Three-dimensional Structure Analysis And Drug Interaction Sites Study Of Parathyroid Hormone Receptor Based On Cryo-electron Microscopy

G protein-coupled receptors(GPCRs)are seven-transmembrane proteins,they are widely expressed on the cell surface.All of them consist of extracellular domain(ECD),transmembrane domain(TMD)and intracellular terminal.The ECD is related to the recognizing and binding of ligands.The binding between receptors and agonist will induce a sharp kink and outward movement of the TM6 to accommodate G proteins.GPCRs can transduce signal through G proteins and other transducers,such as arrestins.The class B GPCRs are receptors for several hormone and they have a relatively larger ECD than other GPCR family members.The large ECD makes the expression and purification of these receptors challenging,which also induces the difficulties in structure determination of the class B GPCRs.As the most important drug targets,class B GPCRs are related to a wide variety of diseases including diabetes,obesity,osteoporosis and cardiovascular disease.Here we focus on the structure determination of the parathyroid hormone receptor-1(PTH1R)and the parathyroid hormone receptor-2(PTH2R),both belong to the class B GPCRs.The former is crucial to calcium homeostasis and bone metabolism,its disfunction can cause the osteoporosis.PTH2 R is mainly expressed in the central nervous system,and involve in the regulation of emotion,body temperature,cell proliferation and cardiovascular function.Despite these two receptors play important roles in the regulation of physiological function,there are still huge challenges for the targeted drug discovery of these two receptors due to the lacking of structural information.To analyze the structure of PTH1 R,we screened different receptor clones,fusion protein tags and expression systems.The initial work flow including the following steps: first purifying the Gs protein;second the purified Gs proteins were used to form the complex with the relevant receptors;third purifying the generated complex for the following structure analysis.Even through the complex was obtained in this way,it is not stable enough to perform the cryo-electron microscopy(cryo-EM).We latter tried to co-express PTH1 R and G proteins in Sf9 insect cells,and added LA-PTH peptide during the protein purification.The stable complex was obtained by this means and we finally got a high-resolution structure after sample collection and data analysis.This is the first reported ligand-PTH1R-Gs complex structure which provides the detail information about the interactions between ligand and receptor,receptor and G proteins.At the same time,it also afforded opportunities for the PTH1 R structural based drug research.During the structural analysis and drug interaction sites study of PTH2 R,we tried different fusion proteins,C-terminal truncations and W mutations to optimize the protein expression and strengthen the receptor-G protein interactions.After coexpressing receptors and G proteins,we added an exogenous ligand to purify the complex.To date,we already got the complex and its stability optimization is ongoing.Although the structure is inaccessible now,the current optimizations had laid foundations for the further study and offered reference for other receptors’ expression and purification.Besides the research about PTH1 R and PTH2 R structure,this thesis also includes high throughput screening(HTS)of methionyl-tRNA synthetase(MRS)inhibitors.Based on the Nano-BiT technique,we developed the screening model and screened 96000 compounds.Though secondary screen,specificity test,toxicity assay and cell proliferation assay,we picked out two compounds WNN1588-A010 and WNN1588-B003 whose IC50 are around tens of micromole and show great potencies of cell proliferation inhibition.This work is the basis for the further functional study and drug discovery.