A molecular modification design method for improving screening efficiency of mutation sites of enzyme molecules

Abstract

The application belongs to the technical field of bioengineering, and provides a molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites, which is based on open source software AutoDock and Pymol, combined with a constructed screening scheme, adopts a full-rational protease design modification method, and performs full-rational design modification of enzyme molecules through the method of site-directed mutagenesis, molecular docking and construction of a mutation site screening scheme, so as to improve the performance of enzyme molecules. The application system established based on the method is convenient for users to test a large number of mutation sites before experiments and to preferentially select mutation sites, thereby improving the screening efficiency of enzyme molecule mutation sites and saving manpower, material resources and financial resources.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a molecular modification design method to improve the screening efficiency of enzyme molecule mutation sites. Background Technology

[0002] Advances in science and technology have led to the gradual maturation of computational simulation theory and a steady improvement in computer performance. However, in many cases, the research results of theoretical computational simulations cannot be well integrated with experimental problems. The successful application of computational biology in enzymology research has not yet been widely adopted, slowing down the continued progress of enzymology research.

[0003] The relationship between enzymes and substrates is one of the core issues in enzyme catalysis research. Current experimental methods require a lengthy, complex, and tedious process to modify a new enzyme, yielding only simple data.

[0004] Directed evolution and rational design are two common strategies for the rational design and modification of enzyme molecules. Directed evolution has been successfully applied to modifying enzyme activity, stability, substrate specificity, stereoselectivity, and other properties, earning it the 2018 Nobel Prize in Chemistry. However, directed evolution requires the construction of large-scale mutant libraries, the establishment of high-throughput screening methods, and consumes significant human, material, and financial resources, while rational design for protein modification is less efficient. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular modification design method to improve the screening efficiency of enzyme molecule mutation sites, aiming to solve the problems existing in the prior art as identified in the background art.

[0006] This invention is implemented as follows: a molecular modification design method to improve the screening efficiency of enzyme molecule mutation sites, the method comprising:

[0007] Protein structures were downloaded from the PDB database and processed using Pymol software; ligand structures were drawn using ChemDraw and then optimized using the MM2 molecular mechanics method in Chem3D software.

[0008] Select the protein sites in the protein structure that you want to mutate after processing the protein structure with software;

[0009] The Mutagenesis module of Pymol was used to construct site-directed mutagenesis at selected protein sites to obtain the mutated protein structure.

[0010] The mutated protein structure and ligand are docked and calculated using AutoDock to generate the complex structure;

[0011] Based on the complex structure, the Binding Energey of the 10 conformations output by each single mutant protein is arranged in ascending order, and the Binding Energey with the lowest score is selected.

[0012] Based on the established catalytic mechanism of the protein, the key amino acids involved in the reaction and their interaction with the ligands are determined. Then, the atoms on the key amino acids and the atoms on the ligands that cause the catalytic reaction are determined. Based on the complex structure of the lowest Binding Energy after docking of each selected single mutant protein, the distance between the atoms on the key amino acids and the atoms on the ligands is measured.

[0013] Compare the lowest-scoring binding energy of all selected mutation sites, sort them in ascending order of score, select the top 20% of lowest-scoring protein mutation sites, and export them to a txt file. Compare the lowest-scoring distances measured by the structures based on the lowest-scoring binding energy after docking all single-probe proteins, sort them in ascending order of score, select the top 20% of lowest-scoring protein mutation sites, and export them to a txt file. Output both schemes to the same txt file, separated by identifiers and statements. If the protein mutation sites of the two schemes overlap, mark the overlapping protein mutation sites with an asterisk (*), and these mutation sites are the preferred selection.

[0014] Preferably, the process of processing the protein structure using Pymol software specifically includes:

[0015] Remove excess water molecules;

[0016] Remove identical subunits;

[0017] Remove the ligands within the protein.

[0018] Preferably, the optimization of the ligand structure using the molecular mechanics method of Chem3D software MM2 specifically includes:

[0019] Choosing Caculations / MM2 / Minimize Energy minimizes chemical energy, thereby optimizing the structure.

[0020] Preferably, the step of generating a complex structure by docking the mutated protein structure with the ligand using AutoDock specifically includes:

[0021] Using the open-source code of AutoDock, a parameter file was constructed to hydrogenate the mutated protein structure. Based on the determined site of action, the coordinates, dimensions, and height of the grid box in AutoDockTools were determined. The interface parameters, i.e. the active site position, were fixed, and the number of docking output conformations was set to 10. Molecular docking calculations were performed between the hydrogenated protein structure and the ligand to obtain the complex structure.

[0022] This invention provides a molecular modification design method to improve the efficiency of enzyme molecular mutation site screening. Based on the enzyme's catalytic mechanism, an enzyme modification design application system was developed. Through site-directed mutagenesis, molecular docking calculations, conformational analysis, and binding energy analysis, a series of enzyme mutation sites are output and the optimal mutation site is screened out, thereby improving the efficiency of enzyme molecular mutation site screening and reducing experimental costs. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of filling in a txt file provided in an embodiment of the present invention;

[0024] Figure 2 A flowchart of a single-point mutation process for sequentially constructing proteins is provided for embodiments of the present invention.

[0025] Figure 3 This is a schematic diagram illustrating the molecular docking calculations performed by the mutated protein and small molecule ligand via AutoDock (open-source software) in an embodiment of the present invention.

[0026] Figure 4 A schematic diagram is provided to output the distances between key acceptor and ligand atoms selected based on the catalytic mechanism into a list.

[0027] Figure 5 The main flowchart of a molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites is provided in an embodiment of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0030] This application, based on the structural information and catalytic mechanism of enzymes, combines computer-aided rational design strategies to design and modify enzymes, thereby improving their thermal stability, affinity, and other properties. The application system developed based on the substrate-selective rational design strategy for enzyme molecules allows users to test a large number of mutation sites before experiments and prioritize their selection, saving manpower, material resources, and financial resources, thus improving research efficiency and enhancing the understanding of protein structure and function.

[0031] like Figure 1 The diagram shown illustrates a molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites, according to an embodiment of the present invention. The method specifically includes the following steps:

[0032] 1) Users find the protein they are studying in the Protein Database (PDB), and use Discovery Studio or Pymol software to perform preprocessing operations on the protein structure, such as removing water, small molecule ligands, and repeating subunits, and save it as a pdb file; users search for and download small molecules from the PubChem database based on small molecule information or draw the three-dimensional structure of small molecules using Chemdraw software based on the two-dimensional structure of small molecules, and then optimize the mechanical structure using Chem3D;

[0033] 2) Create a txt file containing the parameter files needed for site selection, for users to fill in. The mutation and docking parameters include: 1. Folder path of the protein; 2. Protein name; 3. Subunit number of the protein the user wants to mutate; 4. Amino acid number, abbreviation, and the desired amino acid abbreviation. The measurement and selection parameters include: 1. Amino acid name of the protein the user wants to measure; 2. Atomic number; 3. Atomic name; 4. Ligand atomic number the user wants to measure. For example... Figure 2 As shown;

[0034] 3) The protein processed in step 1)

[0035] Using PyMOL (an open-source software for visualizing molecules and proteins), and employing the scripting language of the Mutagenesis module in Python, combined with parameter files and code modifications, single-point mutations of proteins are constructed sequentially based on the user-specified protein chain, selected amino acid sequences and names, and the residues to be mutated. For example... Figure 2 As shown;

[0036] 4) Perform molecular docking calculations on the mutated protein and small molecule ligand from step 2) using AutoDock (open-source software). The steps are as follows: Figure 3As shown, combining the txt file, the first step is to prepare the coordinate files. pdbqt is a coordinate file format specific to AutoDock. Export the protein and small molecule as pdbqt files respectively. For the protein, add hydrogen, calculate the charge, and add atom types; for the small molecule, add hydrogen, calculate the charge, determine the root (torque center), and select rotatable bonds. The second step is to use AutoDockTools to create a grid parameter file (GPF), mainly setting the center coordinates and size of the grid boxes. The third step is to generate a docking parameter file (DPF) in AutoDockTools, including the algorithm and docking parameters. The fourth step is to extract the binding conformation with the minimum binding energy, i.e., the optimal binding conformation, such as... Figure 4 As shown;

[0037] 5) Using Python, write a script to output the lowest binding energy (optimal binding mode) of the complex structure after each mutation to a list, and also output the distance between the key acceptor and ligand atoms selected by the user based on the catalytic mechanism to a list. Finally, use the top 20% of mutation sites with the lowest binding energy and the top 20% with the shortest measured interatomic distances as screening points, such as... Figure 5 As shown;

[0038] Verification Experiment

[0039] To further verify the reliability of this design, the following verification experiments will be conducted.

[0040] By combining reliable literature studies, such as Chen Haiqing et al. [1] using the online prediction software PoPMuSiC to calculate the unfolding free energy change (ΔΔG) of the amino acid substitution sites in the lipase (lipA) protein sequence of Serratia marcescens strain L1 to assist in the design of site-directed mutations of the enzyme protein, in order to predict the changes in enzyme stability after amino acid point mutations, the enzymatic properties were characterized after expression in Escherichia coli and purification of the enzyme protein. The optimal temperature for the two mutant and wild-type lipases was 30 ℃, and the optimal pH value was 8.

[0041] Compared to the wild type, the specific enzyme activity of lipA-Asn86Glu was increased by 8%. This result was experimentally verified using the application system designed in this study. First, the lipA protein and substrate were pretreated. Then, a virtual saturation mutagenesis was performed on Asn86. Next, a series of mutated complex conformations were obtained through molecular docking. Finally, the calculated lowest binding energy and the distance between the lowest binding energy conformations were ranked, and the top 20% of the results were output to a text file. The mutation sites of Asn86Glu were ranked in the top 20%, verifying the reliability of this design scheme.

[0042] The above embodiments of the present invention provide a molecular modification design method to improve the efficiency of enzyme molecular mutation site screening. Based on the enzyme catalytic mechanism, an enzyme modification design application system was developed. Through site-directed mutagenesis, molecular docking calculation, conformation analysis and binding energy analysis, a series of enzyme mutation sites are output and the optimal mutation site is screened out, thereby improving the efficiency of enzyme molecular mutation site screening and reducing experimental costs.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A molecular modification design method to improve the screening efficiency of enzyme molecule mutation sites, characterized in that, The method includes: Protein structures were downloaded from the PDB database and processed using Pymol software; ligand structures were drawn using ChemDraw and then optimized using the MM2 molecular mechanics method in Chem3D software. Select the protein sites in the protein structure that you want to mutate after processing the protein structure with software; The Mutagenesis module of Pymol was used to construct site-directed mutagenesis at selected protein sites to obtain the mutated protein structure. The mutated protein structure and ligand are docked and calculated using AutoDock to generate the complex structure; Based on the complex structure, the Binding Energey of the 10 conformations output by each single mutant protein is arranged in ascending order, and the Binding Energey with the lowest score is selected. Based on the established catalytic mechanism of the protein, the key amino acids involved in the reaction and their interaction with the ligands are determined. Then, the atoms on the key amino acids and the atoms on the ligands that cause the catalytic reaction are determined. Based on the complex structure of the lowest Binding Energy after docking of each selected single mutant protein, the distance between the atoms on the key amino acids and the atoms on the ligands is measured. Compare the lowest binding energy of all selected mutation sites, sort them in ascending order of score, select the protein mutation site with the lowest percentage score, and export it to a txt file; compare the lowest distances measured by the structure based on the lowest binding energy after docking all single mutant proteins, sort them in ascending order of score, select the protein mutation sites with the lowest percentage score, and export them to a txt file; output both schemes to the same txt file and separate them with a set identifier. If the protein mutation sites of the two schemes overlap, mark the overlapping protein mutation sites with *, and this mutation site is the preferred option.

2. The molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites according to claim 1, characterized in that, The specific steps of processing protein structures using Pymol software include: Remove excess water molecules; Remove identical subunits; Remove the ligands within the protein.

3. The molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites according to claim 1, characterized in that, The optimization of ligand structure using the molecular mechanics method of Chem3D software MM2 specifically includes: Choosing Caculations / MM2 / Minimize Energy minimizes chemical energy, thereby optimizing the structure.

4. The molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites according to any one of claims 1-3, characterized in that, The step of docking the mutated protein structure with the ligand using AutoDock to calculate and generate the complex structure specifically includes: Using the open-source code of AutoDock, a parameter file was constructed to hydrogenate the mutated protein structure. Based on the determined action site, the active site position was fixed, and the number of docking output conformations was set to 10. Molecular docking calculations were performed between the hydrogenated protein structure and the ligand to obtain the complex structure.

5. The molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites according to claim 4, characterized in that, The process of fixing the location of the active site based on the determined site of action specifically includes: determining the coordinates, length, width, and height of the GridBox in AutoDockTools.

6. The molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites according to claim 1, characterized in that, The setting identifier includes identifiers and / or statements.

7. The molecular modification design method for improving the screening efficiency of enzyme molecule mutation sites according to any one of claims 1-3, characterized in that, The aforementioned percentage is the top 20%.

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