The Structural Study Of Antagonist Bound 5-HT_1BR And NTSR1-?-arrestin 1 Complex Protein

5-HT_1BR expressed in the serotonergic neurons and non-serotonergic neurons,is the most important one of the 13 GPCR 5-HT receptors.The 5-HT_1BR regulates a wide range of human physiological processes,including modulation of smooth muscle contraction,mood,anxiety and perception through activation by the neuromodulator 5-HT.Triptans are agonists of the 5-HT_1BR,which are among the most frequently prescribed antimigraine medications based on the migraine neurovascular theory.Triptans can make cerebral arteriectasis and arterial pulsation back to normal,reducing headache and playing a significant role in the clinical application.Selective 5-HT_1BR antagonists specifically increase the level of serotonin in the synaptic cleft and serve as potential antidepressant agents.In order to further study the binding of antagonists to 5-HT_1BR and the antagonistic mechanism for 5-HT_1BR by antagonists,we have determined the first structure of a member of the serotonin receptor family in antagonist-bound state,5-HT_1BR in complex with MT,facilitated by replacing ICL3 with a novel optimized variant of BRIL,OB1.OB1 can enhance formation of intramolecular and intermolecular polar interactions,making OB1 a potential general tool for structural studies of membrane proteins.Unlike the agonist ergotamine?ERG?,MT occupies only the conserved orthosteric binding pocket,explaining the wide spectrum effect of MT on serotonin receptors.Compared with ERG,the antagonistbound 5-HT_1BR structure provides a basis for the ligand-mediated switch of 5-HT_1BR activity and reveals a common antagonistic mechanism for class A GPCRs characterized by ligand-induced outward movement of the extracellular end of TM6 through pushing the conserved aromatic residues 6.48,6.50 and 6.51 away from the helix bundle.The outward movement of the extracellular side of TM6 results in the inward shift of the cytoplasmic side of this helix,causing the inactive conformation of GPCRs In this case,the whole TM6 helix moves like a “seesaw” with its middle position serving as pivot point.The desensitization of activated GPCRs through endocytosis,and no-G protein signal pathway are both mediated by arrestins,which has been the focus of GPCR structural biology research.We use NTSR1 as a model protein to develop the structural study of the GPCR-?-arrestins complex protein.We validate the interaction between NTSR1 and ?-arrestin 1 by Alpha screen and Tango assay.Now we are carrying on the structure elucidation of optimized NTSR1-?-arrestin 1 fused proteins by means of protein crystallography and cryo-electron microscopy.At present,we have obtained tiny crystals.Besides we are trying on the data processing and modeling based on the data collected by the 300 Kv Titan Krios.