Transformation method and application of G protein coupled receptor

Abstract

The invention discloses a transformation method and application of a G protein coupled receptor, and belongs to the technical field of protein engineering. In order to improve the molecular weight of membrane protein, enable the enlarged part of the membrane protein and the original membrane protein to form a relatively rigid structure and further meet the structural analysis requirement of a cryoelectron microscope, the invention provides a method for transforming a G protein coupled receptor. The method comprises the following steps: co-expressing fusion protein GPCR (Glycine Polymerase Chain Reaction)-GFP10-11 and GFP1-9 fragments formed by GPCR and GFP10-11 fragments in a eukaryotic cell expression system, and adding GFP-clamp in the purification process; one amino acid at the specific site of GPCR is mutated into cysteine, glutamic acid at the 186th position of the amino acid sequence of GFP-clamp is mutated into cysteine, and a disulfide bond is formed between the amino acid and glutamic acid. The molecular weight of the modified G protein coupled receptor obtained by using the method can be increased to 85 kD or above, and the rigidity of the whole compound space structure is relatively strong, so that the modified G protein coupled receptor can be used for structural analysis of a cryoelectron microscope.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and specifically relates to a method for modifying a G protein coupled receptor and its application. Background Art

[0002] Most drug molecules work by binding to corresponding target proteins in the body. They activate or inhibit the function of target proteins and change the physiological processes of the human body, thereby achieving the purpose of curing diseases. Understanding how these compounds bind to target proteins is of great significance for the development and optimization of drugs. The three-dimensional structure of the complex formed by the target protein and the compound directly provides this information. Therefore, analyzing the three-dimensional structure of the complex formed by the protein and the compound is an important technology in the field of drug research and development.

[0003] G-protein coupled receptors (GPCRs) are a class of target proteins in the human body that have important drug development value. Currently, about 40% of the drugs on the market target GPCRs, and many drugs targeting these proteins are under development. There are a large number of GPCRs in the human body, and more than 800 have been discovered. These GPCRs have certain commonalities in spatial structure: they all contain 7 transmembrane helices; the N-terminus and 3 interhelical loop structures are located on the outside of the cell, and the C-terminus and another 3 interhelical loop structures are located on the inside of the cell. These proteins are located on the cell membrane and act as receptors for signal molecules, transmitting signals to the inside of the cell. External signal molecules bind to the part of the GPCR located on the outside of the cell, causing conformational changes in the transmembrane helices of the receptor, causing the intracellular part of the receptor to bind to the G protein and trigger physiological effects in the cell. GPCRs participate in a wide variety of physiological processes and play an important role in maintaining normal life activities in the human body. Drug molecules can regulate physiological processes inside cells by binding to these receptor proteins and producing stimulatory or inhibitory effects on them. Analyzing the complex structure of these drugs or potential drugs with their target GPCR proteins is of great guiding significance for studying the mechanism of action of these compounds and further drug development.

[0004] At present, the most important methods in structural biology are X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance. As a membrane protein, GPCR has intrinsic properties such as strong hydrophobicity, the need for detergent solubilization, insufficient stability, and a small crystallizable surface. Crystallization is very difficult, and the three-dimensional structure obtained by X-ray crystallography is extremely limited. Nuclear magnetic resonance technology requires a large number of stable and isotope-labeled protein samples, which makes it even more difficult to analyze the structure of membrane proteins such as GPCR. Cryo-electron microscopy does not require protein crystallization, and the amount of sample required is very small. In recent years, it has played an increasingly important role in the structural analysis of proteins, especially membrane proteins. However, cryo-electron microscopy has a major limitation. It has certain requirements for the size of the protein to be analyzed.

[0005] Cryo-electron microscopy uses an electron beam to take photos of frozen protein samples in glassy ice. There are a large number of protein molecule particles in the protein sample, and these protein molecule particles exist in different angles and orientations in the sample. By using a computer to analyze the angles of these particles, the two-dimensional photos obtained by taking photos can be reconstructed to obtain the three-dimensional structure of the protein molecule particles. Because the signal of a single protein molecule particle in a two-dimensional photo is very weak, it is necessary to first compare and classify a large number of particles with the same angle orientation and average them to improve the signal-to-noise ratio. Therefore, accurately determining the angle orientation of the protein molecule particles is a prerequisite for successfully obtaining a high-resolution three-dimensional structure. Because the signal-to-noise ratio of the image decreases with the size of the particle, if the protein molecule particle is too small, the signal-to-noise ratio in the glassy ice is too low, and the structural features are not obvious, it is difficult to accurately determine the angle orientation, resulting in reconstruction failure or low final resolution. A few proteins in the GPCR family have relatively large extramembrane domains, or form dimers with large molecular weights. For most GPCRs, the molecular weight is only 30-60kD, and the structure is mainly transmembrane helices. Detergents are needed to solubilize them when preparing samples. The transmembrane part of the protein particle is wrapped by a layer of dynamic detergent molecules without fixed structural characteristics, so the structural characteristics of the entire particle are even less obvious. Therefore, it is difficult to directly analyze its three-dimensional structure through cryo-electron microscopy technology.

[0006] In order to analyze the three-dimensional structure of small molecular weight membrane proteins through cryo-electron microscopy, one method in practice is to increase the size of the target membrane protein to improve the signal-to-noise ratio of the acquired image. According to the principle of cryo-electron microscopy technology described above, the increase in the size of the membrane protein does not guarantee the analysis of its structure. The enlarged part of the membrane protein must form a relatively rigid structure with the original membrane protein to help accurately determine the angle and orientation of the entire protein particle and strengthen the signal after averaging. If the enlarged part is more flexible than the original membrane protein, the averaged image will be blurred and the quality will deteriorate.

[0007] Currently, there are several methods for analyzing the three-dimensional structure of GPCRs with a molecular weight of 30-60kD using cryo-EM technology:

[0008] 1) Form a larger complex between GPCR and its natural effector protein. In the presence of agonists, GPCR can form a large molecular weight complex with G protein (including α, β, γ subunits), inhibitory protein arrestin or GPCR kinase GRK. Many structures of GPCR and agonist complexes have been resolved by this method. The disadvantages of this method are that it can only be used to resolve the structure of GPCR and agonist complexes. The binding of inhibitory compounds cannot form a complex between GPCR and G protein, so this method cannot be applied; the formed complex is sometimes not stable enough and dissociates during purification and sample preparation, making it impossible to use cryo-electron microscopy technology for structural analysis; the agonist compound needs to be kept present throughout the protein complex purification process, which is very costly for expensive compounds; and only one compound complex can be obtained each time purification is performed.

[0009] 2) Screen and prepare GPCR-specific monoclonal antibodies, intercept the antigen-binding part (Fab, with a molecular weight of about 50kD) on it, and form a complex with the target membrane protein with a larger molecular weight to meet the requirements of cryo-EM technology. Currently, several cryo-EM structures of membrane proteins with molecular weights similar to GPCRs have been resolved using this method. The disadvantages of this method are that the preparation cycle of monoclonal antibodies is very long; the membrane protein is not stable enough, and its transmembrane region needs to be wrapped by lipid molecules or detergents, and the exposed area is relatively small, so the effect as an antigen is poor, and the certainty of successful preparation cannot be guaranteed; the prepared antibody may bind to the flexible part of the membrane protein and cannot form a rigid structure with the GPCR, so it cannot help with the structural analysis; the prepared antibody may hinder the binding of small molecule compounds after binding to the GPCR, and cannot be used to analyze the structure of the small molecule compound complex.

[0010] 3) The third intracellular loop of GPCR is replaced by a small protein BRIL. The N-terminus and C-terminus of BRIL are both helical structures, which form a continuous helical structure with a certain rigidity with the fifth and sixth transmembrane helices of GPCR, and an antibody Fab fragment that specifically binds to BRIL is used to further increase the size of the protein, so as to analyze the structure using cryo-electron microscopy. The disadvantages of this method are that the connection mode of the two continuous helices may impose relatively large restrictions on the conformation of GPCR, affecting its binding with small molecule compounds; BRIL and the antibody fragment bound to it are far away from the main structure of GPCR in space, and the overall flexibility is still relatively large; for a specific GPCR, a lot of exploration of the joint site is required, and there is uncertainty as to whether it can be connected in the form of a continuous helix to assist in structural analysis.

[0011] 4) Use the nanobody that can bind to a specific GPCR in an inactive state for cryo-EM structural analysis of other GPCR structures, transplant the binding part of this nanobody to other GPCRs, and make them bind to the nanobody. The disadvantage of this method is that the nanobody itself is very small, and the increase in the size of GPCR protein particles is limited. The structural analysis has very high requirements for sample preparation conditions and the hardware and software of cryo-EM, and has great uncertainty; and the binding part of the nanobody is a two-segment helical structure. Whether it can match the helical structure of these GPCRs after transplantation is also an uncertain factor; because the nanobody locks the GPCR in an inactive state, this method cannot be used for the analysis of the structure of the GPCR and agonist complex.

[0012] In summary, it is urgent to find a method for modifying GPCR proteins that can not only increase the molecular weight of membrane proteins, but also form a relatively rigid structure with the enlarged part of the membrane protein and the original membrane protein, and solve the above-mentioned technical problems, so that the modified GPCR protein can meet the requirements of cryo-electron microscopy structure analysis. Summary of the invention

[0013] In order to solve the technical problem of analyzing the three-dimensional structure of the complex formed by a small molecular weight GPCR and a compound using the cryo-electron microscopy technology, the present invention provides a method for modifying a G protein coupled receptor, the method comprising co-expressing a fusion protein GPCR-GFP10-11 and a GFP1-9 fragment formed by a G protein coupled receptor and a GFP10-11 fragment in a eukaryotic cell expression system, and adding GFP-clamp-Cys during the purification process to obtain a ternary complex formed by GPCR-GFP10-11, GFP1-9 and GFP-clamp-Cys; the amino acid at position 34.51 of the Ballesteros-Weinstein numbering system in the second intracellular loop of the GPCR is mutated to cysteine, the amino acid sequence of the GFP1-9 fragment is shown in SEQ ID NO.1, the amino acid sequence of the GFP10-11 fragment is shown in SEQ ID NO.2, and the glutamic acid at position 186 of the GFP-clamp-Cys is mutated to cysteine ​​based on the amino acid sequence shown in SEQ ID NO.3.

[0014] It is further defined that the fusion protein GPCR-GFP10-11 is obtained by replacing the third intracellular loop or corner region between the fifth transmembrane helix and the sixth transmembrane helix of GPCR with GFP10-11 through genetic engineering methods.

[0015] It is further defined that 1, 2, 3, 4 or 5 amino acids at the linker of the fusion protein GPCR-GFP10-11 can be replaced.

[0016] It is further defined that the eukaryotic cell is an insect cell or a mammalian cell.

[0017] Preferably, the insect cell is a Spodoptera frugiperda cell Sf-9, and the mammalian cell is a HEK293F cell.

[0018] It is further defined that in the insect cell expression vector, the GPCR-GFP10-11 fusion protein and the GFP1-9 protein with a signal peptide sequence and a maltose binding protein fusion tag at the N-terminus are expressed by the polyhedrin gene promoter and the P10 promoter, respectively.

[0019] It is further defined that the mammalian cell expression vector used for the co-expression has a signal peptide sequence and a maltose binding protein fusion tag at the N-terminus and an internal ribosome entry site downstream.

[0020] It is further defined that the gene sequence of the GFP1-9 fragment is cloned into the internal ribosome entry site of the mammalian cell expression vector.

[0021] Further defined, the ternary complex formed by the G protein coupled receptor fusion protein GPCR-GFP10-11, GFP1-9 and GFP-clamp-Cys is purified by affinity chromatography and molecular sieve chromatography.

[0022] The present invention also provides a ternary complex formed by the G protein coupled receptor fusion protein GPCR-GFP10-11 obtained by the above method, GFP1-9 and GFP-clamp-Cys.

[0023] It is further defined that the total molecular weight of the ternary complex is above 85 kD.

[0024] The present invention also provides the use of the above method or the ternary complex obtained by the above method in analyzing the three-dimensional structure of the complex formed by the compound and the G protein-coupled receptor using cryo-electron microscopy structure.

[0025] It is further defined that the compound is an inhibitor or an agonist.

[0026] The method for modifying the G protein coupled receptor provided by the present invention inserts a short fusion fragment into the GPCR, and utilizes the fusion fragment to combine with the other two partner proteins to make the size of the entire complex particle reach more than 85kD. In addition, the partner protein is close enough to the GPCR to form a structure with a certain rigidity. The amino acids at the adjacent positions on the GPCR and one of the partner proteins are mutated to cysteine ​​to form a disulfide bond between them, and the flexibility of the entire complex is further reduced, so that the three-dimensional structure of the complex formed by the GPCR and the ligand compound can be analyzed by cryo-electron microscopy.

[0027] Specifically, the green fluorescent protein GFP can be divided into two parts, GFP1-9, which contains the first to ninth folded sheets, and GFP10-11, which contains the tenth and eleventh folded sheets, and the two parts can spontaneously combine together. Through genetic engineering methods, the third intracellular loop or corner region between the fifth transmembrane helix (TM5) and the sixth transmembrane helix (TM6) of the GPCR is replaced with GFP10-11. For the A2A adenosine receptor, GFP10-11 is inserted between L208 (5.69a in the Ballesteros-Weinstein numbering system) and L225 (6.27a in the Ballesteros-Weinstein numbering system), and connecting peptides can be added at both ends. For other GPCRs, GFP10-11 can be inserted into the position corresponding to the A2A adenosine receptor (between 5.69a and 6.27 in the Ballesteros-Weinstein numbering system), or 1, 2, 3, 4 or 5 amino acids at the linker can be replaced with other amino acid sequences, such as the corresponding sequence in the A2A adenosine receptor. This GFP10-11 insertion fragment is connected to the fifth and sixth helices of the GPCR in a flexible manner, does not affect the folding of the GPCR into the correct structure, and does not form a continuous helix with the GPCR sequence at the insertion position, so as to reduce the restriction on the GPCR structure. The modified GPCR can still bind to the ligand molecule. The fusion protein (GPCR-GFP10-11) formed by this GPCR and the GFP10-11 fragment is co-expressed in cells with the GFP1-9 fragment. The GFP10-11 fragment in the GPCR-GFP10-11 fusion protein combines with GFP1-9 to form a complete fluorescent GFP molecule. In the subsequent purification process, a GFP-clamp protein with a specific sequence is added to combine with the GFP part. After such modification, the complex formed by some GPCRs such as the A2A adenosine receptor and the companion protein GFP1-9 and GFP-clamp shows strong rigidity as a whole, which can be used for cryo-electron microscopy structure analysis and obtains the high-resolution structure of the complex formed with the inhibitor compound.

[0028] However, for some other GPCRs, after being modified as above and forming a complex with the partner proteins GFP1-9 and GFP-clamp, they still have a certain flexibility and cannot obtain a high-resolution cryo-EM structure. In order to further enhance the rigidity of the entire protein particle, the amino acid at position 34.51 of the Ballesteros-Weinstein numbering system in the second intracellular loop of the GPCR is mutated to cysteine, and the glutamic acid at position 186 on the GFP-clamp is mutated to cysteine, so that a disulfide bond is formed between the GPCR and the GFP-clamp. The formation of the disulfide bond causes cross-linking between the GPCR and the GFP-clamp, and a band with a larger molecular weight is shown in non-reducing SDS electrophoresis, while the disulfide bonds are reduced in reducing SDS electrophoresis, and the respective bands of the two proteins are still shown, which can be used to identify the formation of disulfide bonds. The disulfide bonds designed in this way greatly reduce the flexibility of the entire complex, and the overall structure has strong rigidity, which is suitable for cryo-EM structure analysis. The schematic diagram of the modified G protein-coupled receptor described in the present invention is shown in Figure 1 .

[0029] Beneficial effects of the present invention:

[0030] (1) The modified G protein coupled receptor obtained by the present invention not only has a molecular weight that can be increased to more than 85 kD, but also because the GFP10-11 fragment inserted into the GPCR is simultaneously connected to two sites on the membrane protein, and simultaneously binds to two partner proteins, GFP1-9 and GFP-clamp, as well as the formation of a disulfide bond, the spatial structure of the entire complex is relatively rigid, which is conducive to cryo-electron microscopy structure analysis.

[0031] (2) The GFP10-11 fragment is very short. Its insertion has little effect on the expression and correct folding of GPCRs. It does not form a continuous helical structure with the helices on the membrane protein and has little restriction on the GPCR conformation. It is suitable for the structural analysis of various types of GPCRs with different conformations.

[0032] (3) The stable complex formed by GPCR-GFP10-11 and GFP19 is well expressed in both insect cells and mammalian cells.

[0033] (4) The formed complex is not easy to dissociate during the purification process, and there is no need to conduct a large number of experimental explorations on the conditions for forming the complex and the conditions for purification.

[0034] (5) It can analyze the structure of GPCR-agonist complex as well as the structure of GPCR-inhibitor complex.

[0035] (6) The formation of a complex with the partner protein does not require the addition of a ligand. Expensive ligand compounds can be added to bind to the protein after the sample is purified and frozen during sample preparation, greatly reducing costs.

[0036] (7) Purifying a batch of proteins can prepare a variety of complexes with different small molecule compounds, reducing time, workload, and cost.

[0037] (8) The fluorescence properties of GFP facilitate the use of fluorescence detection size exclusion chromatography for expression screening of small samples, thus saving costs.

[0038] (9) There is no need for a time-consuming and low-certainty antibody screening process. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of GPCR protein engineering;

[0040] Figure 2 This is the identification diagram of GFP-clamp protein after purification; Figure 2 A in the figure is the molecular sieve chromatography result diagram, Figure 2 B in the figure is the result of SDS gel electrophoresis identification;

[0041] Figure 3 This is a screen result diagram of the insertion position of the GFP10-11 fragment (with connecting peptides on both sides) on the third intracellular loop of the A2A adenosine receptor;

[0042] Figure 4 The result of screening for peptide segments connecting the two sides of the GFP10-11 fragment inserted into the third intracellular loop of the A2A adenosine receptor;

[0043] Figure 5 This is a map of the pFastBac4R insect cell expression vector modified from the pFastBac Dual vector;

[0044] Figure 6 This is the purification and identification of the A2A adenosine receptor modified protein complex and the structural analysis of the complex with the small molecule inhibitor ZM241385; Figure 6 A in the figure is the molecular sieve chromatography result diagram, Figure 6 B in the figure is the result of SDS gel electrophoresis identification. Figure 6 C in the figure is the two-dimensional classification result of cryo-EM data particles. Figure 6 D in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data. Figure 6 E in the figure is the overall density of the protein complex reconstructed by cryo-EM three-dimensional reconstruction. Figure 6 F in the figure is the density of ZM241385 molecules reconstructed by cryo-electron microscopy 3D;

[0045] Figure 7 is a map of the mammalian cell expression vector pBacMam4R;

[0046] Figure 8 The purification and identification of the CNR1 cannabinoid receptor modified protein complex and the structural analysis of the complex with the small molecule inhibitor taranabant; Figure 8 A in the figure is the molecular sieve chromatography result diagram, Figure 8 B in the figure is the result of SDS gel electrophoresis identification. Figure 8 C in the figure is the two-dimensional classification result of cryo-EM data particles. Figure 8 D in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data. Figure 8 E in the figure is the overall density of the protein complex reconstructed by cryo-EM three-dimensional reconstruction;

[0047] Fig. 9 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after purification of GFP-clamp-Cys protein; Fig. 9 A in the figure is the molecular sieve chromatography result diagram, Fig. 9 B in the figure is the result of SDS gel electrophoresis identification;

[0048] Fig.10 The purification and identification of the disulfide-crosslinked CNR1 cannabinoid receptor modified protein complex and the structural analysis results of the complex with the inhibitor taranabant; Fig.10 A in the figure is the molecular sieve chromatography result diagram, Fig.10 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions; Fig.10 C in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data; Fig.10 D in the figure is the overall density of the protein complex reconstructed by cryo-EM three-dimensional reconstruction; Fig.10 E in is the molecular density of taranabant;

[0049] Fig.11 The purification and identification of the disulfide-crosslinked NK1R neurokinin receptor modified protein complex and the structural analysis of the complex with the inhibitor aprepitant; Fig.11 A in the figure is the molecular sieve chromatography result diagram, Fig.11 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions; Fig.11 C in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data; Fig.11 D in the figure is the overall density of the protein complex reconstructed by cryo-EM three-dimensional reconstruction; Fig.11 E in is the aprepitant molecular density;

[0050] Fig.12 The purification and identification of the disulfide-crosslinked A2A adenosine receptor modified protein complex and the structural analysis results of the complex with the agonist adenosine molecule; Fig.12 A in the figure is the molecular sieve chromatography result diagram, Fig.12 B in the figure is the result of SDS gel electrophoresis identification. Lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions. Fig.12 C in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data. Fig.12 D in the figure is the overall density of the protein complex reconstructed by cryo-EM. Fig.12 E in is the density of adenosine molecules;

[0051] Fig.13 The purification, identification and structural analysis results of the disulfide-cross-linked CCR8 chemokine receptor modified protein complex; Fig.13 A in the figure is the molecular sieve chromatography result diagram, Fig.13 B in the figure is the result of SDS gel electrophoresis identification. Lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions. Fig.13 C in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data. Fig.13 D in the figure is the overall density of the protein complex reconstructed by cryo-EM three-dimensional reconstruction;

[0052] Fig.14 This is a diagram showing the purification, identification and structural analysis of the disulfide-cross-linked GPRC5D receptor modified protein complex; Fig.14 A in the figure is the molecular sieve chromatography result diagram, Fig.14 B in the figure is the result of SDS gel electrophoresis identification. Lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions. Fig.14 C in the figure is the FSC curve of three-dimensional reconstruction of cryo-EM data. Fig.14 D in the figure is the overall density of the protein complex reconstructed by cryo-EM three-dimensional reconstruction;

[0053] Fig.15 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of GPR52-Cys-GFP10-11 protein after purification; Fig.15 A in the figure is the molecular sieve chromatography result diagram, Fig.15 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0054] Fig.16 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of MC4R-Cys-GFP10-11 protein; Fig.16 A in the figure is the molecular sieve chromatography result diagram, Fig.16B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0055] Fig.17 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after GNRHR-Cys-GFP10-11 protein purification; Fig.17 A in the figure is the molecular sieve chromatography result diagram, Fig.17 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0056] Fig.18 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of PTGDR2-Cys-GFP10-11 protein; Fig.18 A in the figure is the molecular sieve chromatography result diagram, Fig.18 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0057] Fig.19 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after HTR2C-Cys-GFP10-11 protein purification; Fig.19 A in the figure is the molecular sieve chromatography result diagram, Fig.19 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0058] Fig. 20 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of ADRA2B-Cys-GFP10-11 protein after purification; Fig. 20 A in the figure is the molecular sieve chromatography result diagram, Fig. 20 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0059] Fig.21 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after ADRB1-Cys-GFP10-11 protein purification; Fig.21 A in the figure is the molecular sieve chromatography result diagram, Fig.21 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0060] Fig. 22 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after ADRB2-Cys-GFP10-11 protein purification; Fig. 22 A in the figure is the molecular sieve chromatography result diagram, Fig. 22 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0061] Fig.23 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of C5AR1-Cys-GFP10-11 protein; Fig.23 A in the figure is the molecular sieve chromatography result diagram, Fig.23 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0062] Fig.24 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of CCR2-Cys-GFP10-11 protein; Fig.24 A in the figure is the molecular sieve chromatography result diagram, Fig.24 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0063] Fig.25 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of CCR5-Cys-GFP10-11 protein; Fig.25 A in the figure is the molecular sieve chromatography result diagram, Fig.25 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0064] Fig.26 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of CCR6-Cys-GFP10-11 protein; Fig.26 A in the figure is the molecular sieve chromatography result diagram, Fig.26 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0065] Fig. 27 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of CCR7-Cys-GFP10-11 protein; Fig. 27 A in the figure is the molecular sieve chromatography result diagram, Fig. 27 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0066] Fig.28 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after CHRM2-Cys-GFP10-11 protein purification; Fig.28A in the figure is the molecular sieve chromatography result diagram, Fig.28 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0067] Fig.29 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after CLTR2-Cys-GFP10-11 protein purification; Fig.29 A in the figure is the molecular sieve chromatography result diagram, Fig.29 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0068] Fig.30 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of CXCR2-Cys-GFP10-11 protein; Fig.30 A in the figure is the molecular sieve chromatography result diagram, Fig.30 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0069] Fig.31 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of CXCR4-Cys-GFP10-11 protein; Fig.31 A in the figure is the molecular sieve chromatography result diagram, Fig.31 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0070] Fig.32 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after DRD2-Cys-GFP10-11 protein purification; Fig.32 A in the figure is the molecular sieve chromatography result diagram, Fig.32 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0071] Fig.33 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after DRD3-Cys-GFP10-11 protein purification; Fig.33 A in the figure is the molecular sieve chromatography result diagram, Fig.33 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0072] Fig.34 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after GPBAR-Cys-GFP10-11 protein purification; Fig.34 A in the figure is the molecular sieve chromatography result diagram, Fig.34 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0073] Fig.35 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of HRH1-Cys-GFP10-11 protein after purification; Fig.35 A in the figure is the molecular sieve chromatography result diagram, Fig.35 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0074] Fig.36 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after HTR1A-Cys-GFP10-11 protein purification; Fig.36 A in the figure is the molecular sieve chromatography result diagram, Fig.36 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0075] Fig.37 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after HTR1B-Cys-GFP10-11 protein purification; Fig.37 A in the figure is the molecular sieve chromatography result diagram, Fig.37 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0076] Fig.38 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of HTR2B-Cys-GFP10-11 protein after purification; Fig.38 A in the figure is the molecular sieve chromatography result diagram, Fig.38 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0077] Fig.39 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after LPAR1-Cys-GFP10-11 protein purification; Fig.39 A in the figure is the molecular sieve chromatography result diagram, Fig.39 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0078] Fig.40The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of MTNR1B-Cys-GFP10-11 protein after purification; Fig.40 A in the figure is the molecular sieve chromatography result diagram, Fig.40 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0079] Fig.41 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of NPY1R-Cys-GFP10-11 protein after purification; Fig.41 A in the figure is the molecular sieve chromatography result diagram, Fig.41 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0080] Fig.42 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification of OPRD-Cys-GFP10-11 protein after purification; Fig.42 A in the figure is the molecular sieve chromatography result diagram, Fig.42 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0081] Fig.43 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of OX2R-Cys-GFP10-11 protein; Fig.43 A in the figure is the molecular sieve chromatography result diagram, Fig.43 B in the figure is the result of SDS gel electrophoresis identification, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions;

[0082] Fig.44 The figure shows the results of molecular sieve chromatography and SDS gel electrophoresis identification after the purification of PTGDR-Cys-GFP10-11 protein; Fig.44 A in the figure is the molecular sieve chromatography result diagram, Fig.44 B in the figure is the SDS gel electrophoresis identification result diagram, lane 1 is the sample treated under reducing conditions, and lane 2 is the sample treated under non-reducing conditions. DETAILED DESCRIPTION

[0083] The present invention is further described in detail below in conjunction with specific examples and accompanying drawings. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical reagents used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0084] The amino acid sequence involved in the present invention is as follows:

[0085] The amino acid sequences of GFP and GFP-clamp proteins are derived from the structural information of their complex (rcsb protein database number 5MA4).

[0086] The amino acid sequence of GFP is (SEQ ID NO.4):

[0087] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKQA

[0088] The amino acid sequence of GFP-clamp is (SEQ ID NO.3):

[0089] DLGKKLLEAARAGQDDEVRILMANGADVNAADDVGVTPLHLAAQRGHLEIVEVLLKYGADVNAADLWGQTPLHLAATAGHLEIVEVLLKNGADVNARDNIGHTPLHLAAWAGHLEIVEVLLKYGADVNAQDKFGKTPFDLAIDNGNE DIAEVLQKAAGGGSGGGDVNAYDEVGWTPLHKAAWGHLEKVEDLLKNGADVNAADIDGYTPLHLAAFSGHLEIVEVLLKYGADVNADDQAGFTPLHLAAIFGHLEIVEVLLKNGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAA

[0090] The GFP1-9 fragment selected the amino acid sequence of positions 1-198 from the above GFP complete sequence, and modified the position that does not affect the binding interface with GFP-clamp with reference to the split GFP sequence numbered 4KF5 from the protein database of www.rcsb.org. The amino acid sequence is (SEQ ID NO.1):

[0091] MGTRKGEELFTGVVPILIELDGDVNGHKFSVRGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIYFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQNNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLP

[0092] GFP10-11 fragment sequence selection The amino acid sequence at positions 200-229 was selected from the above GFP complete sequence and amino acids 207 and 212 were mutated. The amino acid sequence is (SEQ ID NO.2): HYLSTQTSLSKDLNEKRDHMVLLEFVTAAG

[0093] Example 1: Cryo-EM structure analysis of the complex of A2A adenosine receptor and ZM241385

[0094] (1) GFP-clamp expression and purification

[0095] The DNA sequence encoding the GFP-clamp protein was cloned into the E. coli expression plasmid pMALc2x, with a maltose binding protein fusion tag and a TEV protease cleavage site at the N-terminus and a histidine fusion tag at the C-terminus. TM2 (DE3) competent cells. Pick the positive monoclonal clone into 20mL LB medium with added resistance, and culture the bacteria in small amounts until they are supersaturated; transfer 20mL of the bacteria to 800mL of culture medium containing ampicillin and chloramphenicol resistance for amplification, and when the absorbance of the bacterial solution at 600nm is around 0.6, add 0.5mM isopropyl-β-D-thiogalactoside, and induce overnight at 20℃. Collect the bacterial solution into a large-capacity centrifuge bottle, centrifuge at 4℃, 4000rpm for 30min, remove the supernatant medium, and retain the precipitate. Resuspend the precipitate with a buffer solution (500mM NaCl, 5% glycerol, 20mM imidazole, 50mM Tris-HCl pH 7.8), add appropriate amounts of lysozyme, nuclease and phenylmethylsulfonyl fluoride, and use a cell ultrasonic disruptor to break the bacteria. The above-mentioned ultrasonic suspension is centrifuged again at high speed, 4℃, 20000g, centrifuged for 30min, and the supernatant is retained. Take a suitable volume of Ni-NTA resin and combine it with the supernatant, and rotate and mix at 4℃ for 1.5h. After sufficient combination, centrifuge at 4℃, 2000rpm for 2min, discard the supernatant, and retain the resin bound to the protein. Transfer the Ni-NTA resin to a gravity column, wash the impurities with the above buffer, about 15-20 column volumes, elute the protein with elution buffer (500mM NaCl, 5% glycerol, 500mM imidazole, 50mM Tris-HCl, pH 7.8), collect and measure the protein concentration, add TEV protease at a mass ratio of one-tenth and digest overnight. Dilute with 500mM NaCl, 5% glycerol, 50mM Tris-HCl, pH 7.8 buffer to an imidazole concentration of 20mM, and combine with an appropriate amount of Ni-NTA resin again. The resin was washed with 500 mM NaCl, 5% glycerol, 20 mM imidazole, 50 mM Tris-HCl, pH 7.8, and the protein was eluted with 500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, pH 7.8. The aggregation state and purity of the protein were analyzed by molecular sieve chromatography and SDS-PAGE gel (see Figure 2 ).

[0096] (2) Optimization of the fusion protein formed by A2A adenosine receptor and GFP10-11

[0097] The amino acid sequence of human A2A adenosine receptor was derived from GenBank with the accession number NM_000675.6. The first amino acid at the N-terminus and the C-terminal sequence after 318 were removed. The third intracellular loop was located between L208 and L225. The DNA sequence encoding the GFP10-11 fragment was inserted between these two amino acids by overlapping PCR to replace the original third intracellular loop sequence. In order to optimize the connecting peptide segment to maintain the stability of the fusion protein and reduce flexibility, a series of combinations of positions and linker lengths were made, and the proteins of various combinations were identified and analyzed by fluorescence detection size exclusion chromatography (FSEC).

[0098] First, the connection position between A2A adenosine receptor and GFP10-11 was optimized: GFP10-11 was connected between leucine L, the 208th amino acid in the TM5 transmembrane region of A2A adenosine receptor, and arginine R, the 220th amino acid in the TM6 transmembrane region of A2A adenosine receptor, with a connecting peptide containing 5 amino acids on each side, glycine-serine-glycine-glycine-glycine (GSGGG) on the left and glycine-glycine-serine-glycine-glycine (GGSGG) on the right. This combination is recorded as: L208_GSGGG_GFP10-11_GGSGG_R220(a). Similarly, the modifications of other connection positions are: L208_GSGGG_GFP10-11_GGSGG_A221 (b); L208_GSGGG_GFP10-11_GGSGG_R222 (c); L208_GSGGG_GFP10-11_GGSGG_T224 (d); and L208_GSGGG_GFP10-11_GGSGG_L225 (e). The screening results of the insertion position of the GFP10-11 fragment (with connecting peptides on both sides) on the third intracellular loop of the A2A adenosine receptor are shown in Figure 3 According to the screening results, GFP10-11 was selected to be connected between the 208th amino acid leucine L in the TM5 transmembrane region of the A2A adenosine receptor and the 225th amino acid leucine L in the TM6 transmembrane region of the A2A adenosine receptor.

[0099] Then, the linker connecting A2A adenosine receptor and GFP10-11 was optimized, and different length linkers were designed on both sides of GFP10-11: L208_GSGG_GFP10-11_GGSG_L225; L208_GGG_GFP10-11_GGG_L225; L208_GG_GFP10-11_GGG_L225; L208_GSGGG_GFP10-11_GGSGG_L225; L208_G_GFP1 0-11_G_L225; L208_GGG_GFP10-11_GG_L225; L208_GG_GFP10-11_GGG_L225; L208_GG_GFP10-11_G_L22 5; L208_G_GFP10-11_GG_L225; L208_G_GFP10-11_L225; L208_GFP10-11_G_L225;

[0100] The above-mentioned different A2A adenosine receptor modified sequences were cloned into the mammalian cell expression vector pBacMam4R, with a signal peptide sequence and a maltose binding protein fusion tag at the N-terminus, as well as a TEV protease cleavage site. The vector also has an internal ribosome entry site. After the gene sequence encoding the GFP1-9 fragment is cloned into the internal ribosome entry site, the A2A-GFP10-11 fusion protein and the GFP1-9 fragment can be simultaneously expressed in the cell, and they spontaneously combine to form a complex. The constructed vector DNA was transiently transfected into 293T cells using lipofectamine 2000 transfection reagent. After 40 hours, the cells were collected, weighed, and ultrasonically disrupted. 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) were added to dissolve the cell membrane, and 50 micrograms of GFP-clamp protein were added to every 100 mg of cells. After incubation for 1.5 hours, the mixture was centrifuged at 17000 g and the supernatant was analyzed by fluorescence detection size exclusion chromatography (SEC). The fluorescence of the column effluent was detected by 488 nm excitation light and 510 nm emission light (see Figure 4 ), and judge whether the protein is correctly folded and the properties of the aggregation state according to the position and shape of the fluorescence peak. Finally, it was determined that the L208_G_GFP10-11_L225 sequence can be used for expression and structural analysis, and its amino acid sequence is (SEQ ID NO.5):

[0101] PIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVSLAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIA IRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYLRIFLAARRQLG HYLSTQTSLSKD LNEKRDHMVLLEFVTAAG LQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAG (the underlined sequence is the GFP10-11 fragment sequence, and the wavy line is the short peptide sequence at the joint)

[0102] (3) Cryo-EM structure analysis of the complex between A2A adenosine receptor and ZM241385

[0103] The modified pFastbacDual plasmid has a maltose binding protein sequence with a hemagglutinin signal sequence at the N-terminus in the downstream sequence of the polyhedrin promoter (see Figure 5 ), clone the nucleotide sequence encoding the A2A adenosine receptor fused with the GFP10-11 fragment determined in (2) into the C-terminus of the maltose binding protein, and clone the nucleotide sequence encoding the GFP1-9 fragment into the downstream of the P10 gene promoter. Use the standard Bac-to-Bac baculovirus expression system operation process to produce recombinant baculovirus: the above plasmid is transformed into competent DH10Bac competent cells, and plated with LB agar medium containing kanamycin, gentamicin and tetracycline, as well as IPTG and Bluo-gal, and placed in a 37°C incubator for 48 hours; after blue-white screening, pick the white positive single colony, place it in 5mL LB liquid culture medium for overnight culture, and centrifuge the next day to collect the bacteria and extract the recombinant bacmid DNA from it. Use Cellfectin transfection reagent to transfect the purified recombinant bacmid DNA into Spodoptera frugiperda cells Sf-9, and the P0 generation recombinant baculovirus can be harvested after 4-5 days. High titer baculovirus can be obtained by infecting Sf-9 cells with P0 virus and amplifying them.

[0104] The cell density in good growth condition is 2×10 6 / mL Sf-9 cells were infected with high titer baculovirus, cultured in a 27℃ constant temperature incubator at 125rpm for 48 hours, and the cells were collected by centrifugation. The cells were resuspended in a buffer solution (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8) and disrupted with a cell ultrasonic disruptor. Lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) were added to a final concentration of 1%, and the cell membrane was melted by rotating at 4℃ for 2 hours. High-speed centrifugation at 4℃, 20000g, centrifuged for 30min, the supernatant was mixed with an appropriate volume of amylose resin, and rotated at 4℃ for 1.5h. After full binding, centrifuged at 4℃, 2000rpm for 2min, the supernatant was discarded, and the protein-bound resin was retained. The amylose resin was transferred to a gravity column, and the impurities were washed with a washing buffer (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), and the target protein was eluted with an elution buffer (500mM NaCl, 5% glycerol, 20mM maltose, 50mM Tris-HCl pH7.8, 0.01% LMNG, 0.001% CHS), and the concentration was collected and measured, and then the protein was concentrated to 500μL using a centrifugal filter-10K. The concentrated protein was separated using a Superdex 200Increase 10 / 300GL molecular sieve chromatography column, and the mobile phase was a buffer (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), and the protein in the peak area of ​​the UV absorption peak corresponding to the target protein complex was collected. An appropriate excess of GFP-clamp protein was added to the collected protein to form a complex, and then separated again using a Superdex200Increase 10 / 300GL molecular sieve chromatography column (see Figure 6 A), the mobile phase is buffer (150mMNaCl, 20mM HEPES pH 7.4, 0.005% LMNG, 0.0005% CHS), and the protein in the peak area of ​​the UV absorption peak corresponding to the target protein complex is collected, and the protein is concentrated to 10-15mg / mL using a centrifugal filter. The components of the obtained sample are analyzed and identified by SDS-PAGE gel electrophoresis (see Figure 6 B).

[0105] ZM241385 was dissolved in DMSO at a concentration of 100mM, diluted 100 times with water to a concentration of 1mM, and added to the purified and concentrated protein sample at a volume of 10%, and the final concentration of ZM241385 was 100μM. The Quantifoil Cu 300 1.2 / 1.3 electron microscope grid was hydrophilized using a PELCO easiGlow glow discharger. Using a Vitrobot cryo-sampler, 3μL of protein sample was added to the hydrophilized grid. After a 4-second waiting time, it was blotted at a force of 1-6 and a time of 3-5 seconds, then quickly inserted into liquid ethane pre-cooled by liquid nitrogen for freezing, and then transferred to liquid nitrogen for storage. The prepared frozen sample was loaded into a Titan Krios cryo-EM equipped with a Gatan K2 direct electron detection camera, and image data of protein particles in the sample wells were collected at an accelerating voltage of 300 kV. Data collection used the super-resolution mode, and the specific parameters were pixel size Each image consists of 40 frames, and the total electron dose is

[0106] The collected data were corrected for the displacement caused by the electron beam using MotionCor2, and the contrast transfer function parameters of each image were estimated in the CryoSPARC software. Then, the particles were selected and the local images of the protein particles were extracted. After several rounds of 2D classification and removal of erroneous particles (see Figure 6 C), and construct the initial three-dimensional density model from scratch (ab initio reconstruction). These different reconstructed models are subjected to differential correction (heterogeneous refinement), and the ones with correct shape and best quality are selected. The above two-dimensional classification and three-dimensional differential correction can be repeated for multiple rounds to obtain the best quality protein particle image and density model. Finally, after homogeneous correction (homogeneous refinement) and non-uniform refinement (see Figure 6 D in the figure) to obtain high-quality three-dimensional density (see Figure 6 Based on the three-dimensional density obtained from cryo-EM data, the structural model of the protein complex was built using Coot software, and the molecular structure model of the small molecule compound was built based on the density (see Figure 6 F in). Then the structure was modified using Phenix software to obtain a high-quality structural model.

[0107] Example 2: According to Example 1, human cannabinoid receptor type I (CNR1) was transformed, expressed, purified and structurally analyzed.

[0108] The human cannabinoid receptor type I (CNR1) sequence is derived from GenBank No. NM_001160226.3. The N-terminal and C-terminal regions of the sequence were removed, stability mutations were made based on the existing structure, and the GFP10-11 fragment (underlined) was fused to the third intracellular region. The short peptide sequence corresponding to the A2A adenosine receptor (wavy line mark) was used at the joint. The amino acid sequence of the modified CNR1 receptor-GFP10-11 fusion protein is (SEQ ID NO.6):

[0109] GRGENFMDIECFMVLNPSQQLAIAVLSLTLGTFTVLENLLVLCVILHSRSLRCRPSYHFIGSLAVADLLGSVIFVYSFIDFHVFHRKDSRNVFLFKLGGVTASFT ASVGSLFLAAIDRYISIHRPLAYKRIVTRPKAVVAFCLMWTIAIVIAVLPLLGWUNCEKLQSVCSDIFPHIDKTYLMFWIGVVSVLLLFIVYAYMYILWKARRQLG HYLSTQTSLSKDL NEKRDHMVLLEFVTAAG LQKEIELAKTLVLILVVLIICWGPLLAIMVYDVFGKMNKLIKTVFAFCSMLCLLNSTVNPIIYALRSKDLRHAFRSMFPS

[0110] The nucleotide sequence encoding the CNR1 receptor-GFP10-11 fusion protein was cloned into the mammalian cell expression vector pBacMam4R (see Figure 7), with a signal peptide sequence and a maltose binding protein fusion tag at the N-terminus. The internal ribosome entry site (IRES) downstream of the sequence can co-express the GFP1-9 fragment. The cloned plasmid was amplified and a large amount of plasmid DNA was extracted using an endotoxin-free plasmid extraction kit, and transfected into HEK293F cells in good growth state by PEI-40K. 5mM sodium butyrate was added 24 hours after transfection, and cell fluorescence was observed after another 24 hours and the cells were collected by centrifugation at 2000rpm for 2 minutes. The cells were resuspended in a buffer solution (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8) and disrupted with a cell ultrasonic disruptor. Lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) were added to a final concentration of 1%, and the cell membrane was melted by rotation at 4°C for 2 hours. 4℃ high speed centrifugation, 20000g, centrifugation for 30min, supernatant mixed with appropriate volume of amylose resin, 4℃ rotating mixing for 1.5 hours. After full binding, 4℃, 2000rpm centrifugation for 2min, discard the supernatant, retain the resin bound to the protein. Transfer the amylose resin to a gravity column, wash the impurities with washing buffer (500mM NaCl, 5% glycerol, 50mMTris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), elute the target protein with elution buffer (500mM NaCl, 5% glycerol, 20mM maltose, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), collect and measure the concentration, and then concentrate the protein to 500μL with a centrifugal filter-10K. The concentrated protein was separated by Superdex200Increase 10 / 300GL molecular sieve chromatography column, the mobile phase was buffer (500mM NaCl, 5% glycerol, 50mM Tris-HCl pH 7.8, 0.01% LMNG, 0.001% CHS), and the protein in the peak area of ​​the ultraviolet absorption peak corresponding to the target protein complex was collected. An appropriate excess of GFP-clamp protein was added to the collected protein to form a complex, and then separated again by Superdex 200Increase 10 / 300GL molecular sieve chromatography column, the mobile phase was buffer (150mMNaCl, 20mM HEPES pH 7.4, 0.005% LMNG, 0.0005% CHS), and the protein in the peak area of ​​the ultraviolet absorption peak corresponding to the target protein complex was collected, and the protein was concentrated to 10-15mg / mL with a centrifugal filter. The components of the obtained sample were analyzed and identified by SDS-PAGE gel electrophoresis.

[0111] Molecular sieve chromatography and SDS gel electrophoresis showed that the protein had good properties (see Figure 8A and B in Figure 1), the components contained were consistent with expectations. However, after preliminary data collection and processing using a 200kV Talos Arctica cryo-electron microscope, it was found that there was still a large degree of flexibility between the transmembrane domain of CNR1 and the water-soluble part containing GFP10-11, GFP1-9, and GFP-clamp, and a high-resolution structure could not be obtained (see Figure 8 C, D, E).

[0112] Example 3: Expression and purification of GFP-clamp cysteine ​​mutation and GFP-clamp-Cys mutant protein

[0113] The glutamic acid at position 186 of GFP-clamp was mutated to cysteine ​​(border mark), and the other parts including the maltose binding protein and TEV protease cleavage site at the N-terminus and the histidine tag at the C-terminus remained unchanged. The amino acid sequence of GFP-clamp-Cys is (SEQ ID NO.7):

[0114]

[0115] The plasmid carrying the nucleotide sequence encoding the above GFP-clamp-Cys was transformed into Rosetta TM2(DE3) competent cells. Pick positive monoclones into 20mL LB medium with resistance, and culture the cells in small amounts until they are supersaturated; transfer 20mL of the cells to 800mL of culture medium containing ampicillin and chloramphenicol resistance for amplification, and when the absorbance of the bacterial solution at 600nm is around 0.6, add 0.5mM isopropyl-β-D-thiogalactoside, and induce overnight at 20℃. Collect the bacterial solution into a large-capacity centrifuge bottle, centrifuge at 4℃, 4000rpm for 30min, remove the supernatant medium, and retain the precipitate. Resuspend the precipitate with a buffer solution containing 500mM NaCl, 5% glycerol, 20mM imidazole, 2mM tris(2-carbonylethyl) phosphate hydrochloride (TCEP), 50mM Tris-HCl, pH 7.8, add appropriate amounts of lysozyme, nuclease and phenylmethylsulfonyl fluoride, and use a cell ultrasonic disruptor to break the bacteria. The above-mentioned ultrasonic suspension was centrifuged again at high speed, 4°C, 20000g, centrifuged for 30min, and the supernatant was retained. Take a suitable volume of Ni-NTA resin and combine it with the supernatant, and rotate and mix at 4°C for 1.5h. After sufficient combination, centrifuge at 4°C, 2000rpm for 2min, discard the supernatant, and retain the resin bound to the protein. Transfer the Ni-NTA resin to a gravity column, wash the impurities with the above buffer, wash about 15-20 column volumes, elute the protein with elution buffer (500mM NaCl, 5% glycerol, 500mM imidazole, 50mM Tris-HCl, 2mM TCEP, pH 7.8), collect and measure the protein concentration, and add TEV protease enzyme digestion overnight at a mass ratio of one-tenth. Dilute with a buffer containing 500mM NaCl, 5% glycerol, 50mM Tris-HCl, 2mM TCEP, pH 7.8 to an imidazole concentration of 20mM, and combine with an appropriate amount of Ni-NTA resin again. The resin was washed with 500 mM NaCl, 5% glycerol, 20 mM imidazole, 2 mM TCEP, 50 mM Tris-HCl, pH 7.8, and the protein was eluted with 500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, 2 mM TCEP, pH 7.8. The protein buffer was exchanged with 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8 using a desalting column. The results of molecular sieve chromatography and SDS gel electrophoresis identification after purification of GFP-clamp-Cys protein are shown in Fig. 9 .

[0116] Example 4: Structural analysis of the complex between the cannabinoid receptor type I (CNR1) cysteine ​​mutant and the inhibitor taranabant

[0117] Based on the CNR1 and GFP10-11 fusion protein sequence in Example 2, the leucine at position 222 (Ballesteros-Weinstein numbering system 34.51) in the second intracellular loop was mutated to cysteine ​​(border mark). The amino acid sequence of the modified CNR1-Cys_GFP10-11 is (SEQ ID NO.8):

[0118]

[0119] The nucleotide sequence encoding the above SEQ ID NO.8 was cloned into the mammalian cell expression vector pBacMam4R, with a signal peptide sequence and a maltose binding protein fusion tag at the N-terminus. The internal ribosome entry site (IRES) downstream of the sequence can co-express the GFP1-9 fragment. The cloned plasmid was amplified and a large amount of plasmid DNA was extracted using an endotoxin-free plasmid extraction kit, and transfected into HEK293F cells with good growth status by PEI-40K. 5mM sodium butyrate was added 24 hours after transfection, and cell fluorescence was observed after another 24 hours and the cells were collected by centrifugation at 2000rpm for 2 minutes. The cells were resuspended with a buffer solution (500mMNaCl, 5% glycerol, 50mM Tris-HCl pH 7.8), the purified GFP-clamp-Cys protein was added, and the cells were broken with a cell ultrasonic disruptor. Add 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% cholesterol hemisuccinate (CHS) to a final concentration, rotate at 4°C for 2 hours to dissolve the cell membrane. Centrifuge at 4°C at 20,000g for 30 minutes, mix the supernatant with an appropriate volume of Ni-NTA resin, and rotate at 4°C for 1.5 hours. After sufficient binding, centrifuge at 4°C, 2000rpm for 2 minutes, discard the supernatant, and retain the protein-bound resin. The resin was transferred to a gravity column, and impurities were washed away with a washing buffer (500 mM NaCl, 5% glycerol, 20 mM imidazole, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS), and the target protein was eluted with an elution buffer (500 mM NaCl, 5% glycerol, 500 mM imidazole, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS). The protein eluted from the Ni-NTA resin was then combined with an appropriate volume of amylose resin, mixed by rotation at 4°C for 1.5 hours, and centrifuged at 4°C, 2000 rpm for 2 minutes. The supernatant was discarded, and the amylose resin was transferred to a gravity column. The impurities were washed with a wash buffer (500 mM NaCl, 5% glycerol, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS), and the target protein was eluted with an elution buffer (500 mM NaCl, 5% glycerol, 20 mM maltose, 50 mM Tris-HCl, pH 7.8, 0.01% LMNG, 0.001% CHS). After adding 1 mg of GFP-clamp-Cys, the protein was concentrated to 500 μL using a centrifugal filter-10K and further purified using a Superdex 200Increase 10 / 300GL molecular sieve chromatography column (see Fig.10A), the mobile phase was buffer (150 mM NaCl, 20 mM HEPES pH 7.4, 0.005% LMNG, 0.0005% CHS), and the protein in the peak area of ​​the UV absorption peak corresponding to the target protein complex was collected. The samples were mixed with reducing SDS loading buffer and non-reducing SDS loading buffer, respectively, treated at 37°C for 30 minutes, and the bands of the obtained sample components were analyzed by SDS gel electrophoresis (see Fig.10 In B, sample No. 1 is a sample treated under reducing conditions, and sample No. 2 is a sample treated under non-reducing conditions. In the following embodiments, the SDS gel electrophoresis of different GPCR samples is also that sample No. 1 is a sample treated under reducing conditions, and sample No. 2 is a sample treated under non-reducing conditions).

[0120] Taranabant, an antagonist of CNR1 receptor, was dissolved in DMSO to prepare a mother solution with a concentration of 200 mM. The mother solution was diluted 8 times with DMSO and then further diluted 100 times with water to obtain a taranabant solution with a concentration of 250 μM. It was added to the CNR1 protein sample purified by molecular sieve chromatography at a volume of 10%, and the protein was concentrated to 10-15 mg / mL using a centrifugal filter-10K. The Quantifoil Cu 300 1.2 / 1.3 electron microscope grid was hydrophilized using a PELCO easiGlow glow discharger. Using a Vitrobot freezing sample preparation instrument, 4 μL of protein sample was added to the hydrophilized grid, and after a waiting time of 4 seconds, it was blotted under the conditions of a force of 1-6 and a time of 3-5 seconds, and then quickly inserted into liquid ethane pre-cooled by liquid nitrogen for freezing, and then transferred to liquid nitrogen for storage. The prepared frozen sample was loaded into a Titan Krios cryo-EM equipped with a Gatan K2 direct electron detection camera, and image data of protein particles in the sample well were collected at an accelerating voltage of 300 kV. Data collection used super-resolution mode, with specific parameters such as pixel size Each image consists of 40 frames, and the total electron dose is

[0121] The collected data are corrected for the displacement caused by the electron beam using MotionCor2, and the contrast transfer function parameters of each image are estimated in the CryoSPARC software. Then the particles are selected and the local images of the protein particles are extracted. After several rounds of 2D classification and removal of erroneous particles, the initial 3D density model is reconstructed from scratch (ab initio reconstruction). These different reconstructed models are subjected to heterogeneous refinement, and the ones with correct shape and best quality are selected. The above 2D classification and 3D heterogeneous correction can be repeated for multiple rounds to obtain the best quality protein particle image and density model. Finally, after homogeneous refinement and non-uniform refinement (see Fig.10 C), and obtain high-quality three-dimensional density (see Fig.10 Based on the three-dimensional density obtained from cryo-EM data, the structural model of the protein complex was built using Coot software, and the molecular structure model of the small molecule compound was built based on the density (see Fig.10 Then the Phenix software was used to perform structural modification and obtain a high-quality structural model.

[0122] Example 5: Structural analysis of the complex between the neurokinin receptor NK1R and the inhibitor aprepitant

[0123] The NK1R amino acid sequence is derived from GenBank No. NM_001058.4. The C-terminal region of the sequence was removed, and a stability mutation was made according to the existing structure. The GFP10-11 fragment (underlined) was fused to the third intracellular region. The short peptide sequence corresponding to the A2A adenosine receptor was used at the joint (wavy line mark), and the leucine at position 138 in the second intracellular loop (Ballesteros-Weinstein numbering system 34.51) was mutated to cysteine ​​(border mark). The amino acid sequence of the modified NK1R-Cys_GFP10-11 fusion protein is (SEQ ID NO.9):

[0124]

[0125] The purification and identification method of the modified NK1R-Cys_GFP10-11 fusion protein is shown in Example 4, and the molecular sieve chromatography result of the modified NK1R-Cys_GFP10-11 fusion protein is shown in Fig.11 A in Figure 1, SDS gel electrophoresis identification results are shown in Figure 1 Fig.11 B in.

[0126] The NK1R receptor antagonist aprepitant was dissolved in DMSO to prepare a mother solution with a concentration of 187 mM. The mother solution was diluted 16 times with DMSO and further diluted 100 times with water to obtain a taranabant solution with a concentration of 116 μM. 10% volume was added to the above NK1R protein sample purified by molecular sieve chromatography, and the protein was concentrated to 10-15 mg / ml using a centrifugal filter-10K. The specific method of structural analysis is shown in Example 4. After homogeneity correction and heterogeneity correction (see Fig.11 C), and obtain high-quality three-dimensional density (see Fig.11 Based on the three-dimensional density obtained from the cryo-EM data, the structural model of the protein complex was built using Coot software, and the molecular structure model of the aprepitant was built based on the density of the aprepitant molecule (see Fig.11 Then the Phenix software was used to perform structural modification and obtain a high-quality structural model.

[0127] Example 6: Structural analysis of the complex of A2A adenosine receptor and agonist adenosine

[0128] The leucine at position 110 (Ballesteros-Weinstein numbering system 34.51) in the second intracellular loop of the A2A adenosine receptor sequence fused with the GFP10-11 fragment determined in Example 1 was mutated to cysteine ​​(border mark). The amino acid sequence of the modified A2A-Cys_GFP10-11 fusion protein is (SEQ ID NO.10):

[0129]

[0130] The purification and identification method of the modified A2A-Cys_GFP10-11 fusion protein is shown in Example 4, and the molecular sieve chromatography result of the modified A2A-Cys_GFP10-11 fusion protein is shown in Fig.12 A in Figure 1, SDS gel electrophoresis identification results are shown in Figure 1 Fig.12 B in.

[0131] The A2A adenosine receptor complex purified by molecular sieve chromatography was concentrated to 10-15 mg / ml using a centrifugal filter (molecular weight cutoff 10 kDa). Adenosine was dissolved in pure water to prepare a mother solution with a concentration of 10 mM. 10% of the solution was added to the concentrated protein solution to a final concentration of 1 mM. The specific method of structural analysis is shown in Example 4. After homogeneity correction and heterogeneity correction (see Fig.12 C), and obtain high-quality three-dimensional density (see Fig.12Based on the three-dimensional density obtained from the cryo-EM data, the structural model of the protein complex was built using Coot software, and its molecular structure model was built based on the density of the adenosine molecule (see Fig.12 Then the Phenix software was used to perform structural modification and obtain a high-quality structural model.

[0132] Example 7: Structural analysis of chemokine receptor CCR8

[0133] The chemokine receptor CCR8 sequence is from the amino acid sequence with GenBank number NM_005201.4. The first amino acid is removed from the sequence, and the GFP10-11 fragment (underlined) is fused to the third intracellular region. The short peptide sequence corresponding to the A2A adenosine receptor is used at the joint (wavy line mark). V139 (Ballesteros-Weinstein numbering system 34.51) in the second intracellular loop is mutated to cysteine ​​(border mark). The amino acid sequence of the modified fusion protein is (SEQ ID NO.11):

[0134]

[0135] The purification and identification method of the modified fusion protein is shown in Example 4, and the molecular sieve chromatography result of the modified fusion protein is shown in Fig.13 A in Figure 1, SDS gel electrophoresis identification results are shown in Figure 1 Fig.13 B in.

[0136] The CCR8 protein complex purified by molecular sieve chromatography was concentrated to 6 mg / ml using a centrifugal filter (molecular weight cutoff 10 kDa). The specific method of structural analysis is shown in Example 4. After homogeneity correction and heterogeneity correction (see Fig.13 C), and obtain high-quality three-dimensional density (see Fig.13 D). Based on the three-dimensional density obtained from the cryo-EM data, the structural model of the protein complex was built using Coot software, and then the structure was modified using Phenix software to obtain a high-quality structural model.

[0137] Example 8: Structural analysis of orphan receptor GPRC5D

[0138] The orphan receptor GPRC5D sequence is from the amino acid sequence with GenBank number NM_018654.2. The first amino acid is removed from the sequence, and the GFP10-11 fragment (underlined) is fused to the third intracellular region. The short peptide sequence corresponding to the A2A adenosine receptor is used at the joint (wavy line mark). The 121st amino acid in the second intracellular loop is cysteine ​​(border mark) and no mutation is made. The amino acid sequence of the modified fusion protein is (SEQ ID NO.12):

[0139]

[0140] The purification and identification method of the modified fusion protein is shown in Example 4, and the molecular sieve chromatography result of the modified fusion protein is shown in Fig.14 A in the figure, collect the protein sample at the second UV absorption peak pointed by the arrow, and the SDS gel electrophoresis identification results are shown in the figure Fig.14 B in.

[0141] The CCR8 protein complex purified by molecular sieve chromatography was concentrated to 6 mg / ml using a centrifugal filter (molecular weight cutoff 10 kDa). The specific method of structural analysis is shown in Example 4. After homogeneity correction and heterogeneity correction (see Fig.14 C), and obtain high-quality three-dimensional density (see Fig.14 D). Based on the three-dimensional density obtained from the cryo-EM data, the structural model of the protein complex was built using Coot software, and then the structure was modified using Phenix software to obtain a high-quality structural model.

[0142] The present invention also provides other GPCRs obtained by using the method for modifying G protein coupled receptors of the present invention.

[0143] The amino acid sequences of the following GPCRs were modified in the same manner as the above CNR1 and NK1R: 1. The third intracellular loop of these GPCRs was replaced with a GFP10-11 fragment (SEQ ID NO.13) with a connecting peptide from the A2A adenosine receptor on both sides: RRQLGHYLSTQTSLSKDLNEKRDHMVLLEFVTAAGLQKE; 2. The amino acid at 34.51 in the Ballesteros-Weinstein numbering system in the second intracellular loop was mutated to cysteine. The amino acid sequences of the modified fusion proteins of the 30 GPCRs and GFP10-11 are as follows:

[0144] GPR52-Cys-GFP10-11 (SEQ ID NO.14):

[0145]

[0146] MC4R-Cys-GFP10-11 (SEQ ID NO.15):

[0147]

[0148]

[0149] GNRHR-Cys-GFP10-11 (SEQ ID NO.16):

[0150]

[0151] PTGDR2-Cys-GFP10-11(SEQ ID NO.17):

[0152]

[0153] HTR2C-Cys-GFP10-11(SEQ ID NO.18):

[0154]

[0155] ADRA2B-Cys-GFP10-11(SEQ ID NO.19):

[0156]

[0157] ADRB1-Cys-GFP10-11(SEQ ID NO.20):

[0158]

[0159] ADRB2-Cys-GFP10-11(SEQ ID NO.21):

[0160]

[0161] C5AR1-Cys-GFP10-11(SEQ ID NO.22):

[0162]

[0163] CCR2-Cys-GFP10-11(SEQ ID NO.23):

[0164]

[0165] CCR5-Cys-GFP10-11(SEQ ID NO.24):

[0166]

[0167] CCR6-Cys-GFP10-11(SEQ ID NO.25):

[0168]

[0169] CCR7-Cys-GFP10-11(SEQ ID NO.26):

[0170]

[0171] CHRM2-Cys-GFP10-11(SEQ ID NO.27):

[0172]

[0173] CLTR2-Cys-GFP10-11(SEQ ID NO.28):

[0174]

[0175] CXCR2-Cys-GFP10-11(SEQ ID NO.29):

[0176]

[0177] CXCR4-Cys-GFP10-11(SEQ ID NO.30):

[0178]

[0179]

[0180] DRD2-Cys-GFP10-11(SEQ ID NO.31):

[0181]

[0182] DRD3-Cys-GFP10-11(SEQ ID NO.32):

[0183]

[0184] GPBAR-Cys-GFP10-11(SEQ ID NO.33):

[0185]

[0186] HRH1-Cys-GFP10-11(SEQ ID NO.34):

[0187]

[0188] HTR1A-Cys-GFP10-11(SEQ ID NO.35):

[0189]

[0190] HTR1B-Cys-GFP10-11(SEQ ID NO.36):

[0191]

[0192] HTR2B-Cys-GFP10-11 (SEQ ID NO.37):

[0193]

[0194] LPAR1-Cys-GFP10-11 (SEQ ID NO.38):

[0195]

[0196] MTNR1B-Cys-GFP10-11 (SEQ ID NO.39):

[0197]

[0198] NPY1R-Cys-GFP10-11 (SEQ ID NO.40):

[0199]

[0200] OPRD-Cys-GFP10-11 (SEQ ID NO.41):

[0201]

[0202] OX2R-Cys-GFP10-11 (SEQ ID NO.42):

[0203]

[0204] PTGDR-Cys-GFP10-11 (SEQ ID NO.43):

[0205]

[0206] The nucleotide sequences encoding the modified GPCR and GFP10-11 fusion proteins were cloned into the mammalian cell expression vector pBacMam4R, and transfected into HEK293F cells by the same steps as in Example 1 to co-express the GPCR and GFP10-11 fusion proteins and the GFP1-9 fragment. After the cells were harvested, the GFP-clamp-Cys protein obtained in Example 3 was added and purified by the same steps as in Example 7. The purified proteins were analyzed by molecular sieve chromatography and SDS gel electrophoresis. The results showed that these modified GPCRs also had good properties (see Figure 15-Figure 44 ).

[0207] Because most G protein-coupled receptors have similar overall structures and secondary structure combinations, the GPCRs to which the method for modifying G protein-coupled receptors described in the present invention is applicable include but are not limited to 5-HT1A, 5-HT1B, 5-HT1D, -5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT4, 5HT5A, 5-HT6, 5-HT7, M1, M2, M3, M4, M5, A1, A2A, A2B, A3, ADA1A, ADA1B, ADA1D, ADA2A, ADA2B, ADA2C, ADRB1, ADRB2, ADRB3, C3a, C5a, C5L2, AT1, AT2, and AP J, GPBA, BB1, BB2, BB3, B1, B2, CB1, CB2, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 , CX3CR1, H, GnRH2, H1, H2, H3, H4, HCA1, HCA2, HCA3, KISSR, BLT1, BLT2, CysLT1, CysLT2, OXE, FPR2 / ALX, LPA1, LPA2, LPA3, LPA4, LPA5, S1P1, S1P2, S1P3, S1P4, S1 P5, MCH1, MCH2, MC1, MC2, MC3, MC4, MC5, MT1, MT2, MTLR, NMU1, NMU2, NPFF1, NPFF2, NPS, NPBW1, NPBW2, Y1, Y2, Y4, Y5, NTS1, NTS2, delta, kappa, mu, NOP, OX1, OX2, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14, QRFP, PAF, PKR1, PKR2, PRRP, DP1, DP2, EP1, EP2, EP3, EP4, FP, IP1, TP, PAR1, PAR2, PAR3, PAR4, RXFP1, RXFP2, RXFP3, RXFP4, SST1, SST2, SST3, SST4, SST5, NK1, NK2, NK3, TRH1, TA1, UT, V1A, V1B, V2, OT, CCRL2, CMKLR1, GPR1, GPR3, GPR4, GPR6,GPR12、GPR15、GPR17、GPR18、GPR19、GPR20、GPR21、GPR22、GPR25、GPR26、GPR27、GPR31、GPR32、GPR33、GPR34、GPR35、GPR37、GPR37L1、GPR39、GPR42、GPR45、GPR50、GPR52、GPR55、GPR61、GPR62、GPR63、GPR65、GPR68、GPR75、GPR78、GPR79、GPR82、GPR83、GPR84、GPR85、GPR87、GPR88、GPR101、GPR119、GPR120、GPR132、GPR135、GPR139、GPR141、GPR142、GPR146、GPR148、GPR149、GPR150、GPR151、GPR152、GPR153、GPR160、GPR161、GPR162、GPR171、GPR173、GPR174、GPR176、GPR182、GPR183、LGR4、LGR5、LGR6、LPAR6、MAS1、MAS1L、MRGPRD、MRGPRE、MRGPRF、MRGPRG、MRGPRX1、MRGPRX2、MRGPRX3、MRGPRX4、OPN3、OPN5、OXGR1、P2RY8、P2RY10、SUCNR1、TAAR2、TAAR3、TAAR4、TAAR5、TAAR6、TAAR8、TAAR9、CCPB2、CCRL1、FY、CT、CALRL、CRF1、CRF2、GHRH、GIP、GLP-1、GLP-2、GCGR、SCTR、PTH1、PTH2、PAC1、VPAC1、VPAC2、BAI1、BAI2、BAI3、CD97、CELSR1、CELSR2、CELSR3、ELTD1、EMR1、EMR2、EMR3、EMR4P、GPR56、GPR64、GPR97、GPR98、GPR110、GPR111、GPR112、GPR113、GPR114、GPR115、GPR116、GPR123、GPR124、GPR125、GPR126、GPR128、GPR133、GPR143、GPR144、GPR157、LPHN1、LPHN2、LPHN3、CaS、GPRC6、GABAB1、GABAB2、mGlu1、mGlu2、mGlu3、mGlu4、mGlu5、mGlu6、mGlu7、mGlu8、GPR156、GPR158、GPR179、GPRC5A、GPRC5B、GPRC5C、GPRC5D、frizzled、FZD1、FZD2、FZD3、FZD4、FZD5、FZD6、FZD7、FZD8、FZD9、FZD10、SMO。、

Claims

1. A method for modifying a G protein-coupled receptor, characterized in that: The method comprises the following steps: co-expressing a fusion protein GPCR-GFP10-11 and a GFP1-9 fragment formed by a G protein coupled receptor and a GFP10-11 fragment in a eukaryotic cell expression system, and adding GFP-clamp-Cys during purification to obtain a ternary complex formed by GPCR-GFP10-11, GFP1-9 and GFP-clamp-Cys; the amino acid at position 34.51 in the Ballesteros-Weinstein numbering system in the second intracellular loop of the GPCR is mutated to cysteine, the amino acid sequence of the GFP1-9 fragment is shown in SEQ ID NO.1, the amino acid sequence of the GFP10-11 fragment is shown in SEQ ID NO.2, and the glutamic acid at position 186 of the GFP-clamp-Cys is mutated to cysteine ​​based on the amino acid sequence shown in SEQ ID NO.

3.

2. The method according to claim 1, characterized in that The fusion protein GPCR-GFP10-11 is obtained by replacing the third intracellular loop or corner region between the fifth transmembrane helix and the sixth transmembrane helix of GPCR with GFP10-11 through a genetic engineering method.

3. The method according to claim 2, characterized in that One, two, three, four or five amino acids at the linker of the fusion protein GPCR-GFP10-11 can be replaced.

4. The method according to claim 1, characterized in that: The eukaryotic cells are insect cells or mammalian cells.

5. The method according to claim 1, characterized in that The ternary complex formed by G protein coupled receptor fusion protein GPCR-GFP10-11, GFP1-9 and GFP-clamp-Cys was purified by affinity chromatography and molecular sieve chromatography.

6. A ternary complex formed by the G protein coupled receptor fusion protein GPCR-GFP10-11, GFP1-9 and GFP-clamp-Cys obtained by the method according to any one of claims 1 to 5.

7. The ternary complex according to claim 6, characterized in that The total molecular weight of the ternary complex is above 85 kD.

8. The ternary complex according to claim 6, characterized in that In the ternary complex, the cysteine ​​produced by the mutation at position 34.51 of the Ballesteros-Weinstein numbering system in the second intracellular loop of the G protein coupled receptor forms a disulfide bond with the cysteine ​​at position 186 on GFP-clamp-Cys.

9. Use of the method described in any one of claims 1 to 5 or the ternary complex described in any one of claims 6 to 8 in analyzing the three-dimensional structure of a complex formed by a compound and a G protein-coupled receptor using cryo-electron microscopy structure analysis.

10. The use according to claim 9, characterized in that: The compounds are either inhibitors or agonists.

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