A molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein

Abstract

The present invention discloses a molecular dynamics simulation analysis method for ultrasonic perturbation of the Piezo1 protein, belonging to the field of ultrasonic perturbation. In the molecular dynamics simulation, ultrasonic perturbation is achieved by applying an ultrasonic function to the boundary, and successful ultrasonic loading is verified based on the water density on the X-axis. Furthermore, an analytical method is established to explain the effects of ultrasound on cell membranes from multiple angles, such as cell membrane thickness and lipid-protein interactions, and further study the effects of cell membranes on proteins, thereby determining the effects of ultrasound on the Piezo1 structure. This invention is the first to use ultrasonic loading coarse-grained molecular dynamics simulation to study the conformational changes of the Piezo1 mechanosensitive ion channel, providing important research ideas for future applications in ultrasound therapy, acoustic genetics, and other fields.

Description

Technical Field

[0001] The invention belongs to the fields of ultrasonic perturbation and molecular dynamics simulation, and particularly relates to a molecular dynamics simulation analysis method of Piezo1 protein based on ultrasonic perturbation. Background Art

[0002] Mechanosensitive ion channel proteins are a class of proteins that convert mechanical signals into physiological signals and play a vital role in human life. Among them, the Piezo mechanosensitive ion channel is the largest mechanosensitive ion channel protein discovered to date and includes two members, Piezo1 and Piezo2.

[0003] Piezo1 senses mechanical force, releases cations, induces cell activation and signal transmission, and participates in numerous physiological processes, including regulating blood pressure. In 2018, Prieto and colleagues, using patch-clamp techniques, discovered that Piezo1 activates in response to cell membrane stress induced by continuous ultrasonic sound flow at 43 MHz and 50 W / cm² or 90 W / cm². More recently, Liao et al. also discovered the optimal ultrasound pulse length for activating Piezo1 mechanosensitive ion channels and intracellular calcium responses. Studying the response mechanism of Piezo1 mechanosensitive ion channels under ultrasound perturbations is of great significance for future ultrasound therapy and related drug design.

[0004] Molecular dynamics simulations are currently widely used to study intracellular molecular mechanisms. However, due to limited computing resources, all-atom molecular dynamics methods are difficult to use for studying protein systems with over 7,000 amino acids, such as Piezo1. To conduct molecular dynamics simulations spanning microsecond to millisecond timescales and explore protein conformational changes involving millions of atoms within limited computing resources, coarse-grained modeling can improve simulation efficiency.

[0005] The literature [De Vecchis D, Beech DJ, Kalli A C. Molecular dynamics simulations of Piezo1 channel opening by increases in membrane tension [J]. Biophysical Journal, 2021, 120 (8): 1510-21.] revealed the molecular mechanism by which Piezo1 channels may open in response to increased membrane tension by applying lateral pressure to the membrane; the literature [Botello-Smith WM, Jiang W, Zhang H, et al. A mechanism for the activation of the mechanosensitive Piezo1 channel by the small molecule Yoda1 [J]. Nature Communications, 2019, 10 (1): 4503.] found the Yoda1 binding site and proposed the molecular mechanism by which Yoda1 promotes force-induced conformational changes. However, no one has used ultrasound as a mechanical force to study Piezo1 using molecular dynamics simulation methods. This study, for the first time, uses a molecular dynamics simulation method based on ultrasound perturbations to study the conformational changes of the Piezo1 protein. By simulating the effects of ultrasound on cell membranes, this study further simulates the effects of ultrasound on the Piezo1 protein's conformation. This coarse-grained molecular dynamics simulation method for ultrasound-induced mechanosensitive ion channel proteins represents a significant extension of existing methods and provides important research insights for the application of sonogenetics. Summary of the Invention

[0006] The present invention aims to solve the problems of the existing technology of simulating Piezo1 protein under ultrasonic perturbation, and proposes a molecular dynamics simulation analysis method of Piezo1 under ultrasonic perturbation, specifically using ultrasound waves of different frequencies loaded on the cell membrane, thereby expanding the research method of Piezo1.

[0007] In view of this, the technical solution adopted by the present invention is as follows: a molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein, comprising the following steps:

[0008] Step 1: Establish an ultrasonic model and couple it to Gromacs software;

[0009] In step 2, the missing amino acids of the protein are supplemented and inserted into the cell membrane after coarse-graining, and molecular dynamics simulation analysis is performed using the ultrasound model described in step 1.

[0010] Furthermore, the ultrasonic model is to establish six ultrasonic velocities of the same size but different directions. The velocities are generated at the edges of the six faces of the three-dimensional box, and the velocities of each face are generated at a certain time interval. The six ultrasonic velocities are defined as follows:

[0011]

[0012]

[0013] V i Represents the six ultrasonic velocities along the positive and negative directions of X, Y, and Z, respectively. V max represents the maximum speed of the ultrasonic pulse, m represents the time step, N represents the molecular dynamics simulation step, i as a subscript represents one of the six ultrasonic velocity directions, X0 represents the X coordinate, Y0 represents the Y coordinate, and Z0 represents the Z coordinate.

[0014] Furthermore, a script was written to complete the missing amino acids in specific missing small fragment regions of the protein, and coarse-graining was performed using 4-to-1 mapping. The coarse-grained protein structure was embedded in the cell membrane, and a suitable simulation box with periodic boundaries was constructed. The aforementioned ultrasound model was used to perform ultrasound perturbation molecular dynamics simulations.

[0015] Furthermore, the molecular dynamics simulation analysis includes:

[0016] Observe and analyze the morphological changes of cell membranes, including changes in membrane thickness and membrane area;

[0017] Analyze the interaction time between membrane components and proteins;

[0018] Analysis of protein conformational changes showed that changes in cells under ultrasonic perturbations caused interactions between membrane components and proteins, which further led to conformational changes.

[0019] Furthermore, the calculation of the membrane thickness is as follows: first, the lipids of the inner and outer leaflets of the cell membrane are identified, and the change of the cell membrane thickness with the simulation time is calculated by identifying the peak distance Z of the lipid head group density.

[0020] Furthermore, the membrane area is calculated by 2D Voronoi tessellation, using Freud's local module to perform tessellation of atomic positions.

[0021] Furthermore, the interaction time between the membrane component and the protein is determined by identifying the interaction site between the protein and the membrane component and calculating the residence time of the response site to determine whether the site has a strong interaction or a weak interaction.

[0022] Calculation of lipid (i.e. membrane component) and protein binding sites: For a group of residues that bind to the same lipid molecule at the same time, pylipid takes a distance vector that records the distance between each residue and all lipid molecules as a function of time, and constructs a lipid interaction network whose nodes are protein residues and weights are the Pearson correlation coefficients of residue pairs calculated from their distance vectors.

[0023] Interaction residence time: Since lipids and proteins interact, lipids are no longer diffusing. Therefore, pylipid uses a double exponential to calculate the residence time to account for the long and short decays of lipid relaxation. The residence time is (koff represents the dissociation constant) calculated from the normalized survival function using duration.

[0024] Furthermore, the changes in the protein conformation are observed by using a script to calculate the depth of the dome formed by the Piezo1 protein in the membrane and draw a two-dimensional density map of the protein in the xy plane to observe the structural changes of Piezo1 under ultrasonic perturbation.

[0025] The present invention also provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the molecular dynamics simulation analysis method of ultrasonic perturbation of Piezo1 protein as described above is implemented.

[0026] The present invention has the following advantages:

[0027] (1) This paper uses the method of ultrasonic perturbation coarse-grained molecular dynamics simulation to study the Piezo1 mechanosensitive ion channel for the first time, which can observe conformational changes over a long period of time; by analyzing the cell membrane thickness, protein-lipid interactions and other multi-angle explanations of the effect of ultrasound on the cell membrane, further studying the effect of the cell membrane on the Piezo1 protein.

[0028] (2) An analytical method from the whole to the part was established, that is, by observing the changes in the cell membrane, studying the interaction between cell membrane lipids and proteins, and exploring changes in protein structure.

[0029] (3) Computer simulation of ultrasound perturbations of Piezo1 protein can provide many details that are difficult to obtain experimentally for electrophysiological experiments, and provide certain theoretical guidance for future ultrasound treatment of Piezo1-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the coarse-grained structure diagram of the protein;

[0031] Figure 2 A diagram of a model of protein embedded in a cell membrane;

[0032] Figure 3 is the water density map under ultrasonic disturbance;

[0033] Figure 4 is the cell membrane thickness diagram under different ultrasonic perturbations;

[0034] Figure 5 is a map of protein-lipid interaction sites;

[0035] Figure 6 Protein RMSD plot;

[0036] Figure 7 is a graph of protein density and dome depth;

[0037] Figure 8 It is the overall process framework diagram of the present invention. DETAILED DESCRIPTION

[0038] The following will describe the technical solutions in the embodiments of the present invention in detail with reference to the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention.

[0039] The present invention will be further described below with reference to the accompanying drawings:

[0040] like Figure 8 As shown, a molecular dynamics simulation analysis method of Piezo1 protein based on ultrasonic perturbation includes the following steps:

[0041] Step 1: Use the protein three-dimensional structure comparative modeling tool Modeller to complete the missing amino acids of Piezo1 protein.

[0042] Step 2: Use the Martini coarse-graining tool martinize.py to coarse-grain the protein, use the insane.py tool to build a coarse-grained cell membrane, and then insert the coarse-grained protein into the cell membrane.

[0043] Step 3: Couple the ultrasonic model to the GROMACS software, verify the successful ultrasonic loading through the fluctuation of the lateral water density, and calculate the corresponding frequency.

[0044] Step 4: Referring to the ultrasonic response frequency of Piezo1 demonstrated by Prieto et al., it is intended to simulate ultrasonic perturbations of the Piezo1 protein system at different frequencies of 10 MHz, 20 MHz, and 35 MHz, and compare them with the non-ultrasonic perturbation simulation system.

[0045] Step 5: Analyze the trajectory of the molecular dynamics simulation, including using the lipyphilic tool to analyze the cell membrane morphology and composition, using pylipid to analyze the interaction between lipids and proteins, and using the analysis tools provided by gromacs to analyze the protein structure.

[0046] Furthermore, the step one: using the protein three-dimensional structure comparison modeling tool Modeller to complete the missing amino acids of the Piezo1 protein: first, the Piezo1 multimeric protein is downloaded from the PDB database, and then the protein three-dimensional structure comparison modeling tool Modeller is called by writing a script to perform sequence alignment and residue completion on the protein, and then the structure is optimized in the Modeller software package using the Piezo1 protein three-dimensional structure in the PDB data as a template to ensure that the completed residues have a relatively correct protein tertiary structure. Finally, each chain of the protein model contains fragments 577-717, 782-1365, 1493-1578, 1655-1807 and 1952-2547.

[0047] Furthermore, the step 2: coarse-graining the protein using the Martini coarse-graining tool martinize.py, and constructing the coarse-grained cell membrane using the insane.py tool, and inserting the coarse-grained protein into the cell membrane: First, the protein tertiary structure obtained in step 1 is coarse-grained using the Martini coarse-graining tool martinize.py on the Piezo1 protein, and the coarse-grained structure is as follows: Figure 1 ; Secondly, the coarse-grained membrane was constructed using the insane.py tool, and the coarse-grained protein was inserted into the cell membrane to create a 40×40×40nm 3 The simulation system was added with 0.15 mol / L. The outer leaflet membrane components included: POPC, DPSM, POPE, CHOL, and the inner leaflet membrane components included: POPC, POPE, POPS, CHOL, and POP5. The model is as follows Figure 2 .

[0048] Furthermore, the step three is to use the ultrasonic model coupled to the GROMACS software to verify the successful loading of the ultrasonic wave by plotting the water density on the X-axis and to calculate the frequency of the response: first, an ultrasonic model is established to generate shock waves on six surfaces to irradiate the six surfaces respectively, and each ultrasonic velocity is defined as follows: Formula (1) and Formula (2), and then a 30×30×30nm is established. 3 Simulating a water tank, using the GROMACS built-in tool density to calculate the density fluctuation on the x-axis to verify successful ultrasonic loading, the water density diagram under ultrasonic perturbation is as follows Figure 3 .

[0049]

[0050]

[0051] V i Represents the six ultrasonic velocities along the positive and negative directions of X, Y, and Z, respectively. V max represents the maximum speed of the ultrasonic pulse, m represents the time step, N represents the molecular dynamics simulation step, i as a subscript represents one of the six ultrasonic velocity directions, X0 represents the X coordinate, Y0 represents the Y coordinate, and Z0 represents the Z coordinate.

[0052] Figure 3 The graph shows the change of water density over time at 2nm, 4nm, 6nm, and 8nm on the X-axis. It shows complete periodicity at each X-axis position, indicating that the ultrasound is successfully loaded, and the peaks at different X-axis positions show that the ultrasound also propagates along the X-axis.

[0053] Furthermore, the step four: constructing a non-ultrasonic perturbation simulation system and 10Mhz, 20Mhz, and 35Mhz ultrasonic perturbation simulation systems, and using Gromacs software for molecular dynamics simulation: first, using the ultrasonic model in step 3, establishing 10Mhz, 20Mhz, and 35Mhz ultrasonic perturbation simulation systems and establishing a non-ultrasonic perturbation simulation system; secondly, minimizing the energy of these four simulation systems, and then using the NPT ensemble to balance the four systems for 500ns, and finally using the NPT ensemble to perform 5us dynamic simulation on the four systems.

[0054] Furthermore, the step five: analyzing the molecular dynamics simulation trajectory includes using the lipyphilic tool to analyze the cell membrane morphology and composition, using pylipid to analyze the interaction between lipids and proteins, and using the analysis tools provided by GROMACS to analyze the protein structure: first, the lipyphilic tool is used to analyze the membrane thickness and the area of ​​each lipid of the cell membrane, and then the pylipid tool is used to calculate the binding sites between various lipids and proteins, the residence time of the interaction between lipids and binding sites and single amino acid residues, and finally the GROMACS built-in tools are used to analyze the changes in protein structure.

[0055] Calculation of membrane thickness: First, identify the lipids of the inner and outer leaflets of the cell membrane, and calculate the change of cell membrane thickness with simulation time by identifying the peak distance Z of lipid head group density, such as Figure 4 .

[0056] Calculation of lipid and protein binding sites: For a group of residues that bind to the same lipid molecule at the same time, pylipid writes a distance vector recording the distance between each residue and all lipid molecules as a function of time, and constructs a lipid interaction network whose nodes are protein residues and weights are the Pearson correlation coefficients of residue pairs calculated from their distance vectors.

[0057] Interaction residence time: Since lipids and proteins interact, lipids are no longer diffusing. Therefore, pylipid uses a double exponential to calculate the residence time to account for the long and short decays of lipid relaxation. The residence time is (koff represents the dissociation constant), which is calculated based on the normalized survival function using the duration. It is considered that the residence time exceeds 70% of the simulation time to be a strong interaction. Our simulation trajectory is 5us, so the residence time exceeds 3.5us, so it is considered a strong interaction site. By writing a script to count the interaction sites, Figure 5 , found that CHOL and PIP2 interact strongly with proteins at multiple sites, while POPC, POPE, and POPS interact more weakly. When ultrasonic perturbations were applied at 10 MHz, all lipids had 28 strong interaction sites, 32 at 20 MHz, and 32 at 35 MHz.

[0058] Protein RMSD graph calculation: Using the RMSD calculation tool rms that comes with gromacs, the protein structure of the subsequent simulation frame is compared with the protein of the first frame. The results are as follows Figure 6 shown.

[0059] Protein density and dome depth: The protein density was fitted to the central pore region (residues 2105-2547) of the reference structure protein using the molecular tool densmap provided by gromacs; its dome depth was plotted using the corresponding script. The results are as follows Figure 7 shown.

Claims

1. A molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein, characterized in that: The following steps are involved: Step 1: Establish an ultrasonic model and couple it to Gromacs software; The ultrasonic model is to establish six ultrasonic velocities of the same size and different directions. The velocities are generated at the edges of the six faces of the three-dimensional box, and the velocities of each face are generated at a certain time interval. The six ultrasonic velocities are defined as follows: V i Represents the six ultrasonic velocities along the positive and negative directions of X, Y, and Z, respectively. V max represents the maximum velocity of the ultrasonic pulse, m represents the time step, N represents the molecular dynamics simulation step, i as a subscript represents one of the six ultrasonic velocity directions, X0 represents the X coordinate, Y0 represents the Y coordinate, and Z0 represents the Z coordinate; Step 2: Complement the missing amino acids in the protein, coarse-grain it and insert it into the cell membrane, and perform molecular dynamics simulation analysis using the ultrasound model described in step 1; The molecular dynamics simulation analysis includes: Observe and analyze the morphological changes of cell membranes, including changes in membrane thickness and membrane area; Analyze the interaction time between membrane components and proteins; Analysis of protein conformational changes showed that changes in cells under ultrasonic perturbations caused interactions between membrane components and proteins, which further led to conformational changes.

2. The molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein according to claim 1, characterized in that: The coarse-graining is performed using a 4-to-1 mapping.

3. The molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein according to claim 1, characterized in that: The calculation of the membrane thickness is as follows: first, the lipids of the inner and outer leaflets of the cell membrane are identified, and the change of the cell membrane thickness over the simulation time is calculated by identifying the peak distance Z of the lipid head group density.

4. The molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein according to claim 1, characterized in that: The membrane area was calculated by 2D Voronoi tessellation, using Freud's local module to perform the tessellation of atomic positions.

5. The molecular dynamics simulation analysis method of ultrasonic perturbation of Piezo1 protein according to claim 1, characterized in that: The interaction time between the membrane component and the protein is determined by identifying the interaction site between the protein and the membrane component and calculating the residence time of the response site to determine whether the site has a strong interaction or a weak interaction.

6. The molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein according to claim 1, characterized in that: The changes in protein conformation are observed by using a script to calculate the depth of the dome formed by the Piezo1 protein in the membrane and to draw a two-dimensional density map of the protein in the xy plane to observe the structural changes of Piezo1 under ultrasonic perturbation.

7. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, which, when executed by a processor, implements the molecular dynamics simulation analysis method for ultrasonic perturbation of Piezo1 protein according to any one of claims 1 to 6.

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