A fusion protein for cryo-em structure resolution and a construction method and application thereof

Abstract

This invention discloses a fusion protein for cryo-electron microscopy (cryo-EM) structural analysis, its construction method, and its applications. The method for constructing the fusion protein for cryo-EM structural analysis includes the following steps: inserting WD40 protein into the intracellular or extracellular loop region between adjacent transmembrane helices of the target membrane protein to obtain the fusion protein; the WD40 protein is WDR5 protein, and its amino acid sequence is shown in SEQ ID NO:1. Using the constructed fusion protein for cryo-EM structural analysis can improve particle identifiability features and enhance the quality of two-dimensional classification and three-dimensional reconstruction. The method provided by this invention has good reproducibility and scalability, and is applicable to the cryo-EM structural analysis of different types of membrane proteins.

Description

Technical Field

[0001] This invention relates to the fields of structural biology and protein engineering, and in particular to a fusion protein for cryo-electron microscopy structural analysis, its construction method, and its applications. Background Technology

[0002] Membrane proteins play crucial roles in vital processes such as cellular material transport, signal transduction, and energy metabolism, making them important research subjects in drug development and basic life science research. However, membrane proteins are typically characterized by strong hydrophobicity, high conformational flexibility, and poor in vitro stability, making high-resolution three-dimensional structural determination of them a significant technical challenge for a long time.

[0003] With the development of cryo-electron microscopy (cryo-EM), membrane proteins can be structurally resolved in near-native states without crystallization, making it an important tool for membrane protein structure research. However, in practical applications, especially for membrane proteins with small molecular weights, low symmetry, or existing in monomeric form, problems such as insufficient particle features, severe orientation bias, and difficulties in two-dimensional classification and three-dimensional reconstruction are still common, thus limiting further improvements in the resolution of cryo-EM structure resolution.

[0004] To address these issues, various auxiliary strategies have been proposed in existing technologies. For example, fusion tags or domains can be introduced into the N-terminus, C-terminus, or transmembrane region of the target membrane protein to enhance identifiable features in cryo-electron microscopy. However, the domains introduced in existing technologies have excessive conformational flexibility or interfere with the native conformational stability of the target membrane protein, which is detrimental to high-quality acquisition and three-dimensional reconstruction of cryo-electron microscopy data.

[0005] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a fusion protein for cryo-electron microscopy structure resolution, its construction method and application, aiming to solve the problem of low resolution in existing cryo-electron microscopy structure resolution.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing a fusion protein for cryo-electron microscopy structural analysis, comprising the following steps: WD40 protein is inserted into the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein to obtain a fusion protein; The WD40 protein is the WDR5 protein, and its amino acid sequence is shown in SEQ ID NO:1.

[0008] Preferably, WD40 protein is inserted into the flexible or disordered region of the intracellular loop or extracellular loop between adjacent transmembrane helices of the target membrane protein.

[0009] Optionally, inserting WD40 protein into the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein specifically includes: truncating the flexible or disordered regions in the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein, and inserting the WD40 protein at the truncated position. Preferably, the truncation is a truncation of flexible amino acids.

[0010] Preferably, the step of truncating the flexible or disordered regions in the intracellular or extracellular loop regions between adjacent transmembrane helices of the target membrane protein and inserting the WD40 protein at the truncated position specifically includes: deleting the flexible amino acids between transmembrane helices 2 and 3 of the target membrane protein and inserting the WD40 protein at the deleted position; the target membrane protein is a vesicle GABA transporter.

[0011] Preferably, the step of truncating the flexible or disordered regions in the intracellular or extracellular loop regions between adjacent transmembrane helices of the target membrane protein and inserting the WD40 protein at the truncated position specifically includes: deleting the flexible amino acids between transmembrane helices 8 and 9 of the target membrane protein and inserting the WD40 protein at the deleted position; the target membrane protein is a vesicle GABA transporter.

[0012] Optionally, the target membrane protein is a transmembrane transport protein or an ion channel protein.

[0013] In a second aspect, the present invention provides a fusion protein obtained by the above-described construction method.

[0014] In a third aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned fusion protein.

[0015] In a fourth aspect, the present invention provides a recombinant vector comprising the above-described nucleic acid molecules.

[0016] In a fifth aspect, the present invention provides a host cell comprising the above-described nucleic acid molecule or the above-described recombinant vector.

[0017] In a sixth aspect, the present invention provides the use of the fusion protein, the nucleic acid molecule, the recombinant vector, or the host cell described above in the determination of the structure of a target membrane protein and the screening and validation of drug targets for the target membrane protein.

[0018] Beneficial effects: This invention provides a fusion protein for cryo-electron microscopy structural analysis, its construction method, and its applications. Compared with existing technologies, the advantages of this invention are: 1. Improve the quality of 2D classification and 3D reconstruction First, the construction method provided by this invention does not affect the folding and function of the target membrane protein. Second, since the conformational wobble of the fusion domain (WD40 protein) is effectively restricted, the noise interference caused by conformational heterogeneity during cryo-electron microscopy data acquisition is reduced, which is beneficial to improving the stability and consistency of the constructed fusion protein in two-dimensional classification and further improving the quality and reliability of its three-dimensional reconstruction.

[0019] 2. The fusion domain has a stable conformation and strong versatility. This invention employs a fusion domain (WD40 protein) with a stable folded structure and high conformational rigidity, which can be introduced as an independent rigid module into different types of target membrane proteins, exhibiting good reproducibility and scalability.

[0020] 3. Suitable for membrane proteins with small molecular weight or low symmetry. The construction method provided by this invention can improve the overall molecular weight and structural characteristics of membrane proteins without significantly affecting their native conformation and functional stability. Therefore, it is particularly suitable for membrane proteins with small molecular weight, low symmetry, or existing in monomeric form that are difficult to resolve using traditional cryo-electron microscopy methods. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the method for constructing fusion proteins for cryo-electron microscopy structural analysis of membrane proteins according to the present invention and its application.

[0022] Figure 2 This is a schematic diagram illustrating the construction of the fusion protein used for cryo-electron microscopy structural analysis in Example 1 of the present invention. The WD40 protein (WDR5) is fused to the intracellular loop region between TM2 and TM3 of the target membrane protein (vGAT). This fusion construct is abbreviated as vGAT. EM .

[0023] Figure 3 vGAT fusion protein EM Samples and unfused membrane protein vGAT WT The expression and purification diagrams of the samples include protein gel SDS-PAGE and molecular sieve SEC assays.

[0024] Figure 4 vGAT fusion protein EM Samples and unfused membrane protein vGAT WT Diagram of GABA isotope uptake of cells by vesicles in the sample, 5 independent replicates.

[0025] Figure 5 vGAT fusion protein EM Samples and unfused membrane protein vGAT WT Images showing the two-dimensional particle classification results and three-dimensional reconstruction results from cryo-electron microscopy of the samples.

[0026] Figure 6 vGAT fusion protein EM-1 Three-dimensional reconstruction results of cryo-electron microscopy particles of the fusion proteins vGAT-GFP and vGAT-BRIL. Detailed Implementation

[0027] This invention provides a fusion protein for cryo-electron microscopy structural analysis, its construction method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0028] Current technology still lacks a universal method that can significantly improve the alignment quality and 3D reconstruction resolution of cryo-electron microscopy particles through rational fusion construction while ensuring the native conformation and functional stability of membrane proteins. Developing a membrane protein fusion construction strategy with good reproducibility and scalability has become a pressing technical problem to be solved in the field of cryo-electron microscopy structural biology.

[0029] Based on this, such as Figure 1 As shown, this embodiment of the invention provides a method for constructing a fusion protein for cryo-electron microscopy structural analysis, comprising the following steps: WD40 protein is inserted into the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein to obtain a fusion protein; The WD40 protein is the WDR5 protein, and its amino acid sequence is shown in SEQ ID NO:1. The nucleotide sequence encoding the WDR5 protein is shown in SEQ ID NO:2.

[0030] This invention introduces WD40 protein into a target membrane protein to obtain a fusion protein, and uses the fusion protein for cryo-electron microscopy sample preparation and structural analysis. Verification has shown that this method significantly improves the particle recognition of the target membrane protein in cryo-electron microscopy, thereby significantly improving particle alignment quality and 3D reconstruction resolution. Specifically, the fusion domain used in this invention is the WD40 protein. The WD40 protein domain consists of multiple WD repeat units in a β-propeller folding configuration, which can fold into a typical β-propeller architecture, exhibiting overall conformational stability, high rigidity, and clear spatial anisotropy. The WD40 protein domain provided by this invention has the key structural advantage of its N-terminus and C-terminus being spatially close to each other. By fusing it with the target protein, both can be integrated into a rigid β-sheet framework of a propeller, further improving the particle alignment quality and 3D reconstruction resolution of the target membrane protein in cryo-electron microscopy. In contrast, GFP has a β-barrel structure and can rotate, so GFP as a fusion domain lacks sufficient rigidity and stability; BRIL has a smaller structure and stronger rigidity, but it lacks features. Therefore, BRIL as a fusion domain may interfere with the native conformational stability of the target membrane protein, which is not conducive to high-quality acquisition and three-dimensional reconstruction of cryo-electron microscopy data.

[0031] Preferably, WD40 protein is inserted into the flexible or disordered region of the intracellular loop or extracellular loop between adjacent transmembrane helices of the target membrane protein.

[0032] Because the N-terminus and C-terminus of the β-sheet in the WD40 protein are spatially adjacent and embedded in a rigid β-sheet framework in its native structure, fusion of WD40 with a target protein can integrate them into the rigid β-sheet framework of a propeller. This folding is primarily determined by the WD40 repeat sequence itself, without relying on external binding interfaces. However, if the fusion of WD40 with the target protein occurs in the structural core or rigid region of the target protein, it can easily cause mechanical interference with the closure of the β-sheet at the WD40 terminal. Therefore, selecting flexible or disordered regions in the target protein as fusion sites is a crucial step in ensuring the complete formation of the β-sheet framework. By inserting WD40 into these flexible or disordered regions, the resulting fusion protein does not alter the folding and function of the target membrane protein. Furthermore, appropriate linkers can be used when inserting WD40.

[0033] In this invention, the term "β-sheet" refers to a secondary structure of a protein, formed by parallel or antiparallel β-chains linked by hydrogen bonds. β-sheet frameworks are commonly used to provide protein stability. Specifically, a β-sheet framework refers to a framework in which both the fusion domain (WD40 protein) and the target membrane protein are integrated into a propeller-rigid β-sheet framework, thereby reducing the conformational freedom of the fusion domain, improving the particle recognition properties of the target membrane protein in cryo-electron microscopy, and enhancing the resolution of structural analysis.

[0034] Conformational degrees of freedom refer to the range of spatial rotations or alterations a molecule or protein can undergo. In some cases, excessively high degrees of conformational freedom can lead to structural instability or make data difficult to resolve. In this invention, by inserting the WD40 protein, the conformational changes of the fusion domain in cryo-electron microscopy analysis are reduced, thereby improving the resolution of its structural resolution.

[0035] In addition, the present invention can also use a variety of WD40 proteins, such as RACK1 and Gβ, which have folding stability, high conformational rigidity and obvious spatial anisotropy of β-propeller structure features. They can also be introduced into the target membrane protein as independent rigid body modules to provide stable structural features for cryo-electron microscopy particle alignment and three-dimensional reconstruction.

[0036] It is understood that this invention discloses a method that can significantly improve the structural resolution of target membrane proteins in cryo-electron microscopy. In this method, WD40 protein is fused into different positions of the target membrane protein, all of which significantly improve the structural resolution.

[0037] In some embodiments, inserting WD40 protein into the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein specifically includes: truncating the flexible or disordered regions in the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein, and inserting the WD40 protein at the truncated position.

[0038] In this embodiment, the method of introducing WD40 protein into the target membrane protein specifically includes the following two steps: Step 1, Determination of the structural regions of the target membrane protein: By analyzing the amino acid sequence and structural characteristics of the target membrane protein, transmembrane regions, intracellular loop regions, extracellular loop regions, and N-terminal or C-terminal regions can be determined. Regions that do not significantly affect the native conformation or biological function of the membrane protein (i.e., flexible or disordered regions) are selected as potential fusion sites. Step 2, Selection of fusion sites: The fusion domain (WD40 protein) is attached to the intracellular loop region between adjacent transmembrane helices of the target membrane protein. By rationally selecting the fusion site, the fusion domain provides additional morphological features in cryo-electron microscopy while avoiding adverse effects on the bulk structure of the membrane protein. Therefore, this invention introduces a fusion domain by selecting regions that do not affect the native conformation of the membrane protein as fusion sites, thereby not affecting the folding and function of the target membrane protein.

[0039] In some preferred embodiments, the truncation refers to truncating flexible amino acids in intracellular or extracellular loops.

[0040] In some preferred embodiments, the flexible amino acids of the intracellular loop between adjacent transmembrane helices of the target membrane protein are truncated, and the WD40 protein is fused at the truncated position.

[0041] Those skilled in the art will understand that the "flexible amino acid" is typically an amino acid with a small side chain that does not affect the conformation of the fusion protein. The flexible amino acid in this invention can be asparagine, alanine, glycine, serine, etc., and will not be listed here.

[0042] In this embodiment, flexible amino acids were chosen to be deleted because these regions inherently possess significant flexibility. Deleting these amino acids provides space for the insertion of the fusion protein without affecting the function of the target membrane protein. Therefore, when the WD40 domain is introduced at this location, the key structural advantage of the WD40 domain's N-terminus and C-terminus being spatially close allows the fusion domain to be stably integrated into the membrane protein. By integrating the WD40 protein into the rigid β-sheet framework of the membrane protein's propeller, the conformational freedom of the fusion domain is significantly reduced, improving the overall structural stability of the target membrane protein, thereby providing clearer and more stable structural data for cryo-electron microscopy analysis. This structural optimization not only enhances the identifiability of the membrane protein but also improves the accuracy of image alignment and the resolution of 3D reconstruction during cryo-electron microscopy reconstruction.

[0043] In this invention, the "intracellular loop (ICL)" refers to the loop-shaped structure located on the intracellular side (i.e., the cytoplasmic side) of a membrane protein and connecting adjacent transmembrane helical regions. Membrane proteins are typically composed of multiple transmembrane regions (transmembrane helices), and the intracellular loop is located between these transmembrane regions, directly exposed to the cytoplasmic environment.

[0044] In this invention, the "extracellular loop (ECL)" refers to the loop-shaped structural portion of a membrane protein located on the extracellular side (i.e., the side of the extracellular environment) connecting adjacent transmembrane helical regions. The transmembrane helical portion of the membrane protein crosses the cell membrane, while the extracellular loop is exposed outside the cell.

[0045] In other embodiments, when inserting the WD40 protein into the target membrane protein, the present invention may use a linker structure (such as a linker peptide) to connect the two.

[0046] In this embodiment, the connecting structure can be used to restrict the orientation change of the fusion domain relative to the target membrane protein during cryo-electron microscopy data acquisition, thereby providing a stable and identifiable structural feature. Specifically, the fusion domain and the target membrane protein can be connected by a connecting structure. By reducing the relative conformational freedom between the fusion domain and the membrane protein through the connecting structure, the flexible wobbling of the fusion domain in cryo-electron microscopy is reduced, thereby improving the stability of particle alignment.

[0047] In some preferred embodiments, the step of truncating the flexible or disordered regions in the intracellular or extracellular loop regions between adjacent transmembrane helices of the target membrane protein and inserting the WD40 protein at the truncated position specifically includes: deleting the flexible amino acids between transmembrane helices 2 and 3 of the target membrane protein and inserting the WD40 protein at the deleted position; the target membrane protein is a vesicle GABA transporter.

[0048] In this embodiment, the amino acid deletion region comprises all flexible regions between TM2 and TM3 of the vesicular GABA transporter. Amino acid deletions can be performed as needed without affecting the basic helix structure of the vesicular GABA transporter. In this invention, the modification site is the deletion of asparagine from position 174 (N-terminus) to glycine from position 177 (N-terminus) of the vesicular GABA transporter. The modified fusion protein retains the helix characteristics of the wild-type vesicular GABA transporter while avoiding damage to the overall structure of the target protein after fusion.

[0049] In some preferred embodiments, the step of truncating the flexible or disordered regions in the intracellular or extracellular loop regions between adjacent transmembrane helices of the target membrane protein and inserting the WD40 protein at the truncated position specifically includes: deleting the flexible amino acids between transmembrane helices 8 and 9 of the target membrane protein and inserting the WD40 protein at the deleted position; the target membrane protein is a vesicle GABA transporter.

[0050] In this embodiment, the amino acid deletion region comprises all flexible regions between TM8 and TM9 of the vesicular GABA transporter. Amino acid deletions can be performed as needed without affecting the basic helix structure of the vesicular GABA transporter. In this invention, the modification site involves the deletion of glycine at position 413 and serine at position 414, starting from the N-terminus of the vesicular GABA transporter. The modified fusion protein retains the helix characteristics of the wild-type vesicular GABA transporter while avoiding damage to the overall structure of the target protein after fusion.

[0051] In some embodiments, the target membrane protein is a transmembrane transport protein or an ion channel protein.

[0052] The construction method provided by this invention is applicable to different types of target membrane proteins, including transmembrane transport proteins and ion channel proteins. The target membrane protein is not limited to having a specific number of transmembrane domains or a specific length of inter-helical loop region. As long as the target membrane protein has an exposed region that does not significantly affect its native conformation and functional stability, it can be used as the target of the fusion construction method described in this invention. The construction method provided by this invention combines fusion sites and fusion domains (WD40 protein) for systematic design, significantly improving the identifiable features of membrane proteins in cryo-electron microscopy without significantly affecting the native conformation and functional stability of the target membrane protein. This improves the two-dimensional classification effect and the three-dimensional reconstruction quality, thereby increasing the structural resolution. This is because the construction method provided by this invention can improve the overall molecular weight and structural features without significantly affecting the native conformation and functional stability of the membrane protein. Therefore, this construction method is particularly suitable for membrane proteins with small molecular weights, low symmetry, or existing in monomeric form that are difficult to resolve using traditional cryo-electron microscopy methods.

[0053] An embodiment of the present invention provides a fusion protein, which is obtained by the above-described construction method.

[0054] In this invention, a fusion protein is defined as a new protein containing multiple functional regions formed by combining two or more different protein sequences through genetic engineering. In some applications, fusion proteins are commonly used for labeling, functional enhancement, or protein structural studies.

[0055] An embodiment of the present invention provides a nucleic acid molecule that encodes the above-mentioned fusion protein.

[0056] An embodiment of the present invention provides a recombinant vector comprising the above-described nucleic acid molecules.

[0057] In this invention, "recombinant vector" can also be referred to as "expression vector," which refers to an expression vector capable of expressing the target protein in a suitable host cell. It is a gene construct containing operably linked basic regulatory elements, wherein the operably linked basic regulatory elements enable the expression of the inserted gene. Preferably, the recombinant vector is constructed to carry a nucleic acid molecule or fragment thereof encoding the target membrane protein and WD40 protein of this invention. The recombinant vector can be transformed or transfected into host cells.

[0058] The expression vector of the present invention can also be obtained by linking (inserting) the nucleic acid molecule of the present invention into a suitable vector. There are no particular limitations on the vector into which the gene of the present invention will be inserted, as long as it can replicate within the host. For example, plasmid vectors, bacteriophage vectors, viral vectors, etc., can be used. Specifically, commercially available expression vectors can be used, as can animal viruses such as retroviruses, adenoviruses, and vaccinia viruses, and insect viruses such as baculoviruses. The plasmids that can be used in the present invention are not limited to the examples described.

[0059] In order to operatively link the nucleic acid molecules of the present invention to a vector, the vector of the present invention may contain, in addition to the promoter and the polynucleotide of the present invention, cis elements such as enhancers, splicing signals, poly A addition signals, selection markers and ribosome binding sequences.

[0060] An embodiment of the present invention provides a host cell comprising the above-described nucleic acid molecule or the above-described recombinant vector.

[0061] The constructed vector can be introduced into host cells via transformation (or transfection). Transformation can be performed using any method. Common transformation methods include: CaCl2 precipitation; electroporation; calcium phosphate precipitation; protoplasmic fusion; silica cellulose-mediated transformation; Agrobacterium-mediated transformation; PEG-mediated transformation; and transformation using dextran sulfate, cationic liposomes (ipofectamine), and drying / inhibition methods. Using the vector described above and transfection with that vector, the gene encoding the GRAB probe of this invention can be introduced into host cells.

[0062] There are no particular limitations on the host cell used in this invention, as long as it can express the target membrane protein and WD40 protein of this invention. For example, the host cell can be bacteria, yeast, mammalian cells, etc. In a preferred embodiment, the host cell is HEK293S GNTI. - cell.

[0063] The embodiments of the present invention provide the application of the above-described fusion protein, the above-described fusion protein, the above-described nucleic acid molecule, the above-described recombinant vector, or the above-described host cell in the determination of target membrane protein structures and the screening and validation of target membrane protein drug targets.

[0064] This invention provides a general method that can significantly improve the alignment quality and 3D reconstruction resolution of cryo-electron microscopy particles by rationally fusing and constructing structures while ensuring the native conformation and functional stability of membrane proteins. This method can significantly improve the identifiable features of particles and enhance the quality of 2D classification and 3D reconstruction.

[0065] Furthermore, this invention establishes a membrane protein fusion construction strategy with good reproducibility and scalability. This invention has wide applicability and is widely applicable to different types of membrane proteins; it is particularly suitable for membrane proteins with small molecular weight, low symmetry, or existing in monomeric form that are difficult to resolve using traditional cryo-electron microscopy methods.

[0066] In summary, the technical solution adopted in this invention specifically includes the following steps: Step S1: Determination of the target membrane protein structural region The amino acid sequence and structural features of the target membrane protein are analyzed to identify transmembrane regions, intracellular loop regions, extracellular loop regions, and N-terminal or C-terminal regions. Regions that do not significantly affect the native conformation or biological function of the membrane protein are selected as potential fusion sites.

[0067] Step S2: Selection of fusion sites The fusion domain is attached to the intracellular loop region between adjacent transmembrane helices of the target membrane protein. By carefully selecting the fusion site, the fusion domain provides additional morphological features in cryo-electron microscopy while avoiding adverse effects on the protein's bulk structure.

[0068] Step S3: Selection of fusion domain The fusion domain is selected from protein domains with stable folding structures. By introducing this fusion domain, the overall characteristic density of the target membrane protein in cryo-electron microscopy is increased.

[0069] Step S4: Design of the connection structure The fusion domain is connected to the target membrane protein via a linker structure that provides conformational constraint. This linker structure reduces the relative conformational freedom between the fusion domain and the membrane protein, thereby minimizing the flexible wobbling of the fusion domain in cryo-electron microscopy and improving particle alignment stability.

[0070] Step S5: Expression of the fusion protein The fusion gene (a nucleic acid molecule encoding a fusion protein) is cloned into an expression vector, and the fusion protein is expressed using host cells.

[0071] Step S5: Cryo-electron microscopy structural analysis Samples were prepared using the fusion protein, and cryo-electron microscopy data acquisition, two-dimensional classification, and three-dimensional reconstruction analysis were performed to obtain the three-dimensional structural information of the target membrane protein.

[0072] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all standard procedures widely used in their respective fields. Definitions and explanations of the relevant terms are provided below.

[0073] In this invention, amino acid residues can be represented by a single letter or a three-letter symbol, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0074] In this invention, the term "wild type" has the meaning commonly understood by those skilled in the art, referring to the typical form of an organism, strain, or gene, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from natural sources and has not been intentionally modified by humans.

[0075] In this invention, cryo-electron microscopy is a technique for observing rapidly frozen biological samples using an electron microscope. At low temperatures, the water in the sample is frozen, preventing structural damage under conventional microscopy and enabling the provision of high-resolution three-dimensional structural images at the molecular level.

[0076] In this invention, the term "two-dimensional classification" is a step in cryo-electron microscopy data processing that involves grouping a large number of two-dimensional images according to their similarity for subsequent three-dimensional reconstruction. This step is crucial for improving reconstruction accuracy.

[0077] In this invention, the term "three-dimensional reconstruction" is the final step in cryo-electron microscopy data processing, which provides a detailed spatial layout of the target molecule by reconstructing multiple two-dimensional images into a three-dimensional structure.

[0078] To verify the effectiveness of the fusion protein construction method described in this invention in cryo-electron microscopy structure resolution of membrane proteins, and to evaluate the improvement effect of this method on two-dimensional classification and three-dimensional reconstruction of cryo-electron microscopy particles, the invention will be further illustrated below through specific embodiments.

[0079] Example 1 This embodiment provides a fusion protein vGAT EM The methods for constructing and expressing the fusion protein are as follows: The target membrane protein vGAT was selected (in this embodiment, vesicle GABA transporter was used as the research object, and the amino acid sequence of vesicle GABA transporter is shown in SEQ ID NO:3). The fusion site can be selected from the intracellular loop region between adjacent transmembrane helix 2 (TM2) and transmembrane helix 3 (TM3) of the target membrane protein vGAT. The flexible amino acids between TM2 and TM3 of the membrane protein vGAT (i.e., asparagine from position 174 to position 177 of SEQ ID NO:3) were deleted. The N-terminal asparagine and C-terminal serine of WDR5 (as shown in SEQ ID NO:1) were connected to glutamate at position 173 and position 178 of vGAT, respectively, to form a fusion domain vGAT-WD40 (abbreviated as vGAT) with a stable folded structure. EM A key structural advantage of utilizing this domain in fusion constructs lies in the spatial proximity of its N-terminus and C-terminus, both integrated within the rigid β-sheet framework of a propeller. This enhances structural stability and effectively restricts the conformational freedom of domain WD40 relative to the target membrane protein (e.g., Figure 2 (As shown).

[0080] fusing the vGAT gene EM (Its nucleotide sequence is shown in SEQ ID NO:5) was cloned into the mammalian cell expression vector pEGBac-mam, and the protein was expressed using the Bac-Mam system. Specifically: a) 40 μg of recombinant Bacmi plasmid was transfected with liposome PEI25K at a mass ratio of 1:3 into a 10 mL volume cell with a density of 0.6 × 10⁻⁶ mcg. 6Sf9 insect cells, cultured at 27°C for 4 days, can produce P1 generation virus. b. Transfect 10 mL of the cells with P1 generation virus at a volume ratio of 1:10, resulting in a density of 0.6 × 10⁻⁶ cells / mL. 6 Sf9 cells cultured at 27°C for 3 days yielded P2 generation virus. c. Further infection with the P2 generation virus at a volume ratio of 1:100 until a volume of 80 mL and a density of 2.0 × 10⁻⁶ cells were achieved. 6 Sf9 cells were cultured at 27°C for 3 days to obtain P3 generation virus. 80 mL of the generated P3 generation virus was then used to infect 800 mL cells at a density of 3.0 × 10⁻⁶ cells / day at a volume ratio of 1:10. 6 HEK293S GNTI per mL - Cells were cultured at 37°C. After 12 hours, 10 mM sodium butyrate was added, and the cells were transferred to 30°C for further culture for 60 hours. The fusion protein vGAT was obtained. EM Cells (the amino acid sequence of which is shown in SEQ ID NO:4).

[0081] Example 2 This embodiment describes the fusion protein vGAT obtained in Example 1. EM Cells were purified to obtain the fusion protein vGAT. EM The sample is as follows: 0.8 μL of 293F cells were re-vortexed in 40 mL buffer A (20 mM HEPES, 200 mM NaCl, pH 7.5), and 60 μL of a protease inhibitor mixture (1 / 500 ratio) and 10 μL of 1 mg / mL DNAase were added. The cells were transferred to a beaker and sonicated on an ice pack for 7 minutes (10 mm probe, 10% power, 1 s sonication followed by a 2 s pause). 1% LMNG was added to the lysed product, and the mixture was magnetically stirred at 4°C for 60 min. The extract was transferred to a 40 mL centrifuge tube, balanced, and centrifuged at 70000 g / 6°C for 30 min. The supernatant was transferred to a new 50 mL conical tube, and the insoluble residue was filtered out using a syringe filter. 2 mL of STarm Streptactin Beads 4FF gel was added to the conical tube and incubated at 4°C with rotation for 30 min. Pour the STarm Streptactin Beads 4FF gel into the column and allow the supernatant to flow down by gravity. Wash the STarm Streptactin Beads 4FF gel with 40 mL of buffer B (20 mM HEPES, 200 mM NaCl, pH 7.5, 0.05% LMNG). Add 3 mL of buffer C (20 mM HEPES, 100 mM NaCl, pH 7.5, 0.025% LMNG, 5 mM biotin) to the column, incubate at room temperature for 3 minutes, and collect the eluent. The fusion protein vGAT is obtained. EM Samples. As a control, a sample of the membrane protein vGAT with unfused domains was obtained using the same expression and purification conditions.

[0082] like Figure 3 As shown, GAT EM The molecular sieve absorption peaks of the fusion protein and the unfused vGAT protein were similar, indicating that the introduced fusion domain did not affect vGAT folding. Simultaneously, GABA isotope cellular uptake experiments showed that the vGAT fusion protein exhibited similar GABA transport activity to the unfused vGAT protein, indicating that the fusion domain did not interfere with vGAT function (e.g., Figure 4 (As shown).

[0083] Example 3 This embodiment provides a fusion protein vGAT EM-1 Compared with Example 1, the difference is that the fusion protein provided in this example is constructed by fusion at different insertion sites, as detailed below: A fusion domain was introduced into the intracellular loop region between adjacent transmembrane helices 8 (TM8) and 9 (TM9) of the target membrane protein vGAT. The flexible amino acids between TM8 and TM9 of vGAT (i.e., glycine at position 413 and serine at position 414 of SEQ ID NO:3) were deleted. The N-terminal asparagine and C-terminal serine of WDR5 (as shown in the amino acid sequence of SEQ ID NO:1) were then linked to glutamate at position 412 and arginine at position 415 of vGAT, respectively, to obtain the fusion protein, which was labeled vGAT. EM-1 .

[0084] For the fusion protein vGAT EM-1 Purification was performed using the same method as in Example 2 to obtain the fusion protein vGAT. EM-1 sample.

[0085] Comparative Example 1 This comparative example provides a fusion protein vGAT-GFP, which differs from Example 1 in that it is constructed using a GFP fusion domain, as detailed below: Under the same target membrane protein conditions as in Example 1, an attempt was made to introduce another protein domain with higher conformational flexibility, GFP, into the same fusion site of the target membrane protein vGAT from Example 1. Specifically, the flexible amino acid between TM2 and TM3 of the membrane protein vGAT (i.e., asparagine from position 174 to glycine in SEQ ID NO:3) was deleted, and the N-terminal serine and C-terminal histidine of GFP2-231 were linked to glutamate at position 173 and glutamate at position 178 of vGAT, respectively. The resulting fusion protein vGAT-GFP was obtained.

[0086] The fusion protein vGAT-GFP was purified using the same method as in Example 2, resulting in a vGAT-GFP fusion protein sample.

[0087] Comparative Example 2 This comparative example provides a fusion protein vGAT-BRIL, which differs from Example 1 in that it is constructed using a BRIL fusion domain, as detailed below: In another example, the fusion domain BRIL was also introduced into the same fusion site of the target membrane protein vGAT from Example 1. Specifically, the flexible amino acid between TM2 and TM3 of the membrane protein vGAT (i.e., asparagine from position 174 to glycine in SEQ ID NO:3) was deleted, and the N-terminal alanine and C-terminal leucine of BRIL were linked to glutamate at position 173 and 178 of vGAT, respectively. This region exhibited higher conformational flexibility or proximity to the functionally critical domain compared to the fusion site selected in Example 1. The resulting fusion protein vGAT-BRIL was obtained.

[0088] The fusion protein vGAT-BRIL was purified using the same method as in Example 2, resulting in a vGAT-BRIL fusion protein sample.

[0089] Test Example 1 This test example uses the unfused membrane protein vGAT sample as a control. The fusion protein vGAT prepared in Example 2 was examined by cryo-electron microscopy. EM The sample underwent structural analysis, as detailed below: 1. Sample preparation for cryo-electron microscopy The fusion protein vGAT was synthesized using a Vitrobot Mark IV instrument. EM The sample and the unfused membrane protein vGAT sample were prepared into cryo-electron microscopy samples. The specific process is as follows: (1) The fusion protein vGAT was prepared into cryo-electron microscopy samples. EM (1) The sample and the unfused membrane protein vGAT sample were concentrated to 8-10 mg / mL. (2) The gold mesh was subjected to glow discharge treatment at a power of 25 W for 50 s. (3) The cryo-electron microscopy sample was prepared using a Vitrobot instrument. The sample preparation container was pre-cooled with liquid nitrogen and ethane was introduced to cool the ethane to a solid-liquid coexistence state. (4) The sample preparation container was placed in the Vitrobot, the hydrophilicized gold mesh was picked up, and the sample preparation program was run (blot time 5 s, wait time 8, temperature 4°C, humidity 100%, etc.). 4.5 μL of sample was added to the surface of the R1.2 / 1.3 300-mesh copper mesh. After the sample preparation was completed, it was immediately stored in liquid nitrogen.

[0090] 2. Cryo-electron microscopy data collection and processing (1) Data collection: Batch data collection was performed using SerialEM software on a Titan Krios electron microscope (300 kV) equipped with a Gatan K3 camera. The parameters were set as follows: sample plane magnification was 105000 x, original collected image pixel size was 0.855 Å, total electron dose was approximately 55 e / Å2, and defocus value was set between -1.0 and -2.3 μm. (2) Data processing: The original image data was drift corrected and electron-weighted using the MotionCorr2 software program, and then the contrast transfer function (CTF) of the images was evaluated using the Ctfind4 software. The corrected images were imported into cryoSPAR for further processing. First, the images were manually screened, and images with better quality were automatically selected based on particle size using blob-pick and subjected to multiple rounds of two-dimensional classification. The selected particle types are subjected to ab-initio reconstruction to reconstruct the initial model. The initial model is then used as a template to perform multiple rounds of 3D reconstruction on the particles. The structural differences between each type are compared and it is determined whether multiple reconstruction images exist.

[0091] 3. Experimental Results: The results show that, compared with the unfused membrane protein vGAT, the fusion protein vGAT obtained by the fusion protein construction method described in this invention has higher efficiency. EM It exhibits significant advantages in cryo-electron microscopy two-dimensional classification and particle alignment, effectively improving the identifiable features of particles (such as...). Figure 5 (As shown). This demonstrates that the fusion protein construction method provided by this invention can improve the feasibility and data quality of cryo-electron microscopy structure resolution of membrane proteins without significantly interfering with the native conformation of membrane proteins, and has good practical value.

[0092] Test Example 2 The fusion proteins prepared in Example 3 and Comparative Examples 1-2 were subjected to structural analysis, as follows: 1. Experimental methods: The experimental methods for sample preparation, data collection and processing for cryo-electron microscopy are the same as those in test example 1.

[0093] 2. Experimental Results: (1) such as Figure 6 As shown in Figure a, the fusion protein vGAT prepared in Example 3 EM-1 This study demonstrates that fusion with WD40 at different insertion sites enhances the identifiable features of membrane proteins in cryo-electron microscopy, thereby improving two-dimensional classification performance.

[0094] (2) such as Figure 6As shown in Figure b, after using the fusion protein vGAT-GFP prepared in Comparative Example 1 for cryo-electron microscopy sample preparation and data acquisition, it was found that the fusion domain exhibited large conformational wobble during cryo-electron microscopy data acquisition, resulting in obvious conformational heterogeneity of particles in two-dimensional classification and failing to effectively improve the quality of two-dimensional classification and three-dimensional reconstruction.

[0095] (3) such as Figure 6 As shown in Figure c, in the fusion protein vGAT-BRIL prepared in Comparative Example 2, the fusion domain interfered with the overall stability of the target membrane protein to some extent. The fusion protein exhibited poor particle uniformity during the preparation of cryo-electron microscopy samples, which was not conducive to subsequent two-dimensional classification and three-dimensional reconstruction analysis.

[0096] In summary, this invention provides a fusion protein for cryo-electron microscopy structural analysis, its construction method, and its applications. The construction method of this invention selects a region that does not affect the native conformation of the membrane protein as the fusion site, introduces a fusion domain (WD40 protein), and leverages the key structural advantage of WD40 protein's N-terminus and C-terminus being spatially close to each other, both integrated within the rigid β-sheet framework of a propeller, thereby reducing the conformational freedom of the WD40 protein. Using this fusion protein for cryo-electron microscopy structural analysis can improve particle identifiability and enhance the quality of two-dimensional classification and three-dimensional reconstruction.

[0097] While GFP and BRIL each have their own characteristics, their flexibility or rigidity is insufficient to provide adequate stability, especially in cryo-electron microscopy analysis. The flexibility of GFP leads to significant conformational wobble, failing to effectively stabilize membrane proteins; while the rigidity of BRIL, although offering some benefit, may interfere with the stability and homogeneity of membrane proteins, affecting data quality. Therefore, this invention addresses the common problems in existing cryo-electron microscopy structural analysis of target membrane proteins, such as insufficient particle features, alignment difficulties, and limited resolution, without affecting the folding and function of the target membrane protein.

[0098] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for constructing a fusion protein for cryo-electron microscopy structural analysis, characterized in that, Includes the following steps: WD40 protein is inserted into the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein to obtain a fusion protein; The WD40 protein is the WDR5 protein, and its amino acid sequence is shown in SEQ ID NO:1; Preferably, WD40 protein is inserted into the flexible or disordered region of the intracellular loop or extracellular loop between adjacent transmembrane helices of the target membrane protein.

2. The construction method according to claim 1, characterized in that, The insertion of WD40 protein into the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein specifically includes: truncating the flexible or disordered regions in the intracellular loop region or extracellular loop region between adjacent transmembrane helices of the target membrane protein, and inserting the WD40 protein at the truncated position.

3. The construction method according to claim 2, characterized in that, The step of truncating the flexible or disordered regions in the intracellular or extracellular loop regions between adjacent transmembrane helices of the target membrane protein and inserting the WD40 protein at the truncated position specifically includes: deleting the flexible amino acids between transmembrane helices 2 and 3 of the target membrane protein and inserting the WD40 protein at the deleted position; the target membrane protein is a vesicle GABA transporter.

4. The construction method according to claim 2, characterized in that, The step of truncating the flexible or disordered regions in the intracellular or extracellular loop regions between adjacent transmembrane helices of the target membrane protein and inserting the WD40 protein at the truncated position specifically includes: deleting the flexible amino acids between transmembrane helices 8 and 9 of the target membrane protein and inserting the WD40 protein at the deleted position; the target membrane protein is a vesicle GABA transporter.

5. The construction method according to claim 1, characterized in that, The target membrane protein is a transmembrane transport protein or an ion channel protein.

6. A fusion protein, characterized in that, The fusion protein is obtained by the construction method described in any one of claims 1-5.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 6.

8. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 7.

9. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 7 or the recombinant vector of claim 8.

10. The application of the fusion protein of claim 6, the nucleic acid molecule of claim 7, the recombinant vector of claim 8, or the host cell of claim 9 in the determination of target membrane protein structures and the screening and validation of target membrane protein drug targets.

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