Structural modification and optimization method of PC-PYY peptide based on molecular dynamics simulation and product thereof

Abstract

The invention discloses a structural modification and optimization method of a PC-PYY peptide based on molecular dynamics simulation and a product thereof. The method specifically comprises the following steps: respectively constructing a PC-PYY peptide model and a candida albicans model; establishing a simulation box, solvating the model, adding Na < + > and C1 <-> to neutralize the system, and performing energy minimization; simulating a finished product and optimizing a polypeptide amino acid sequence; carrying out molecular dynamics simulation on the modified PC-PYY peptide and the candida albicans model again; comparing and analyzing data results of the initial PC-PYY peptide and the modified PC-PYY peptide; the obtained peptide chain enhances the selective binding capacity to a fungal membrane and improves the antifungal efficacy of the peptide chain.

Description

Technical Field

[0001] The invention relates to molecular dynamics simulation technology and biomedical fields, and in particular to a method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation, and also to a product prepared by the method. Background Art

[0002] Candida albicans is one of the common pathogens in the human body, which can cause oral, genital and systemic infections. The current treatment for Candida albicans infection is to use broad-spectrum antibiotics. However, the widespread use of antibiotics has led to a significant increase in bacterial resistance, so there is an urgent need to develop new drugs to meet this challenge. Traditional antibiotics exert their antibacterial effects by passing through the cell membrane and interfering with the physiological processes within the cell. When bacteria adjust their permeability to antibiotics or undergo target mutations, the antibiotics will lose their target and fail to work. Antimicrobial peptides can directly interact with the cell membrane, affect the stability of the membrane, and then cause the membrane structure to be incomplete, thereby exerting their antibacterial activity. Therefore, antimicrobial peptides can work without relying on specific protein recognition, which gives antimicrobial peptides a high degree of resistance to drug resistance, making them candidates for the development of new drugs. Among antimicrobial peptides, peptides containing α-helical structures have been widely studied because of their significant antibacterial activity. Peptide YY (PC-PYY) secreted by human intestinal Paneth cells is a peptide with potential antifungal activity. Compared with other α-helical peptides, PC-PYY peptide shows specific targeting effect on Candida albicans cell membrane. However, its original structure has deficiencies in stability and selectivity, which limits its wide application in drug development. Molecular dynamics simulation technology has significant advantages in optimizing peptide structure, predicting its interaction with biological membranes, and designing highly effective antifungal peptides. It can capture the dynamic behavior of peptides and membranes in real time at atomic resolution, revealing its adsorption, membrane penetration, and interaction mechanism with specific phospholipid molecules.

[0003] Therefore, it is necessary to develop a structural modification and optimization scheme for PC-PYY peptide based on molecular dynamics simulation to improve its antifungal activity and selectivity through rational design and optimization of the key domains of the peptide. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation; a second object of the present invention is to provide a peptide chain obtained by the method.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation, comprising the following steps:

[0007] (1) Construct PC-PYY peptide and Candida albicans models respectively;

[0008] (2) Create a simulation box, solvate the model, and add Na + 、C1 - To neutralize the system and minimize the energy;

[0009] (3) Finished product simulation and optimization of peptide amino acid sequence;

[0010] (4) The modified PC-PYY peptide was again subjected to molecular dynamics simulation with the Candida albicans model;

[0011] (5) Compare and analyze the data results of the initial PC-PYY peptide and the modified PC-PYY peptide.

[0012] Preferably, in the present invention, step (1) is to use GROMACS to construct a three-dimensional initial structure model of the PC-PYY peptide and Candida albicans according to the known amino acid sequence of the PC-PYY peptide, and the initial structure of the PC-PYY polypeptide is YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY.

[0013] Preferably, step (2) of the present invention is to place the polypeptide in a physiological solution environment for equilibrium simulation to achieve a stable state; then establish a simulation box of the PC-PYY peptide and the Candida albicans membrane model, solvate the model, and finally perform energy minimization, equilibrium treatment, and finished product simulation. Molecular dynamics simulation calculations are performed to observe the structural changes of the PC-PYY peptide during the simulation process, and the MM / PBSA method is used to quantify the binding free energy of the PC-PYY peptide and the Candida albicans membrane.

[0014] Preferably, the Candida albicans membrane is composed of four phospholipids including but not limited to 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoglyceride, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate in proportion, and the proportion can be adjusted according to demand.

[0015] Preferably, in the present invention, step (3) is to use VMD software to load the PDB structure of the original PC-PYY peptide, modify amino acids 1-12, and modify the negatively charged amino acids at the N-terminus to positively charged amino acids by VMD tools to obtain the optimized peptide chain sequence.

[0016] Preferably, in the present invention, step (5) is to compare and analyze the data results of the initial PC-PYY peptide and the modified PC-PYY peptide, analyze the effect of the modified PC-PYY peptide on the fluidity, permeability and local curvature of the Candida albicans membrane and calculate its binding free energy.

[0017] 2. The peptide chain obtained by the method is characterized in that (1) amino acids 1 to 12 are modified; or the negatively charged amino acid at the N-terminus is replaced by a positively charged amino acid; (2) the optimized peptide chain sequence is obtained according to method (1).

[0018] The beneficial effects of the present invention are as follows: the present invention significantly enhances the selective binding ability of PC-PYY peptide to fungal membranes, especially the binding efficiency to anionic phospholipids (such as PLPG), through precise analysis based on molecular dynamics simulation and targeted structural modification, by optimizing the structure, and has the following advantages:

[0019] 1) Thereby improving its antifungal efficacy.

[0020] 2) It can significantly improve the fluidity and water molecule permeability of the membrane and cause local curvature changes, thereby destroying the stability of the fungal membrane.

[0021] 3) The self-aggregation effect of PC-PYY peptide at high concentrations was avoided by design, ensuring that the peptide still maintained excellent antifungal activity at high concentrations.

[0022] 4) The optimized PC-PYY peptide has higher specificity and can act on the target more accurately, reducing the impact on other non-target cells or tissues and reducing potential side effects. The optimized PC-PYY peptide has a wide range of application potentials in the medical and industrial fields, especially in the development of highly effective antifungal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0024] Figure 1 Flow chart of structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation;

[0025] Figure 2 The three-dimensional initial structure model of PC-PYY peptide and Candida albicans strain;

[0026] Figure 3Binding free energy analysis results (A: Binding free energy ΔG of different items in an original PC-PYY polypeptide system, items with ΔG of 0 Kal / mol are replaced by OTHER, where OTHER items include: ΔBOND, ΔANGLE, ΔDIHED, ΔUB, ΔIMP, ΔCMAP, Δ1-4VDW, Δ1-4EEL, ΔEDISPER; B: Free energy ΔG contribution of each amino acid residue in an original PC-PYY polypeptide system, red represents positive ΔG, blue represents negative ΔG, the ΔG of the amino acid not shown is 0 Kcal / mol, and the numbers after the colon represent the residue numbers of the amino acids; C: Free energy ΔG contribution of each phospholipid in an original PC-PYY polypeptide system, red represents positive ΔG, blue represents negative ΔG, and the numbers after the colon represent the residue numbers of the phospholipids);

[0027] Figure 4 The radial distribution functions (RDF) of the peptide C backbone relative to the P atom in the phospholipid head group in a PC-PYY peptide system. The red line represents PLPG, the blue line represents PLPE, the green line represents PLPA, and the yellow line represents POPA.

[0028] Figure 5 Three-dimensional schematic diagram of PC-PYY before and after modification (A: three-dimensional schematic diagram of PC-PYY before modification, B: three-dimensional schematic diagram of PC-PYY after modification). DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] Example 1: PC-PYY peptide and its structural modification by molecular dynamics simulation

[0031] The structural modification and optimization process of PC-PYY peptide by molecular dynamics simulation is as follows Figure 1 As shown, the specific steps are as follows:

[0032] (1) Using GROMACS, a three-dimensional initial structural model of PC-PYY peptide and Candida albicans was constructed based on the known amino acid sequence of PC-PYY peptide. Figure 2 As shown, the initial structure of the PC-PYY polypeptide was YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO. 1).

[0033] (2) To ensure the structural stability of the peptide and eliminate environmental interference, the peptide was placed in a physiological solution environment for equilibrium simulation for 20 ns to achieve a stable state. Then, a simulation box of the PC-PYY peptide and Candida albicans membrane model was established, and the model was solvated by adding 0.15 M sodium chloride solution for energy minimization and equilibrium treatment. The simulation parameters were as follows: temperature was 311 K, pressure was 1 bar, CHARMM36m force field was used, short-range interaction and electrostatic interaction were cut off at 1.2 nm, and the finished product simulation time was 300 nanoseconds. Molecular dynamics simulation calculations were performed to observe the structural changes of the PC-PYY peptide during the simulation process, and the MM / PBSA method was used to quantify the binding free energy of the PC-PYY peptide and the Candida albicans membrane.

[0034] The distance distribution and binding mode between key residues (such as ARG) and PLPG were evaluated by radial distribution function. Figure 3 As shown in the figure, through binding free energy analysis, it was found that the amino acid residue ARG located on the peptide chain plays a key role in antibacterial activity. In addition, the 15th and 16th amino acids GLU have positive charges, which leads to repulsion when binding to the negatively charged Candida albicans membrane. Through molecular dynamics simulation trajectories, it was found that the N-terminal 1-12 amino acids have steric hindrance when binding to the Candida albicans membrane, and hinder the formation of the polypeptide α-helix structure. Figure 4 As shown, in the radial distribution function of the phospholipid head group P atom centered on the mass center of the PC-PYY polypeptide C backbone, the peak value of the PLPG head group is the largest. Therefore, the PC-PYY peptide has a higher binding tendency to the PLPG head group in the phospholipid membrane, which indicates that PLPG is the key phospholipid for the interaction between the PC-PYY peptide and the Candida albicans membrane.

[0035] In this step, the composition and proportion of the Candida albicans membrane are mainly composed of four phospholipids: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoglyceride (PLPG), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (PLPE), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate (PLPA), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), and the ratio is 4.3:

[0036] 3.5:1.1:1.1. The phospholipid bilayer membrane was constructed using genmixmem. The constructed phospholipid bilayer membrane contained 1152 phospholipid molecules and the XY plane size was 18nm×18nm.

[0037] (3) According to the analysis results of step (2), the finished product simulation is performed and the polypeptide amino acid sequence is optimized. Specifically, amino acids 1 to 12 are cut, and GLU at positions 15 and 16 are mutated to ARG; the PDB structure of the original PC-PYY peptide is loaded using VMD software, amino acids 1 to 12 are deleted, and GLU at positions 15 and 16 are modified to ARG at the N-terminus using the VMD tool; the mutated structure may have unreasonable bond lengths or collisions, so it is necessary to use pdb2gmx in GROMACS to regenerate the force field topology file, and minimize the energy of the mutant PC-PYY to optimize its structure. The optimized peptide chain sequence is SPRRLSRYYASLRHYLNLVTRQRY (SEQ ID NO. 2), and its three-dimensional structure is as follows: Figure 5 .

[0038] (4) Based on the optimization results of step (3), the modified PC-PYY peptide structure is again subjected to molecular dynamics simulation with the Candida albicans model. During the simulation, the overall structural stability of the peptide chain, the binding mode with the Candida albicans membrane surface, and the binding free energy are monitored. The specific steps include:

[0039] System preparation: The modified PC-PYY peptide structure was loaded into the Candida albicans membrane model and energy minimized using PACKMOL. Solvent was added to the system and the system was adjusted to achieve electroneutrality.

[0040] Energy minimization and equilibration: After the system was constructed, energy minimization was performed and the simulation parameters were set to be consistent with (1), followed by equilibration under the NPT ensemble (5 ns) to stabilize the system.

[0041] Finished product simulation: Run a 300ns molecular dynamics simulation to record the binding process and dynamic changes of the peptide to the membrane.

[0042] (5) The data results of the initial PC-PYY peptide and the modified PC-PYY peptide were compared and analyzed, and the effects of the modified PC-PYY peptide on the fluidity, permeability, and local curvature of the Candida albicans membrane were analyzed and its binding free energy was calculated. The results obtained by molecular dynamics simulation were compared and analyzed with the experimental data. By comparing the binding characteristics and binding stability of the PC-PYY peptide with the simulated negatively charged Candida albicans membrane surface before and after mutation, as well as the effect of the PC-PYY peptide on the stability of the Candida albicans membrane, the reliability of the molecular dynamics simulation results and the effectiveness of the mutation scheme were further verified.

[0043] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation, characterized in that: The steps include: (1) Construct PC-PYY peptide and Candida albicans models respectively; (2) Create a simulation box, solvate the model, and add Na + 、C1 - To neutralize the system and minimize the energy; (3) Finished product simulation and optimization of peptide amino acid sequence; (4) The modified PC-PYY peptide was again subjected to molecular dynamics simulation with the Candida albicans model; (5) Compare and analyze the data results of the initial PC-PYY peptide and the modified PC-PYY peptide.

2. The method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation according to claim 1, characterized in that: Step (1) is to use GROMACS to construct a three-dimensional initial structure model of PC-PYY peptide and Candida albicans according to the known amino acid sequence of PC-PYY peptide, and obtain the initial structure of PC-PYY polypeptide as YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY.

3. The method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation according to claim 1, characterized in that: Step (2) is to place the peptide in a physiological solution environment for equilibrium simulation to achieve a stable state; then establish a simulation box for the PC-PYY peptide and the Candida albicans membrane model, solvate the model, and finally perform energy minimization, equilibrium treatment, and finished product simulation. Molecular dynamics simulation calculations are performed to observe the structural changes of the PC-PYY peptide during the simulation process, and the MM / PBSA method is used to quantify the binding free energy of the PC-PYY peptide and the Candida albicans membrane.

4. The method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation according to claim 3, characterized in that: The Candida albicans membrane is composed of phospholipids including but not limited to 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoglyceride, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate in proportion, and the proportion can be adjusted according to needs.

5. The method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation according to claim 1, characterized in that: Step (3) is to use VMD software to load the PDB structure of the original PC-PYY peptide, modify amino acids 1-12, and modify the negatively charged amino acids at the N-terminus to positively charged amino acids using the VMD tool to obtain the optimized peptide chain sequence.

6. The method for structural modification and optimization of PC-PYY peptide based on molecular dynamics simulation according to claim 1, characterized in that: Step (5) is to compare and analyze the data results of the initial PC-PYY peptide and the modified PC-PYY peptide, analyze the effect of the modified PC-PYY peptide on the fluidity, permeability and local curvature of the Candida albicans membrane and calculate its binding free energy.

7. A peptide chain obtained by the method according to any one of claims 1 to 6, characterized in that: (1) Modify amino acids 1 to 12; or replace the negatively charged amino acid at the N-terminus with a positively charged amino acid; (2) obtain the optimized peptide chain sequence according to method (1).

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