell research (DOI: 10.1038/s41422-019-0256-2) This is another important breakthrough of Xu Huaqiang's team in this field after the landmark research published in nature in 2015 and cell in 2017, which provides an important basis for functional research and design optimization of preferred ligands based on GPCR structure GPCR, as the largest cell membrane receptor protein family in human body, contains more than 800 members, and is the target of more than one third of clinical and drug research GPCR mainly carries out signal transduction by coupling downstream G protein and repressor protein Repressors include visual repressors (arrestin1 and 4) and non visual repressors (arrestin2 and 3) As early as 2015, Xu Huaqiang's team used the world's strongest X-ray free electron laser technology to obtain the crystal structure of arrestin1 rhodopsin complex with high resolution The three-dimensional structure for the first time shows the binding mode of repressor protein and GPCR, which is quite different from the interaction between G-protein and GPCR, laying an important foundation for further understanding the downstream signal transduction pathway of GPCR Non visual repressor proteins are involved in the regulation of more than 800 other downstream GPCR signaling pathways, not only blocking the binding of receptor and G protein, but also mediating the internalization of receptor and a series of non-G protein dependent signaling pathways The research on the structure of non visual repressor protein and GPCR complex has always been the focus of GPCR field However, the interaction between non visual repressor protein and GPCR is weak, and the assembled complex is highly flexible, which brings great challenges to structural analysis After several rounds of screening, Xu Huaqiang's team determined that Ntsr1 is a model receptor After eight years of efforts, we systematically explored various factors that enhance the interaction between arrestin2 and ntsr1 and improve the stability of the complex, including the introduction of repressor 3A mutant, the joint use of forward allosteric agonist ml314 and endogenous ligand nts, the assistance of conformational stable antibody fragment fab30, the fusion expression of the complex, the co expression of Grk to promote receptor phosphate Chemical and detergent screening The stable arrestin2-ntsr1 complex was obtained, and its structure of frozen electron microscope was analyzed This structure and the The structure of arrestin1 rhodopsin complex is similar to that of arrestin1, but arrestin2 has a 90 ° rotation conformation difference compared with arrestin1, which makes TM5, TM6 and ICL3 of the receptor in front of the N-terminal domain of arrestin2 This conformation is more conducive to the recruitment of arrestin2 by replacing the C-terminal of the receptor with ICL3, which can be used to explore the interaction between non visual repressor protein and GPCR The second model of Yin Wanchao, postdoctoral student of Xu Huaqiang research group, Yin Yuling, doctoral student of Yu Xuekui research group, Li Zhihai, postdoctoral student of Yu Xuekui research group, and Jin Mingliang, doctoral student of Congyao research group of national Protein Science Center (Shanghai) of Shanghai Institute of Biochemistry and cell, Chinese Academy of sciences are the co first authors of this paper The partners of this study include the Van Andel Research Institute, Zhejiang University and Harbin University of technology The research was supported by the Ministry of science and technology of the people's Republic of China, the National Natural Science Foundation of the people's Republic of China, the major scientific and technological infrastructure open research project of the Chinese Academy of Sciences, the Shanghai Municipal Science and Technology Commission, and the National Institutes of health of the United States.