Structural Biology Studies Of Orphan Receptor GPR35

Background:GPR35 belongs to class A(rhodopsin-like)G protein-coupled receptors(GPCRs).It is mainly expressed in gastric and intestinal epithelial cells,dendritic cells,and macrophages in the small intestine and colon.GPR35 plays a crucial role in regulating the gastrointestinal tract’s homeostasis and provides an important link between metabolism,immunity,and the gut microbiome.Abnormal signaling of GPR35 is strongly associated with an increased risk of inflammatory bowel diseases(IBDs),including ulcerative colitis,Crohn’s disease and primary sclerosing cholangitis.GPR35 activation may have important implications for related gastrointestinal diseases.GPR35,as a drug target for gastrointestinal diseases,has a very broad application prospect in inflammatory bowel disease.However,as orphan G protein-coupled receptors(o GPCR),the lack of tool ligands has hindered the understanding of GPR35’s ligand recognition and signal transduction mechanisms.Furthermore,the low sequence homology of GPR35 with other class A GPCRs(less than 20%)has impeded a deeper understanding of its structure and the discovery of tool ligands.Therefore,deciphering the structure of GPR35 in its activated state could provide clues to further elucidate its function in IBD and provide a structural basis for the design of small molecule and peptide drugs targeting GPR35.Objective:This study aims to use single-particle cryo-electron microscopy(cryo-EM)technology to elucidate the complex protein structure of GPR35 binding downstream G protein under different ligand activation.The specific ligand binding mode of GPR35 and the signal transduction mechanism of biased activation were elucidated based on the cryo-electron microscopy structure.Finally,a theoretical basis for drug design was provided using the cryo-electron microscopy structure.Methods:In this study,we used the Bac-to-Bac insect cell expression system to co-express the complex protein of GPR35 in complex with downstream G proteins under ligand activation.We employed a series of clone optimization techniques,including fusion tag screening,adding fusion proteins,conservative amino acid site mutation,Nano Bi T method,tryptophan mutation,and G protein GTP binding site mutation,to determine the optimal clone and purification conditions.We then expressed and purified a large amount of complex protein of GPR35 bound to different G proteins under various ligand activation states.After determining the properties of the complex protein by molecular exclusion chromatography,SDS-PAGE,Western Blot,and 120 k V negative staining electron microscopy,we collected data using single-particle cryo-EM technology and processed and analyzed the data using cryo SPARC v3.3.2.Result:This study utilized a cloning optimization strategy to identify a wild-type full-length GPR35 clone with N-terminal BRIL fusion,C-terminal Lg Bi T and 10×His tags,which was subsequently cloned into a modified p Fast Bac1 vector containing an N-terminal HA signal peptide and Flag tag.This clone was used for expression and purification of GPR35 complexes,which showed optimal stability,homogeneity,and yield.The introduction of conservative and tryptophan mutations at certain residues did not significantly stabilize the complex.However,introducing five GTP-binding site mutations in Gα_i protein was found to effectively stabilize the complex.Moreover,the engineering chimeric mini Gα_q could form stable complexes with GPR35,whereas the wild-type Gα_q could not efficiently assemble the complex.The Nano Bi T method significantly improved the stability and homogeneity of the complex,and the functional experiments showed that the introduction of Lg Bi T could enhance the basal activity of GPR35 and facilitate complex assembly.After determining the optimal clones for each complex component,large-scale expression and purification were performed,resulting in the successful purification of stable,homogeneous,and high-quality Lodoxamide-GPR35-DNG_i,5-HIAA-GPR35-DNG_i,and Lodoxamide-GPR35-mini G_q complex proteins.Finally,the cryo-electron microscopy structure of Lodoxamide-GPR35-DNG_iwas obtained at 3.39?resolution,using single-particle data collection and structural analysis.Conclusion:In this study,Lodoxamide-GPR35-DNG_i,5-HIAA-GPR35-DNG_iand Lodoxamide-GPR35-mini G_q complex proteins were successfully expressed and purified.Finally,the single-particle cryo-EM data were collected and the structure of Lodoxamide-GPR35-DNG_i was resolved to 3.39?.These results provide a structural basis for studying the mechanism of GPR35 ligand binding and its interaction with downstream different G proteins,as well as for the development of structure-based targeted drugs for GPR35.