A modified kifc1 protein, kifc1-az82-microtube protein complex and application thereof

Abstract

This invention relates to the field of biotechnology, specifically to a modified KIFC1 protein, a KIFC1-AZ82-Microtube protein complex, and their applications. This invention provides a modified KIFC1 protein, the amino acid sequence of which is shown in SEQ ID NO.1. This invention successfully constructed a method for in vitro assembly and paclitaxel stabilization of microtubules. After pre-incubation of KIFC1 with AZ82, it was combined with Microtubes, resulting in the first preparation of a KIFC1-AZ82-Microtube ternary complex. This complex was then applied to cryo-electron microscopy for structural analysis, clearly revealing the tubular helical backbone structure of the microtubules and the binding interface between KIFC1 and Microtubes, clarifying the interaction conformation between KIFC1 and Microtubes in the presence of AZ82.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a modified KIFC1 protein, a KIFC1-AZ82-Microtube protein complex, and their applications. Background Technology

[0002] Microtubules are core cytoskeletal structures formed by the dynamic polymerization of α / β-tubulin, playing an irreplaceable regulatory role in key life activities such as cell morphology maintenance, intracellular substance transport, and spindle assembly during mitosis. Abnormalities in microtubule assembly homeostasis are closely related to the occurrence and development of tumors; therefore, microtubules and their interacting proteins have become important targets for anti-tumor drug development.

[0003] Kinesin family member C1 (KIFC1) is a molecular motor protein belonging to the kinesin-14 family, specifically capable of moving towards the negative end of microtubules. KIFC1 binds to microtubules through its motility domain and utilizes energy generated from ATP hydrolysis to move along the microtubules towards the negative end. During cell division, it mediates centrosome aggregation, spindle assembly, and maintains bipolar spindle stability. KIFC1 is highly expressed in various tumor cell types, especially in tumor cells with centrosome expansion. KIFC1 maintains cell survival by assembling multipolar spindles, making it an important potential target for tumor-targeted therapy.

[0004] AZ82 is a highly selective small molecule inhibitor that specifically targets KIFC1. It can bind to KIFC1 and block its ATPase activity and interaction with microtubules, thereby inhibiting tumor cell division and showing good drug potential. However, the precise binding mode of AZ82 to KIFC1 and the molecular mechanism by which it regulates KIFC1-microtubule interactions are still unclear. Currently, it mainly relies on molecular docking and computer simulation prediction, and lacks direct validation with high-resolution structural data.

[0005] Currently, studies have reported on the binding characteristics of KIFC1 to microtubules, the inhibitory effect of AZ82 on KIFC1, and the regulatory mechanisms of microtubule dynamic assembly. However, no reports have been found on whether KIFC1, AZ82, and microtubules can form a stable ternary complex. Due to the dynamic nature of the interaction between KIFC1 and microtubules and their limited binding affinity, coupled with the inherently poor stability of microtubules, it is difficult to obtain KIFC1-microtubule complexes with good homogeneity and high stability. Furthermore, this fails to meet the stringent requirements of sample purity and homogeneity for cryo-electron microscopy structural analysis. Additionally, the small molecular weight of KIFC1 protein alone makes it difficult to directly use cryo-electron microscopy single-particle analysis techniques to resolve its three-dimensional structure.

[0006] Therefore, there is an urgent need in this field for a stable KIFC1-AZ82-microtubule ternary complex that can reveal the interaction conformation between KIFC1 and microtubules in the presence of AZ82 through high-resolution structural analysis, thereby providing a precise structural template and molecular basis for the rational design of anti-tumor drugs targeting KIFC1. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a modified KIFC1 protein, a KIFC1-AZ82-Microtube protein complex and its applications.

[0008] The present invention achieves the above objectives through the following technical solutions: The present invention provides a modified KIFC1 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] As a further optimization of the above invention, the modified KIFC1 protein further includes a fusion tag and an enzyme cleavage site, and the amino acid sequence of the modified KIFC1 protein containing the fusion tag and enzyme cleavage site is shown in SEQ ID NO.2.

[0010] This invention provides a method for preparing a modified KIFC1 protein, comprising the following steps: modifying KIFC1 with Uniprot number Q9BW19 using gene editing techniques to obtain a gene encoding the modified KIFC1 protein; ligating the gene into an expression vector and transforming it into host cells for induced expression; and purifying the modified KIFC1 protein by affinity chromatography and gel filtration chromatography.

[0011] The present invention provides a polynucleotide that encodes the modified KIFC1 protein as described above, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0012] The present invention provides a recombinant expression vector comprising the polynucleotides described above.

[0013] As a further optimization of the above invention, the expression vector is pET-28a.

[0014] This invention provides the application of modified KIFC1 protein in the preparation of KIFC1-AZ82-Microtube protein complex, comprising the following steps: Microtubes were resuspended in G-PEM buffer containing 5% glycerol and 1 mM GTP, centrifuged to remove the precipitate, and incubated at 37°C. Then, paclitaxel stock solution was added stepwise to achieve final concentrations of paclitaxel of 1 μM, 5 μM, 14 μM, and 20 μM, with an interval of 5-10 minutes between each step. After the final concentration of paclitaxel was achieved, incubation was continued to obtain Microtubes. The modified KIFC1 protein was mixed with AZ82 and incubated at room temperature to form the KIFC1-AZ82 complex; After mixing and incubating the Microtube prepared in step (1) with the KIFC1-AZ82 complex prepared in step (2), the KIFC1-AZ82-Microtube protein complex was obtained.

[0015] As a further optimization of the above invention, the incubation time in step (1) is 1-3 min, with an interval of 5-10 min between each step, and the incubation continues for 38-42 min; in step (2), the modified KIFC1 protein is mixed with AZ82 at a molar ratio of (1~2):(1~2), and the incubation time is 10-30 min; in step (3), the incubation time is 30s~2 min.

[0016] This invention provides a method for resolving the three-dimensional structure of KIFC1 protein using cryo-electron microscopy. The three-dimensional structure of the KIFC1-AZ82-Microtube protein complex described above is obtained by cryo-electron microscopy single-particle analysis, and the resolution of the three-dimensional structure is 4 Å.

[0017] This invention provides an application of the KIFC1-AZ82-Microtube protein complex in the rational design of antitumor drugs targeting KIFC1.

[0018] The beneficial effects of this invention are as follows: This invention successfully constructed a method for in vitro assembly and paclitaxel stabilization of microtubules. After pre-incubation of KIFC1 and AZ82, the microtubules were bound together, resulting in the first preparation of a KIFC1-AZ82-Microtube ternary complex. This complex was then applied to cryo-electron microscopy for structural analysis, obtaining a three-dimensional electron density map with a resolution of 4 Å. The tubular-helical backbone structure of the microtubules and the binding interface between KIFC1 and the microtubules were clearly revealed, clarifying the interaction conformation between KIFC1 and the microtubules in the presence of AZ82. This invention provides crucial structural biology evidence for elucidating the molecular functional mechanism of KIFC1 and revealing the inhibitory mechanism of AZ82, laying an important foundation for the rational design of antitumor drugs targeting KIFC1 and the development of novel microtubule regulation tools. Based on the above-mentioned KIFC1-AZ82-Microtube protein complex and the three-dimensional structural information obtained from its analysis, this invention can be directly applied to the rational design of antitumor drugs targeting KIFC1, for example, as a structural template for molecular docking, virtual screening, and structure-based drug optimization, demonstrating significant scientific research value and application prospects. Attached Figure Description

[0019] Figure 1 To modify the affinity chromatography results of KIFC1 protein; Figure 2 To modify the affinity chromatography results after KIFC1 protease digestion; Figure 3 To modify the results of KIFC1 protein gel filtration chromatography; Figure 4 To modify the QC detection results of KIFC1 protein; Figure 5 For comparison, use the affinity chromatography results of KIFC1 protein; Figure 6 The results of KIFC1 protein gel filtration chromatography are for reference. Figure 7 The results of KIFC1 protein QC testing are used as a reference. Figure 8 Three-dimensional structure diagram of the modified KIFC1-AZ82-Microtube protein complex; Figure 9 The image shows the three-dimensional structure of the KIFC1-AZ82-Microtube protein complex for comparison. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] 1. Materials Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0022] 2. Method 2.1 Gene Synthesis of KIFC1 Using KIFC1 (Uniprot ID Q9BW19) as a template, a truncated version of amino acids 305-673 was constructed. The target gene sequence was obtained through gene synthesis and, after verification by sequencing, was cloned into the pET-28a expression vector. A 6His-TEV tag was added to the N-terminus to construct the recombinant plasmid pET-28a-6His-TEV-KIFC1(Q305-K673). Here, "6His" is the affinity purification tag, and "TEV" is the protease cleavage site used for subsequent tag removal. All synthesized genes were verified by a sequencing company. The amino acid sequence of the modified KIFC1 protein is shown in SEQ ID NO.1. The amino acid sequence of the modified KIFC1 protein with the fusion tag is shown in SEQ ID NO.2. The nucleotide sequence encoding the target gene was synthesized and is shown in SEQ ID NO.3.

[0023] Simultaneously, a previously reported KIFC1 protein was constructed as a control (hereinafter referred to as the control KIFC1 protein). This control protein was derived from PDB ID 5WDH, namely pFastBac1-6His-Thrombin-KIFC1 (L307-C663) (T368P). Here, "6His" is the affinity purification tag, and "Thrombin" is the protease cleavage site used for subsequent tag removal. The synthesized genes were verified by a sequencing company. The coding sequence of the control KIFC1 protein with the fusion tag is shown in SEQ ID NO.4.

[0024] 2.2 Expression and purification of KIFC1 2.2.1 Expression of KIFC1 A. Expression of modified KIFC1 protein The correctly sequenced KIFC1 recombinant plasmid was transformed into T7 Express competent cells and cultured overnight at 37°C. Single colonies were picked and inoculated into 50 mL LB medium, cultured overnight at 37°C, and then transferred to 1 L LB medium at a 1:100 ratio. The cells were cultured at 37°C until the OD600 reached 0.6–0.8, then 0.5 mM IPTG was added, and the cells were induced at 15°C for 16 hours. The cells were collected by centrifugation at 5000 rpm for subsequent purification.

[0025] B. Control of KIFC1 protein expression The control KIFC1 protein (derived from PDB ID 5WDH, amino acid L307-C663, containing the T368P mutation) was expressed using an insect cell-baculovirus expression system. The specific steps are as follows: The recombinant plasmid pFastBac1-6×His-Thrombin-KIFC1(L307-C663) (T368P) was transformed into DH10Bac competent cells (Bomaide, BC112-01) and cultured at 37°C for 48 hours. Positive clones were obtained by blue-white screening, and positive clones were picked and cultured overnight in LB medium. Recombinant Bacmid (baculovirus plasmid) was extracted.

[0026] Insect Sf9 cells (Thermo, 11496015) were diluted to 1×10⁻⁶. 6 Cells / mL. Take 15 μL of recombinant Bacmid and add it to 100 μL of Grace insect medium (Thermo, 10902104) containing 7 μL of X-treme transfection reagent (Roche). Mix well and incubate for 15 minutes. Transfect the mixture into Sf9 cells, and harvest the P0 generation recombinant baculovirus after 4 days of culture. Add 1 mL of P0 virus to 50 mL of Sf9 cells, culture for 3 days, centrifuge for 5 minutes, and collect the supernatant as P1 virus. Add 4 mL of P1 virus to 200 mL of Sf9 cells, culture for 3 days, centrifuge for 5 minutes, and collect the supernatant as P2 virus.

[0027] The prepared P2 generation virus was transfected into Hi-5 cells (Thermo, B85502) at a ratio of 3 μL / 0.5 mL, with a total expression volume of 1.6 L. After culturing at 27°C for 48 hours, the cells were collected for subsequent purification.

[0028] 2.2.2 Purification of KIFC1 A. Purification of modified KIFC1 The collected bacterial cells were weighed and added to lysis buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM PMSF) at a 1:10 ratio. The cells were homogenized by high-pressure autoclaving and the supernatant was collected by centrifugation at 16,000 rpm. Purification was performed using a His FF affinity chromatography column with an N-terminal His tag. The column was first equilibrated to 10 column volumes with washing buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol). The lysis supernatant was then loaded onto the column, and the target protein was eluted with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, 500 mM imidazole). The results are shown below. Figure 1 As shown, KIFC1 was successfully eluted.

[0029] To obtain the tag-free protein, TEV enzyme was added to the elution buffer and the mixture was digested overnight at 4°C. The digestion product was then loaded again onto a His FF affinity chromatography column. The tag-free KIFC1 protein permeated out, and the permeate was collected. Figure 2 Samples from each step were subjected to SDS-PAGE analysis.

[0030] The collected permeate was concentrated and subjected to HiLoad 16 / 600 Superdex 200 pg gel filtration chromatography. The SEC buffer was 20 mM Tris-HCl pH 8.0, 5% glycerol, and 250 mM NaCl. The target protein Peak 2 was collected based on the peak chromatogram. Figure 3 ).

[0031] The purified protein underwent quality testing, including SDS-PAGE purity analysis, mass spectrometry analysis, and analytical molecular sieve detection. The results are as follows: Figure 4 As shown, SDS-PAGE results indicate that the purity of KIFC1 protein is >99%; mass spectrometry confirms that the molecular weight is consistent with the target protein; analytical molecular sieve results show that the protein has good aggregation and uniformity, and is in a monomeric state in solution.

[0032] B. Purification of the control KIFC1 sample The collected bacterial cells were weighed and added to lysis buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM PMSF, 2 mM β-ME) at a 1:10 ratio. The cells were homogenized by high-pressure autoclaving and the supernatant was collected by centrifugation at 16,000 rpm. Purification was performed using a His FF affinity chromatography column with an N-terminal His tag. The column was first equilibrated to 10 column volumes with washing buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 2 mM β-ME). The lysis supernatant was then loaded onto the column, and the target protein was eluted with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 2 mM β-ME, 500 mM imidazole). The results are shown below. Figure 5 As shown, KIFC1 was successfully eluted.

[0033] The collected eluent was concentrated and subjected to gel filtration chromatography using a HiLoad 16 / 600 Superdex 200 pg column. The SEC buffer consisted of 20 mM Tris-HCl pH 8.0, 5% glycerol, 150 mM NaCl, and 2 mM DTT. Peak 1 (the target protein) was collected based on the peak chromatogram. Figure 6 ).

[0034] The purified protein underwent quality testing, including SDS-PAGE purity analysis, mass spectrometry analysis, and analytical molecular sieve detection. The results are as follows: Figure 7 As shown, SDS-PAGE results indicate that the purity of KIFC1 protein is >99%; mass spectrometry confirms that the molecular weight is consistent with the target protein; analytical molecular sieve results show that the protein has good aggregation and uniformity, and is in a monomeric state in solution.

[0035] 2.3 Preparation of Microtubules Take 1 mg of tubulin (purchased from Shanghai Unimicron Biotechnology Co., Ltd., Cat. No. T240-B), add G-PEM buffer (80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, pH 7.0), 5% glycerol and 1 mM GTP, resuspend to 5 mg / mL, and centrifuge to remove the precipitate. Incubate the tubulin solution at 37°C for 2 minutes, then add 2 mM paclitaxel stock solution stepwise to achieve final concentrations of 1 μM, 5 μM, and 14 μM, with an 8-minute incubation interval between each step, until the final paclitaxel concentration reaches 20 μM. Continue incubation for 40 minutes to stabilize the microtubes, and store at room temperature after preparation. The tubulin protein consists of tubulin α and tubulin β, the amino acid sequence of which is shown in SEQ ID NO.5, and the amino acid sequence of tubulin β is shown in SEQ ID NO.6.

[0036] 2.4 Cryo-electron microscopy structural analysis of the KIFC1-AZ82-Microtube complex 2.4.1 Cryo-electron microscopy sample preparation of the KIFC1-AZ82-Microtube protein complex The purified KIFC1 protein or KIFC1 control sample (10 mg / mL) obtained in step 2.2.2 was mixed with AZ82 (CAS No. 1449578-65-7) diluted to 0.77 mM with DMSO at a molar ratio of 1:1 and incubated at room temperature for 10-30 min to form the KIFC1-AZ82 complex. The microtubes prepared in step 2.3 were diluted to 2.5 mg / mL with G-PEM buffer and added to a grid (QUANTIFOIL R 1.2 / 1.3 on Au 300 mesh). After incubation for 30 seconds, excess liquid was blotted off with filter paper, and then the KIFC1-AZ82 complex was added. The mixture was incubated at room temperature for 30 seconds to 2 minutes (final concentration of KIFC1 or KIFC1 control sample: 4 mg / mL; concentration of AZ82: 0.1 mM). Unbound excess protein and inhibitors were washed away with G-PEM buffer to reduce background noise, resulting in the KIFC1-AZ82-Microtube protein complex sample.

[0037] 2.4.2 Data Collection and Structure Analysis The prepared sample was placed on a 300 kV Titan Krios cryo-electron microscope for data collection, and the magnification was adjusted to ensure a balance between the number of microtube bundles and the resolution. The KIFC1-AZ82-Microtube protein complex of this invention was then reconstructed in three dimensions. Figure 8Image acquisition results showed that the microtubules were uniformly distributed in a filamentous pattern, with the KIFC1 protein bound to the microtubule surface. After processing, a three-dimensional electron density map with a resolution of 4 Å was obtained, clearly revealing the tubular-helical backbone structure of the microtubules and the binding interface between KIFC1 and the microtubules. This clarified the interaction conformation between KIFC1 and microtubules in the presence of AZ82, providing a crucial high-resolution structural basis for elucidating the molecular functional mechanism of KIFC1 and optimizing drugs targeting KIFC1.

[0038] Under identical experimental conditions, the KIFC1-AZ82-Microtube protein complex of the control sample was reconstructed in three dimensions. Figure 9 The results showed that although uniformly distributed microtubule filamentous structures could be observed, only individual tubulin was found after processing, and the KIFC1 control sample was not present in the structure. This indicates that the modified KIFC1 protein of this invention can form a stable complex with AZ82 and Microtube, while the previously reported KIFC1 protein (L307-C663, T368P) derived from PDB ID 5WDH could not form a good complex under the same conditions.

[0039] in conclusion This invention successfully constructed a truncated KIFC1 (Q305-K673) protein and established an efficient expression and purification method, obtaining KIFC1 protein with a purity >99% and good homogeneity. Based on this, a method for in vitro microtubule assembly and paclitaxel stabilization was established. After pre-incubation with AZ82, KIFC1 was bound to microtubules, successfully preparing the KIFC1-AZ82-Microtube ternary complex. Cryo-electron microscopy structural analysis of this complex yielded a high-resolution three-dimensional electron density map, clearly revealing the tubular-helical backbone structure of the microtubules and the binding interface between KIFC1 and the microtubules, clarifying the interaction conformation between KIFC1 and microtubules in the presence of AZ82. This invention represents the first high-resolution structural resolution of the KIFC1-AZ82-Microtube ternary complex, providing crucial structural biology evidence for elucidating the molecular functional mechanism of KIFC1 and revealing the inhibitory mechanism of AZ82. It also lays an important foundation for the rational design of antitumor drugs targeting KIFC1 and the development of novel microtubule regulation tools.

[0040] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A modified KIFC1 protein, characterized in that, The amino acid sequence of the modified KIFC1 protein is shown in SEQ ID NO.

1.

2. The modified KIFC1 protein according to claim 1, characterized in that, The modified KIFC1 protein also includes a fusion tag and an enzyme cleavage site, and the amino acid sequence of the modified KIFC1 protein containing the fusion tag and enzyme cleavage site is shown in SEQ ID NO.

2.

3. A method for preparing the modified KIFC1 protein as described in any one of claims 1-2, characterized in that, Includes the following steps: The KIFC1 gene of Uniprot number Q9BW19 was modified using gene editing techniques to obtain the gene encoding the modified KIFC1 protein; the gene was ligated into an expression vector and transformed into host cells for induced expression; the modified KIFC1 protein was obtained by affinity chromatography and gel filtration chromatography purification.

4. A polynucleotide, characterized in that, The polynucleotide encodes the modified KIFC1 protein as described in claim 2, and its nucleotide sequence is shown in SEQ ID NO.

3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 4.

6. The recombinant expression vector according to claim 5, characterized in that, The expression vector is pET-28a.

7. The use of the modified KIFC1 protein as described in any one of claims 1-2 in the preparation of the KIFC1-AZ82-Microtube protein complex, characterized in that, Includes the following steps: Microtubes were resuspended in G-PEM buffer containing 5% glycerol and 1 mM GTP, centrifuged to remove the precipitate, and incubated at 37°C. Then, paclitaxel stock solution was added stepwise to achieve final concentrations of paclitaxel of 1 μM, 5 μM, 14 μM, and 20 μM, with an interval of 5-10 minutes between each step. After the final concentration of paclitaxel was achieved, incubation was continued to obtain Microtubes. The modified KIFC1 protein was mixed with AZ82 and incubated at room temperature to form the KIFC1-AZ82 complex; After mixing and incubating the Microtube prepared in step (1) with the KIFC1-AZ82 complex prepared in step (2), the KIFC1-AZ82-Microtube protein complex was obtained.

8. The application according to claim 7, characterized in that, The incubation time in step (1) is 1-3 minutes. Each step is incubated for 5-10 minutes, and then incubated for 38-42 minutes. In step (2), the modified KIFC1 protein is mixed with AZ82 at a molar ratio of (1~2):(1~2), and the incubation time is 10-30 minutes. In step (3), the incubation time is 30 seconds to 2 minutes.

9. A method for resolving the three-dimensional structure of KIFC1 protein using cryo-electron microscopy, characterized in that, The three-dimensional structure of the KIFC1-AZ82-Microtube protein complex according to any one of claims 7-8 was obtained by cryo-electron microscopy single-particle analysis, and the resolution of the three-dimensional structure was 4 Å.

10. The application of the KIFC1-AZ82-Microtube protein complex as described in claim 7 in the rational design of antitumor drugs targeting KIFC1.

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