Molecular dynamics simulation method for OSCA protein ion selectivity

Abstract

The invention discloses a molecular dynamics simulation method for OSCA protein ion selectivity, and belongs to the field of neuroscience and the field of molecular dynamics. The structural characteristics of OSCA ion channel protein ion selectivity are discussed by using a stretching molecular dynamics method, the motion trails of different ions under stretching molecular dynamics are analyzed from the microscopic perspective, the dynamic change of selectivity is observed, and the hindering site of an ion selective filter is judged. A distance-time chart in the ion transmission process is drawn by stretching a molecular dynamics track, PMF is calculated through umbrella-shaped sampling, energy barriers at different positions of a channel are observed, energy barrier differences of different ions passing through the same barrier point are compared, and the ion selectivity difference of the OSCA ion channel is judged. The characteristics of the ion selective filter can be intuitively analyzed through molecular dynamics, so that the rule of ion selectivity is summarized, and the ion selective filter can be favorably applied to basic research and clinical application.

Description

Technical Field

[0001] This invention belongs to the field of neuroscience and molecular dynamics, specifically relating to a simulation method for the ion selectivity of OSCA protein based on molecular dynamics. Background Technology

[0002] Ion channels in biological membranes are pathways for the passive transport of various inorganic ions across membranes. Ion channels on cell membranes are crucial for the exchange of substances between living cells and their external environment. Organisms must precisely regulate the concentrations of various ions in intracellular and extracellular fluids, allowing ions of specific sizes to pass through through ion-selective filters, transmitting signals to maintain homeostasis. Many drugs also exert their effects by regulating the activity of ion channels, highlighting their vital role in organisms. OSCA ion-selective channel proteins were first discovered in plants and play a crucial role in plant growth and development, helping plants sense changes in their environment and respond accordingly. OSCA ion-selective channel proteins are widely distributed in various plants and are divided into four branches. Members of each branch play specific roles in different environments and physiological processes, influencing the entire process of plant growth and development. For example, the calcium-selective signaling of OSCA channel proteins can respond to osmotic changes and play a role in stomatal closure, organ and fruit development, and reproductive processes.

[0003] Current main methods for studying ion channels include cryo-electron microscopy, nanophospholipid disks, patch-clamp techniques, ultrasound stimulation, and molecular dynamics simulation. Experimental methods can characterize the instantaneous conformation of proteins at a given moment, while molecular dynamics simulation can establish dynamic simulation processes using computers, capturing dynamic change data. Molecular dynamics simulation (MD) is a method based on Newtonian mechanics to simulate the physical trajectories and states of molecules. It is used to simulate the motion and interactions of atoms and molecules over a given time to study the structure and properties of molecules or molecular systems. Through computer-aided methods, it connects the properties of microscopic and macroscopic systems, allowing observation of the dynamic changes in proteins. OSCA proteins are ion channel proteins that allow cations to pass through. Using molecular dynamics simulation, the dynamic process of different ions passing through OSCA ion channels can be observed from a microscopic perspective. This allows for the identification of obstruction sites in ion-selective filters, observation of ion channel structure and analysis of pore size, comparison of the ability of different cations to pass through the ion channel, and observation of structural changes in proteins during ion passage, which is beneficial for analyzing differences in ion selectivity.

[0004] Currently, the study of ion-selective filters in OSCA ion channel proteins using molecular dynamics for protein structure research is not yet in-depth. While molecular dynamics simulations can obtain a simple conformation of proteins after equilibrium, the details of the conformation of protein ion-selective filters are not accurately understood. By obtaining the equilibrium state of proteins through molecular dynamics simulations, and then calculating the free energy using stretching molecular dynamics simulations and umbrella sampling, accurate calculations of free energy changes can be obtained. This allows for observation of the dynamic changes of ions passing through the ion-selective filter, leading to a more accurate analysis of the ion selectivity of OSCA ion channels and comparison of differences in ion selectivity. Using molecular dynamics simulations and stretching molecular dynamics simulations to calculate free energy allows for the determination of the structural characteristics and gating mechanisms of OSCA protein ion-selective filters. Analyzing the differences in ion selectivity of OSCA ion channels for different cations is beneficial for exploring the structural features of OSCA protein ion-selective filters. Understanding the structural characteristics of ion-selective filters is beneficial for applications in basic research and clinical practice. It is also crucial for plants to sense mechanical forces in their internal and external environments and respond accordingly to promote growth and development. Furthermore, it is extremely important for the study of the OSCA / TMEM63 channel family. Ion-selective filters are crucial for living organisms, regulating ion concentrations in intracellular and extracellular fluids by controlling the ions entering the body, controlling signal transmission, maintaining homeostasis, and facilitating drug delivery. This invention analyzes the characteristics of ion-selective filters from a microscopic perspective, using stretched molecular dynamics to explore the structural features of various ion channel proteins and investigate their ion-selective filter characteristics. Summary of the Invention

[0005] To compare the ability of different cations to pass through OSCA protein ion channels, determine differences in ion selectivity, and observe the details of residue interactions within the ion-selective filter, this invention proposes a molecular dynamics simulation method for OSCA protein ion selectivity. The technical solution of this invention is as follows:

[0006] A molecular dynamics simulation method for the ion selectivity of OSCA proteins, comprising the following steps:

[0007] Step 1: Obtain the protein crystal structure of OSCA protein containing two peptide chains from the PDB database;

[0008] Step 2: Embed the OSCA protein into the cell membrane environment on the CHARMM-GUI online website, set the system force field, and prepare the molecular dynamics simulation system;

[0009] Step 3: Perform molecular dynamics simulations using NAMD software until the system reaches equilibrium.

[0010] Step 4: Analyze the trajectory files, log files, and extended system configuration files after the molecular dynamics simulation using the VMD TK console and Analysis module.

[0011] Step 5: Extract the representative conformation of the protein after molecular dynamics trajectory equilibrium, add different cation solutions to remodel, and calculate the free energy using stretching molecular dynamics and umbrella sampling methods respectively.

[0012] Furthermore, in step one, the crystal structure of the OSCA protein containing two peptide chains is obtained from the PDB database.

[0013] Furthermore, in step two, the Membrane Builder module in the CHARMM-GUI online website is used to select one chain of the OSCA dimer protein to start modeling. The monomer protein is embedded in the cell membrane environment, a suitable pH value is selected, missing residues are added, and protonation states are assigned. In CHARMM-GUI, the monomer is embedded in a POCP membrane, 0.1 mol / L CaCl2 solution is added, the entire monomer is encapsulated in a periodic box, and the entire monomer is loaded into a CHARMM36 force field.

[0014] Furthermore, step three, using NAMD software to perform molecular dynamics simulations, specifically includes: writing the configuration file required for the NAMD simulation; using the steepest descent method to minimize the energy of the system and bring it to a relatively stable state; first, performing the simulation under a canonical ensemble (NVT) where the system temperature, volume, and ion number remain constant to reduce errors caused by temperature fluctuations; after reaching the specified temperature, performing the simulation for a sufficient time under constant-pressure, constant-temperature (NPT) conditions where the particle number, pressure, and temperature remain constant until the protein structure tends to stabilize and the system reaches a stable state in terms of density and temperature.

[0015] Further, in step four, the molecular dynamics simulation trajectory is analyzed using the VMD TK console and Analysis module. In the molecular dynamics simulation, each atom in the system moves continuously at each time step according to its initial velocity. As time progresses, the atomic motion trajectory of the entire system is completely simulated until the set simulation time is reached. Information related to atomic motion is recorded in log files, trajectory files, and extended system configuration files. VMD software is used to analyze the dynamic conformational changes of the ion-selective filter of the protein throughout the simulation, calculate the root mean square deviation (RMSD), determine the structural stability of the OSCA ion channel, and analyze the interactions, angles, and distances of relevant residues within the OSCA protein ion channel. This reflects which regions within the OSCA ion-selective filter hinder ion passage and which regions facilitate it.

[0016] Furthermore, the molecular dynamics log files were analyzed using the VMD analysis interface. This analysis included: the relevant settings and force field files used throughout the simulation process; the output of temperature, pressure, and energy information during the simulation; and the use of VMD software to analyze whether the temperature and pressure remained stable at the set values, how energy and volume changed, and whether the OSCA protein maintained a relatively stable conformation throughout the simulation.

[0017] Furthermore, step five, calculating the free energy using stretched molecular dynamics and umbrella sampling, specifically includes:

[0018] After molecular dynamics simulation and equilibrium, the protein structure is clustered, and the representative conformation of the clustered protein is extracted as the initial model. Solutions containing different cations are added to the representative protein conformations, and the cations are moved to the channel opening to constrain the stretched cations. After energy minimization and equilibrium, NAMD software is used to simulate the process of ions passing through the ion channel from one side to the other at a constant speed along the Z-axis, and a tension-time curve is plotted to infer the ion selectivity. Then, the free energy is calculated using umbrella sampling; PMF curves are obtained, and the energy barrier differences of different cations passing through the same obstacle site are compared. The obstacle site of the ion selective filter is determined based on the PMF curves, and the ion selectivity of the OSCA ion channel for different cations is compared. In the SMD research method, a hypothetical atom is first set as the force-applying atom. The force-applying atom is connected to an atom in the system through a hypothetical spring. During the stretching, the force-applying atom moves at a constant speed, pulling the atoms in the system. This is constant-speed stretching. However, the velocity of the atoms being pulled in the system is not constant; the tension they experience is determined by the deformation and elastic coefficient of the spring, and it obeys Hooke's Law.

[0019]

[0020] Umbrella sampling is an enhanced sampling method used in molecular dynamics simulations to calculate free energies. The basic idea is to first divide the reaction coordinates into multiple windows, ensuring a sufficient number of windows cover the entire reaction coordinate space, and then add a bias potential to each window. Each umbrella sampling window is then simulated separately, and the umbrella histograms from each sampling window are combined to represent the probability distribution of conformations along the reaction coordinates under the influence of the bias potential.

[0021] The weighted histogram analysis method (WHAM) was used to combine the umbrella sampling simulation results to obtain the mean force (PMF).

[0022] The advantages and beneficial effects of this invention are as follows:

[0023] (1) This invention uses molecular dynamics simulations of the OSCA protein to elucidate the ion selectivity of the OSCA ion selective filter. Compared with previous experimental studies, this invention elucidates the interactions of relevant residues in the OSCA protein ion channel and the entire process of helical changes in the ion channel from a more microscopic perspective, providing a more intuitive analysis of the characteristics of the ion selective filter.

[0024] (2) Using constant-speed stretching molecular dynamics simulation, compare the magnitude of the stretching force required for different ions to pass through the same channel and the same obstruction site at the same speed, calculate the PMF using their trajectories, and compare the energy barrier required for different ions at the same channel and the same obstruction site to determine the ion selectivity of the ion selective filter. Attached Figure Description

[0025] Figure 1 This is the periodic box model of protein monomers in this invention;

[0026] Figure 2 This is the RMSD image of the protein in this invention;

[0027] Figure 3 This is a representative protein structure diagram of the present invention;

[0028] Figure 4 This is a static overall view of the protein ion selective filter of the present invention;

[0029] Figure 5 This is a protein electrostatic surface potential diagram of the present invention;

[0030] Figure 6 This is a flowchart illustrating the overall process framework of the present invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and thoroughly described below. The described embodiments are only a part of the embodiments of the present invention.

[0032] The technical solution of the present invention to solve the above-mentioned technical problems is:

[0033] To achieve the above objectives, the present invention adopts the following technical solution: a molecular dynamics simulation method for the ion selectivity of OSCA proteins, comprising the following steps:

[0034] Step 1: Obtain the crystal structure of the OSCA protein mutant open state 8XRY containing two peptide chains from the PDB database.

[0035] Step 2: Embed the OSCA dimer protein structure in the cell membrane environment on the CHARMM-GUI online website, select one strand to construct the initial model; add missing residues; embed the monomer into a periodic box containing POPC, TIP, and 0.1 mol / L CaCl2 solution on the CHARMM-GUI online website, and load the monomer into the force field of CHARMM36.

[0036] Step 3: Molecular Dynamics Simulation; Compile the configuration file required for NAMD simulation. First, use the steepest descent method to minimize the energy of the system and bring it to a relatively stable state. Simulate under a canonical ensemble (NVT) where the system temperature, volume, and ion number remain constant to reduce errors caused by temperature fluctuations. After reaching the required temperature of 310 K, simulate for a sufficient time under constant-pressure, constant-temperature (NPT) conditions until the protein structure tends to stabilize and the system reaches a stable state in terms of density and temperature.

[0037] Step 4: Analyze the molecular dynamics trajectory using the VMD TK console and Analysis module. In the molecular dynamics simulation, each atom in the system moves continuously at each time step according to its initial velocity. As time progresses, the atomic trajectories of the entire system are completely simulated until the set simulation time is reached. Information related to atomic motion is recorded in log files, trajectory files, and extended system configuration files. VMD software is used to analyze the changes in the ion-selective filter of the protein throughout the simulation, calculate the root mean square deviation (RMSD), determine the structural stability of the OSCA ion channel, and analyze the interactions, angles, and distances of relevant residues within the OSCA protein ion channel. This reveals which regions within the OSCA ion-selective filter hinder ion passage and which regions facilitate it.

[0038] Step 5: Calculate Free Energy Simulation. Free energy describes the thermodynamic quantity of a system in equilibrium. In stretched molecular dynamics simulations, an external force is applied to drive ions through ion channels, and the change in the system's free energy with respect to ion position is calculated to analyze the differences in ion selectivity. During the simulation, the simulation velocity is often greater than the actual velocity of ions through the ion channels. An enhanced sampling method—umbrella sampling—is used. By introducing a bias potential, the system is confined to a specific region for sampling, and the free energy difference of ions at different positions is calculated, thus improving the calculation accuracy. Umbrella sampling simulation can find the equilibrium state within a set of umbrella sampling windows and obtain a relatively accurate PMF curve by reweighting the constant bias potential trap along the reaction coordinates.

[0039] First, download the open-state dimeric crystal structure of the OSCA protein mutant named 8XRY from the PDB database.

[0040] The crystal structure was manipulated using the Membrane Builder module on the CHARMM-GUI online website. One chain of the protein was selected to start modeling, and it was embedded in the cell membrane environment, with residues 253-286 deleted. The pH was set to 7.2. The initial model was then embedded in a POPC lipid bilayer and immersed in a periodic box containing TIP and 0.1 mol / L CaCl2 solution. The entire monomer was then loaded into the force field of CHARMM36m.

[0041] All simulations used the CHARMM36 force field. During the simulation, the system was first minimized using the steepest descent method over 10,000 steps. Then, the system was heated in the NVT ensemble to a temperature of 310 K. Next, the system was balanced in the NPT ensemble by partially restraining the dihedral angles and planes of the protein; and the magnitude of the harmonic restraint was gradually decreased during equilibrium.

[0042] Finally, the NPT ensemble was used to perform a 250 ns molecular dynamics simulation of the system under isothermal and isobaric conditions without any restrictions. Throughout the simulation, the Nose-Hoover Langevin piston method was used to keep the pressure constant at 1.01325 bar, and Langevin kinetics was used to keep the temperature constant at 310 K.

[0043] OSCA ion channel protein opening and related helical movement within the ion channel, as well as the disruption of hydrophobic barriers, are closely related. Therefore, understanding the helical rotation and residue distribution of OSCA protein ion channels is crucial. VMD (Vibration Dynamics Method) is used to observe the interactions, angles, and distances of related residues within the channel, analyze the channel helices to understand the ion channel composition, identify sites that impede ion passage and sites that facilitate ion passage, and compare differences in ion selectivity.

[0044] The representative conformation after equilibrium, extracted from protein clustering analysis following molecular dynamics simulation, is used as the initial conformation for stretched molecular dynamics. + and Ca 2+ Stretching molecular dynamics simulations were performed to determine the differences in ion selectivity of the OSCA ion-selective filter for different ions. Representative protein conformations were extracted using cluster analysis and manipulated in the MembraneBuilder module of the CHARMM-GUI online website. The first model was embedded in a cell membrane environment with a pH of 7.2. The initial model was then embedded in a POPC lipid bilayer and immersed in a periodic box containing a TIP and 0.1 mol / L KCl solution. The entire monomer was then loaded into a CHARMM36m force field. The second model was also embedded in a cell membrane environment with a pH of 7.2. The initial model was then embedded in a POPC lipid bilayer and immersed in a periodic box containing a TIP and 0.1 mol / L CaCl2 solution. The entire monomer was then loaded into a CHARMM36m force field.

[0045] In the first model, select K to apply the external force. + Move it to the channel opening to prepare for the stretching molecular dynamics simulation. Before stretching, first move K to the channel opening. + After applying certain constraints to maintain the system at equilibrium, K + The stretching operation is performed near the channel opening, followed by energy minimization and equilibrium simulations. Once the system reaches equilibrium, stretching molecular dynamics simulations are initiated, with K artificially introduced during the simulation. +An imaginary external force is applied to cause ions to move along ion channels, and detailed coordinate information of the ions throughout the stretching process is obtained. Important information about the ion conduction process is obtained by analyzing energy and structure. Similarly, in the second model, Ca is selected as the target of the external force. 2+ The Ca2+ was moved to the channel opening in preparation for the stretching molecular dynamics simulation. Before stretching, the Ca2+ was first moved to the channel opening. 2+ After applying certain constraints to maintain the system at equilibrium, Ca 2+ The stretching operation was performed near the channel opening, followed by energy minimization and equilibrium simulations. Once the system reached equilibrium, stretching molecular dynamics simulations were initiated, in which Ca was artificially introduced. 2+ An imaginary external force is applied to cause ions to move along ion channels, and detailed coordinate information of the ions throughout the stretching process is obtained. Important information about ion conduction is obtained by analyzing energy and structure. All stretching molecular dynamics simulations used above maintain a constant velocity. To compare the capabilities of different cation channels, different cations are selected for constant-velocity stretching molecular dynamics simulations to determine the ability of different ions to pass through the ion-selective filter. Information about the magnitude of the SMD force is extracted from the NAMD output file in the VMD TK console log file, and a stretching-time plot is generated using Ogin. This plot is then compared with the K at the same site. + and Ca 2+ The magnitude of the pulling force is used to initially determine the ion selectivity of the OSCA ion-selective filter, and then an umbrella sampling simulation is initiated using an SMD trajectory. The reaction coordinates used in the umbrella sampling simulation are along the z-distance of the channel axis, and the reaction coordinate space is spaced at intervals of... Divide a window and use the above stretching molecular dynamics simulation to obtain K. + Ca 2+ The structure near the center of each window is extracted from the channel trajectory file as the initial conformation for each independent window. Then, a 1 ns molecular dynamics simulation is performed on each independent window to obtain its trajectory file. Finally, the merged window data is analyzed using the weighted histogram analysis (WHAM) method to obtain the PMF, and K0 is plotted using Oorgin. + and Ca 2+ PMF plot, compared with K at the same site + and Ca 2+ By determining the energy required to overcome this point, the obstruction site of ion-selective filtration can be identified. Combined with the tension-time diagram obtained from SMD, the ion selectivity of different cations passing through the OSCA ion-selective filter can be determined. The characteristics of the ion-selective filter can be analyzed intuitively, and the laws of ion selectivity can be summarized.

Claims

1. A molecular dynamics simulation method for the ion selectivity of OSCA channel proteins, characterized in that, Includes the following steps: Step 1: Obtain the protein crystal structure of OSCA protein containing two peptide chains from the PDB database; Step 2: Embed the OSCA protein into the cell membrane environment on the CHARMM-GUI online website, set the system force field, and prepare the molecular dynamics simulation system; Step 3: Perform molecular dynamics simulations using NAMD software until the system reaches equilibrium. Step 4: Analyze the trajectory files, log files, and extended system configuration files after the molecular dynamics simulation using the VMD TK console and Analysis module. Step 5: Extract the representative protein conformation after molecular dynamics trajectory equilibrium, and remodel it by adding different cation solutions. Calculate the free energy using stretched molecular dynamics and umbrella sampling methods respectively. Based on the magnitude of the mean force (PMF), the differences in the selective passage of various ions through the OSCA channel protein can be distinguished.

2. The molecular dynamics simulation method for OSCA protein ion selectivity according to claim 1, characterized in that, Step one involves obtaining the protein structure of OSCA protein containing two peptide chains from the PDB database, or directly obtaining the protein structure containing only two complete peptide chains from the PDB database, excluding other lipid molecules.

3. The molecular dynamics simulation method for OSCA protein ion selectivity according to claim 1, characterized in that, Step two involves using the CHARMM-GUI online website to select one chain of the OSCA dimer protein to begin modeling, embedding the monomer protein into the cell membrane environment, selecting an appropriate pH value, adding missing residues, and assigning protonated states. In the CHARMM-GUI Membrane Builder module, the monomer is embedded into a POPC membrane, 0.1 mol / L CaCl2 solution is added, the entire monomer is encapsulated in a periodic box, and the entire monomer is loaded into a CHARMM36 force field.

4. The molecular dynamics simulation method for OSCA protein ion selectivity according to claim 1, characterized in that, Step three involves performing molecular dynamics simulations using NAMD software. Specifically, this includes minimizing the energy of the system using the steepest descent method to bring the system to a relatively stable state. The simulation is first performed under a canonical ensemble (NVT) where the system temperature, volume, and ion number remain constant to reduce errors caused by temperature fluctuations. After reaching the specified temperature, the simulation is performed for a sufficient time under constant-pressure and constant-temperature (NPT) conditions until the protein structure tends to stabilize and the system reaches a stable state in terms of density and temperature.

5. The molecular dynamics simulation method for OSCA protein ion selectivity according to claim 1, characterized in that, Step four involves analyzing the molecular dynamics simulation trajectory using the VMD TK console and Analysis module. In the molecular dynamics simulation, each atom in the system moves continuously at each time step based on its initial velocity. As time progresses, the atomic trajectories of the entire system are fully simulated until the set simulation time is reached. Information related to atomic motion is recorded in log files, trajectory files, and extended system configuration files. VMD software is used to analyze the dynamic conformational changes of the ion-selective filter throughout the simulation, calculate the root mean square deviation (RMSD), determine the structural stability of the OSCA ion channel, and analyze the interactions, angles, and distances of relevant residues within the OSCA protein ion channel. This analysis reveals which regions within the OSCA ion-selective filter hinder ion passage and which regions facilitate it.

6. The molecular dynamics simulation method for OSCA protein ion selectivity according to claim 1, characterized in that, Step five, calculating the free energy using stretched molecular dynamics and umbrella sampling, specifically includes: After molecular dynamics simulations and equilibrium, protein structures are clustered, and representative conformations of the clustered proteins are extracted as initial models. Solutions containing different cations are added to these representative conformations, and the cations are moved to the channel openings. Using NAMD software, the process of simulating ions passing through the ion channel from one side to the other at a constant speed along the Z-axis is simulated, and a pull-time plot is generated to infer ion selectivity. Then, the free energy is calculated using umbrella sampling to obtain PMF curves, and the energy barrier differences for different cations passing through the same barrier site are compared to determine the ion selectivity of the OSCA ion channel for different cations. In the SMD research method, a hypothetical atom is first set as the force-exerting atom, connected to an atom in the system by a hypothetical spring. During the pull, the force-exerting atom moves at a constant speed, pulling the atoms in the system—this is constant-speed pull. However, the speed of the pulled atom in the system is not constant; the pull force is determined by the deformation and elastic coefficient of the spring and satisfies Hooke's Law. Umbrella sampling is an enhanced sampling method used in molecular dynamics simulations to calculate free energies. The basic idea is to use the trajectory file obtained from SMD (Simplified Method Analysis) to first divide the trajectory file into multiple windows based on the reaction coordinates, ensuring enough windows cover the entire reaction coordinate space. A bias potential is added to each window. Then, each umbrella sampling window is simulated separately, and the umbrella histograms from each sampling window are combined to represent the probability distribution of the conformation along the reaction coordinates under the influence of the bias potential. Finally, the weighted histogram analysis method (WHAM) was used to combine the umbrella sampling simulation results to obtain the potential of mean force (PMF).

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