Thermostabilizing Mutations Assisted The Structural Study Of G Protein-coupled Receptors GLP-1R And GPR124

G protein-coupled receptors（GPCRs）constitute the protein family with the most members in the human genome,participate in a variety of physiological and biochemical reactions in the human body,and are also the targets of many blockbuster drugs.However,due to the hydrophobicity of the seven transmembrane domain（7TM）and its multiple remodeling properties,it is difficult to obtain high-purity,monodisperse,stable and active protein in vitro for crystallization.The introduction of thermostabilizing mutations is one of the main measures to overcome this difficulty,but currently,large-scale screening methods are mainly used,which is time-consuming and labor-intensive.The first part of this thesis studies the structure of the class B1 secretin GPCR.For a long time,the class B1 GPCR has been a template for peptide hormone recognition and signal transduction.Glucagon-like peptide-1 receptor（GLP-1R）,one of the members of this family,is an important target of anti-diabetic drugs.A variety of peptide drugs act on this receptor are already on the market.We have previously analyzed the crystal structure of inactive GLP-1R combined with two negative allosteric modulators.The structure contains ten thermally stable mutations,which were selected from a total of 98 mutations designed earlier.In this study,we first systematically summarized all 98 mutations tested before.The results showed that the mutation strategy introducing aromatic amino acids to enhance the hydrophobic interaction between the helices had the highest success rate,while the introduction of disulfide bonds or salt bridges and other strategies,due to strict geometric restrictions on chemical bonds,had the lowest success rate.Four back mutations were further studied through fluorescence thermal stability detection experiments,crystallization and molecular dynamics simulations.It was found that the mutation I196^2.66bF increased the thermal stability of the protein itself,the mutation S271^4.47bA was conducive to crystal packing（decreased entropy）,and mutation S193^2.63bC and M233^3.36bC are dispensable for crystallization because they do not form disulfide bonds.The above results provide general mutation design strategies for B1 type GPCR or other membrane protein structure research:1)lock the receptor in a specific conformation;2)increase the interaction force within or between molecules（for crystal packing）;3)enhance the rigidity of the transmembrane helix;4)strengthen the ligand-receptor binding interface.The second part of this thesis conducts structural biology research on the class B2 adhesion GPCR（aGPCR）.aGPCR has a very large extracellular domain（ECD）and a characteristic 7TM of GPCRs.The ECD of aGPCR is composed of a variety of domains,including the very conserved GPCR autoproteolysis-inducing domain（GAIN）and the GPCR proteolysis site（GPS）.The GPS of some receptors are hydrolyzed during the maturation process,and some are not hydrolyzed,but in both cases the ECD and 7TM of aGPCR are bound together.Current studies have confirmed that GPS hydrolyzable aGPCR can expose a small peptide（stachel）at the N-terminus of its transmembrane region after binding to a ligand and activate its own 7TM;while GPS unhydrolyzed aGPCR may activate its 7TM through the relative position changes between different domains under the action of mechanical force.We chose GPR124,which has a higher expression level among the non-cleavable receptors,for structural study,and first tried to analyze the crystal structure of its transmembrane region.After obtaining a better 7TM expression fragment,we conducted the thermostabilizing mutation screening,and we have obtained several potential mutations.At the same time,we also tried to express and purify different fragments of the receptor,co-purify the receptor with extracellular ligand integrinαvβ3 or intracellular linker G protein,and use nanodisc for reassembly to prepare samples for single-particle cryo-electron microscopy analysis.The results show that we have screened out a medium molecular weight,stable,multi-domain GPR124 expression clone and can be packaged with nanodisc,but currently it is necessary to further optimize the complex with extracellular ligand or G protein to obtain a sufficiently large and monodisperse sample for cryo-EM analysis.Although the structure of high-resolution GPR124 has not yet been obtained,our research provides very useful information for the subsequent analysis of its structure and a clearer understanding of its possible activation mechanism.