Studies On Yeast Mating Pheromone Receptors And Their Complexes In Saccharomyces Cerevisiae

Most of the physiological processes in cells are related to the largest family of cell surface receptors : G protein-coupled receptors(GPCRs).GPCR is divided into five families : A,B,C,D and F.The Class D family receptors are only found in fungi,and they can regulate the survival and reproduction of fungi.S.cerevisiae mating pheromone receptors Ste2 and Ste3 belong to this family of GPCRs,which can interact with mating pheromone α factor and a factor produced by opposite types of cells to activate the common downstream heterotrimeric G protein Gpa1-Ste4-Ste18,thereby mediating a series of cascade reactions.At present,there are few studies on the receptor structure of this family.The analysis of the receptor structure can provide a design template for new drugs targeting fungal GPCRs to treat a variety of refractory fungal diseases.In this paper,X-ray crystal diffraction and cryo-electron microscopy were used to analyze the structure of Ste2 and Ste3 receptors.Ste2 crystal structure mainly improves the thermal stability of the receptor by truncation of the flexible region at the end of the receptor,insertion of fusion protein and amino acid mutation,can do LCP crystallization experiment with inhibitory ligands.At the same time,in order to study the activated structure of its binding to G protein,we tested the protein purification effect of G protein in different expression systems and modified G protein by mutation and truncation to increase the interaction between receptor and G protein to form a stable complex.Ste3 has no inhibitor,and a fully activated crystal structure is obtained by inserting a subunit / fragment stable receptor.The activated state structure of cryo-EM was constructed by connecting Ste3 and Ste4 to form chimeric protein and Nano Bi T construction strategy.It is expected to increase the binding probability of receptor and G protein by the strong interaction between αβ subunit and Lg Bi T,Hi Bi T.The Anti-BRIL-Fab cloning strategy was selected for the inhibitory structure of Ste3 to increase protein mass.Although we obtained a high yield of receptor protein with uniform properties on Ste2,crystallization and complex attempts were unsuccessful.In addition,we have obtained a certain composite sample after several rounds of cloning optimization on Ste3,but its yield and uniformity need to be improved,which has not reached the level of cryo-EM data collection.Although we have not yet obtained the high-resolution structure of S.cerevisiae mating pheromone receptors,we have fully understood the characteristics of the two receptors through a series of tests,and obtained better expression clones,which provides an important basis for subsequent experiments and receptor structure and activation mechanism.