CN110634533A

CN110634533A - Method for obtaining controllable TRPV5 variant based on computer simulation

Info

Publication number: CN110634533A
Application number: CN201910793867.XA
Authority: CN
Inventors: 沈勇; 谭俊杰
Original assignee: National Sun Yat Sen University
Current assignee: National Sun Yat Sen University
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2019-12-31
Anticipated expiration: 2039-08-27
Also published as: CN110634533B

Abstract

The invention discloses a method for obtaining a controllable TRPV5 variant based on computer simulation. The method firstly adopts CHARMM-GUI to perform mutation treatment on a channel protein, then uses a molecular dynamics simulation module pmemd.cuda in Amber16 to simulate cations of the TRPV5 channel protein variant under different states, and finally combines the analysis of the movement path of the cations by using a cPptraj module in VMD and Amber16 to verify the channel performance of the TRPV5 channel protein variant. The invention can research and analyze the motion trail of the whole simulation system, overcomes the difficulty that the intermediate state is difficult to continuously observe, and can intuitively observe the motion change of the cation in a very short time scale. The whole calculation process has high efficiency, short time consumption and simple operation, and is beneficial to wide application in the fields of biochemistry, protein engineering and the like.

Description

Method for obtaining controllable TRPV5 variant based on computer simulation

Technical Field

The invention belongs to the technical field of molecular dynamics computer simulation. More particularly, it relates to a method for obtaining regulatable TRPV5 variants based on computer modeling.

Background

Transient Receptor Potential cation channel proteins (TRP channels) are a class of cation channel proteins that are widely found in organisms, wherein TRPV5 is a member of the TRPV subfamily of members of the Transient Receptor Potential (TRP) channel superfamily, and is a cation channel protein of which the subfamily is V and member 5. The channel proteinConsisting of 729 amino acids and having a molecular weight of about 83 kDa. The corresponding gene consists of 15 exons arranged on chromosome 7q34, and is mainly distributed in kidney, osteoclast, placenta, etc. Among them, the distribution of renal tubules is high, and it is mainly expressed in apical cell membranes of renal epithelial cells in distal tubules and collecting tubules, and also expressed in osteoclasts, and its expression level is not as high as that in kidneys, but it is suggested that TRPV5 is an important channel protein mediating osteoclast bone resorption. The research proves that the calcium-calcium complex has the effect of reducing Ca in the kidney²⁺Active reabsorption is relevant, TRPV5 is a unique calcium-selective ion channel in the TRP family. First, when this channel is open, TRPV5 allows calcium ions in urine to enter the cell along a concentration gradient, and after calcium ions enter the cell through TRPV5, they diffuse to the basolateral membrane bound to calbindin D28K. Mice knockout of the relevant TRPV5 gene in the mouse study all showed varying degrees of in vivo ion imbalance and urine calcium overload. Studies with different populations also show that the low renal stone risk of african americans is also closely related to TRPV5 expression. Therefore, the active reabsorption process of TRPV5 calcium ions in the epidermal cells of kidney plays an important role in human body. Research on mechanism discussion, structural modification, regulation and the like of TRPV5 is beneficial to promoting the cognition of modern medicine on kidney diseases, and provides a new research angle for curing diseases caused by calcium ion imbalance.

At present, econazole (Econazol) and calcitonin (camodulin, CaM) are mostly used to be combined with residues at an outlet of TRPV5, calcium ion conduction of TRPV5 is regulated through experimental means such as sol electrophoresis, and the structure, electrophysiological properties and regulatory factors of TRPV5 are researched. However, experimental research has certain limitations, influence guidance is single, reverse reaction operability is poor, substances such as econazole, calcitonin and the like are not easy to separate once being combined with an outlet of TRPV5, ideal regulation and control effects can be achieved only by combining a plurality of compounds and residues, and cost is high. The research on the variant of the TRPV5 channel protein is complicated in experiment, long in culture time and difficult to operate, and becomes a research bottleneck of the TRPV 5. In addition, the process of conducting calcium ions by the TRPV5 is very short, the ion conduction process is difficult to capture experimentally, and the dynamic regulation process of the TRPV5 cannot be continuously observed, so that the research on the conduction performance and the protein structure design of the TRPV5 is limited.

The method for constructing the protein structure model by using computer simulation can save the experiment cost and has high efficiency. However, the research of constructing the TRPV5 channel protein variant structure by computer simulation of protein engineering and enzyme-catalyzed residue phosphorylation modification technology is not reported. Theoretically, a complex protein variant final structure in an ion uptake process can be constructed by simulating a protein and ion reaction process through a computer based on a reaction molecular dynamics method, but due to the fact that the number of variants is large, the final structures of the variants are only used for comparison, the performances of different variants are difficult to evaluate comprehensively, and meanwhile, the technical problems that the ion conduction continuous state is difficult to observe, the reaction affinity is uncontrollable, the simulation time is long, the calculated amount is large, the speed is slow and the like exist, and further research on the TRPV5 in the science of biochemistry, protein engineering and the like is not facilitated.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides a method for acquiring a TRPV5 channel protein variant capable of being manually regulated and controlled based on computer simulation, which can more intuitively observe the ion uptake process of the TRPV5 channel protein by step-by-step simulation and continuous observation of a simulation system by virtue of multiple visualization tools such as VMD (VMD) and cpptraj modules.

The above purpose of the invention is realized by the following technical scheme:

a method for obtaining an artificially regulated TRPV5 channel protein variant based on computer simulation, comprising the following steps:

s1, analyzing key sites of a TRPV5 protein structure, carrying out mutation modification on the key sites to obtain a TRPV5 channel protein mutant A1 which loses the ability of taking calcium ions, and closing a channel;

s2, carrying out phosphorylation modification on the mutated sites of the mutant A1 described in S1 to obtain a plurality of TRPV5 channel protein mutants A2 capable of taking in calcium ions, and opening the channel;

s3, respectively placing calcium ions to be ingested near channel inlets of mutants A1 and A2 of S1 and S2, and respectively constructing cell membrane structure models of mutants A1 and A2 of S1 and S2 by using CHARMM-GUI software; the cell membrane structure model comprises variant A1 or variant A2, calcium ions to be taken in, solute ions, phospholipid bilayers and solvents;

s4, respectively performing energy minimization optimization on the cell membrane structure model of S3 by using a kinetic simulation module in molecular kinetic simulation software and combining a steepest descent method and a conjugate gradient method to obtain an energy optimized system;

s5, carrying out position limitation on protein heavy atoms and calcium ions in the energy-optimized system obtained in the step S4, and then carrying out pre-balance simulation on the system by using a dynamic simulation module in molecular dynamics simulation software to obtain a pre-balance system;

s6, performing molecular dynamics simulation on the pre-equilibrium system obtained in the S5 by using a dynamics simulation module in molecular dynamics simulation software to obtain a calcium ion motion track;

s7, combining a VMD program and a software ranging tool, observing whether calcium ions enter an ion channel of the different variants at channel inlets, analyzing the calcium ion movement situation according to the calcium ion movement track obtained in S6, and obtaining initial optimal variants in different variation routes;

s8, after obtaining the initial optimal variant, carrying out kinetic simulation (including energy minimization optimization, pre-equilibrium simulation and molecular dynamics simulation, and all parameters are consistent with the previous simulation) again so as to further test the calcium ion intake capacity, obtaining a motion trail file containing a plurality of crystal structure output files and all atoms of a system after the kinetic simulation is finished, and further verifying whether the calcium ions can be taken by the channel protein mutant at a farther distance;

s9, firstly, converting the crystal structure output files of S8 into PDB files suitable for observation by using an ambdbb module, then observing the final positions of calcium ions in the crystal structure output files by using VMD visualization software, analyzing the motion trail files of S8 by using an analysis module in molecular dynamics simulation software, and selecting and determining the final optimal TRPV5 channel protein variant according to the analysis result.

At present, experimental researches are concentrated on residues at an outlet of a TRPV5 ion channel, and because variant research steps are various and time is long, the research on the disability modification at an inlet of a TRPV5 channel protein is rarely carried out; in addition, the existing experimental observation and computer simulation both focus on the intermediate state structure of the capture system, and the ion uptake in the protein channel is a continuous process, so that the defect that the continuous state of ion conduction is difficult to observe exists. The method firstly adopts CHARMM-GUI to carry out mutation treatment on the channel protein, and then uses a molecular dynamics simulation module pmemd.cuda in Amber16 to simulate the TRPV5 channel protein variant cation under different states, and the reactivity is controllable; and finally, analyzing the motion path of the cation by combining VMD molecular visualization software and a Cpptraj data extraction module in Amber16, and analyzing the motion track of the whole simulation system on the atomic level, so that the problem that the intermediate state is difficult to continuously observe is solved. The whole simulation process is short in time, high in calculation process efficiency, short in time consumption, high in efficiency and simple in operation, and can make up for the defects of experimental research.

In a preferred embodiment of the invention, the critical position at S1 is aspartic acid at position 542 (Asp 542).

Further, in a preferred embodiment of the present invention, the mutational modification of the key site is: aspartic acid at position 542 of the TRPV5 channel protein was mutated into a serine mutant (D542S), a threonine mutant (D542T) and a tyrosine mutant (D542Y), respectively.

In a preferred embodiment of the present invention, the phosphorylation modification at S2 is: residue number 542 of said variant A1 was phosphorylated by CHARMM-GUI software.

Further, in the preferred embodiment of the present invention, the phosphorylation modification described in S2 resulted in a phosphorylated serine mutant (D542S-Phos), a phosphorylated threonine mutant (D542T-Phos), and a phosphorylated tyrosine mutant (D542Y-Phos).

In a preferred embodiment of the present invention, the cell membrane structure model of S2 is electrically neutral; the solute ions include chloride ions and sodium ions. The invention adopts the most common palmitoyl oleoyl phosphatidylcholine (POPC, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine) to build a phospholipid bilayer. The invention selects sodium ion and chloride ion (Na)⁺/Cl^-) The system is adjusted to maintain the system electrically neutral.

In the preferred embodiment of the present invention, the VMD software is used in S3 to determine the coordinate information of the mutant channel entry, and the location of calcium ion is set by modifying the PDB formatted mutant crystal structure file.

The dynamic simulation optimization comprises energy minimization optimization, pre-equilibrium simulation and dynamic simulation. The method specifically comprises the following steps: optimizing the system by using a kinetic simulation module in molecular kinetic simulation software to limit the minimization of the performance quantity; carrying out pre-equilibrium simulation on the system, and gradually releasing the limitation on protein and phospholipid; and (3) simulating different variant structures by adopting an NPT (non-point-to-point) ensemble and a dynamics simulation module in molecular dynamics simulation software to obtain an initial optimal variant structure.

In a preferred embodiment of the present invention, the energy minimization optimization of S4 includes optimizing the positions and structures of phospholipid molecules, hydrogen atoms of proteins, and solution molecules; the method comprises the steps of running a CHARMM36m force field in a Pmem module in an Amber16 dynamics simulation tool, sequentially adopting a steepest descent method and a conjugate gradient method to optimize a cell membrane structure model for 5000-20000 steps, preferably 5000-15000 steps, so as to obtain an optimal energy optimized system.

In order to make the simulation process as close to the real situation as possible, according to the motion requirements of different structures, the following position limitation is firstly carried out on protein heavy atoms, phospholipid molecules and calcium ions in the structure optimization simulation and the pre-equilibrium simulation before the beginning of the formal molecular dynamics simulation, so that the system can be fully adapted to the simulated environmental conditions. Through multiple experiments, the position restriction weight can accurately simulate the motion conditions of protein heavy atoms, phospholipid molecules and calcium ions, so that the interaction process of the calcium ions and biomolecules such as proteins can be accurately predicted.

Further, in the preferred embodiment of the present invention, the energy minimization optimization process of S4 is performed by

The weight of (A) limits the heavy atoms of the protein, preferably

The weight of (a) limits the protein heavy atoms; by using

The weight of (A) limits calcium ion, preferablyThe phospholipid molecules have a position-limiting weight of

It is preferable that

In the preferred embodiment of the present invention, the pre-balance simulation of S5 is performed on 375-450 ps by using pmemd. cuda module of Amber 16; the pre-equilibrium simulation is carried out in six steps, and the position limitation of protein heavy atoms and phospholipid molecules is gradually released at the ambient temperature of 303.15-330.5K.

In a preferred embodiment of the present invention, the pre-equilibrium simulated protein heavy atom position limit weight of S5 isIt is preferable that

PhospholipidsThe position-limiting weight of the molecule is

It is preferable that

The calcium ion has a position-limiting weight ofIt is preferable that

In a preferred embodiment of the present invention, the molecular dynamics simulation pressure control in S6 is performed by Monte Carlo pressure control method, the temperature control is performed by Langevin method, the environment is set to 303.15-307.5K, NPT ensemble of one atmosphere, and the cutoff value of non-bond interaction is set to 10 angstroms.

In the preferred embodiment of the present invention, the track file of S8 is saved as a NetCDF format file.

In the preferred embodiment of the present invention, S9 uses Cpptraj module in Amber16 to parse the track file, and uses distance instruction to obtain the distance data between the calcium ion and the center of the channel entrance.

Compared with the prior art, the invention has the following beneficial effects:

(1) the method for artificially obtaining the TRPV5 channel protein variant through computer simulation has the advantages of high efficiency and simple operation, and overcomes the limitations of complex traditional experimental process and long experimental time.

(2) The invention utilizes CHARMM-GUI to carry out different variations on TRPV5 channel protein, thereby being convenient for researchers to fully research various variation possibilities in a short time and having high operation flexibility.

(3) The invention uses pmemd. cuda module in Amber16, carries out molecular dynamics simulation by means of a Graphics Processing Unit (GPU), and has higher working efficiency compared with the conventional molecular dynamics simulation by using a Central Processing Unit (CPU), thereby solving the problem of overlong simulation time of the conventional molecular dynamics calculation simulation in millisecond time scale and being beneficial to wide application in biochemistry and protein engineering.

(4) The method can adjust the time length of energy minimization optimization, pre-equilibrium simulation and kinetic simulation in the process according to the size of the simulation system, so the method can be simultaneously suitable for a large system with a large number of molecules and a small system with a small number of molecules, and has wide applicability and wide application space.

(5) The invention combines VMD molecular visualization software and a Cpptraj data extraction module in Amber16 to analyze the motion trail of the whole system on the atomic level, and compared with the observation means of the traditional experiment, the invention overcomes the difficulty that the intermediate state is difficult to continuously observe, and can more intuitively observe the whole process of ion motion of the cations in a very short time scale.

Drawings

FIG. 1 is a schematic flow chart of the present invention.

Fig. 2 is a schematic design diagram of TRPV5 protein structure.

Fig. 3 is a schematic diagram of a TRPV5 channel protein variant.

FIG. 4 is a schematic diagram of calcium ion entry into the ion channel inlet after a period of computational simulation of a phosphorylated threonine mutant (D542T-Phos).

FIG. 5 is a schematic diagram showing that calcium ions do not enter the ion channel entrance and flow into the peripheral cavity after a period of computational simulation of the threonine mutant (D542T).

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way.

Example 1

The invention provides a method for obtaining a TRPV5 channel protein variant based on computer simulation, which comprises the following steps:

s8, after the initial optimal variant is obtained, performing kinetic simulation again to further test the calcium ion intake capacity, obtaining a motion track file containing a plurality of crystal structure output files and each atom of the system after the kinetic simulation is finished, and further verifying whether the calcium ions can be taken by the channel protein mutant at a farther distance;

The tool software used by the invention is molecular dynamics simulation software, the type of the molecular dynamics simulation software is not limited in the invention, as long as the molecular dynamics simulation can be realized, and Amber16 calculation software is adopted in the implementation process of the invention.

The process of the invention is as shown in figure 1, firstly, using CHARMM-GUI to perform residue variation or phosphorylation modification on TRPV5 protein file, inserting calcium ions by modifying protein variant PDB file after determining entry coordinates by using VMD visualization software, and finally using CHARMM-GUI to construct cell membrane structure model for protein variant; then, performing a series of molecular dynamics simulations, including energy minimization, equilibrium state simulation and dynamics simulation; and finally, analyzing each final structure to obtain the feasibility of the protein engineering scheme, and providing a new idea for the artificial regulation scheme of TRPV 5.

In the present invention, the crystal structure Data of the TRPV5 channel Protein is derived from the PDB database (collectively called Protein Data Bank), and the crystal structure of the desired Protein channel is downloaded from the database.

The present invention provides residue variation and phosphorylation of TRPV5 entry residues in CHARMM-GUI (http:// www.charmm-GUI. org /). According to previous researches, the TRPV5 channel protein is a tetrameric protein, and the entrance of the TRPV5 channel protein is composed of four aspartic acids (Asp542 or D542 for short), so that the TRPV5 channel protein can be subjected to protein engineering at the position to endow the TRPV5 channel protein with artificial regulation function which is consistent with the experimental expectation.

The TRPV5 temporarily loses the capacity of taking calcium ions from the environment by means of residue variation, and the TRPV5 plays a role in closing the entrance of a channel. Therefore, as shown in fig. 2 and 3, aspartic acid 542 of TRPV5 channel protein is changed to isoserine (Ser, S), threonine (Thr, T) and tyrosine (Tyr, Y), respectively, and it is possible to satisfy the artificial regulation purpose because only the above three amino acids can be subjected to the next phosphorylation. After mutation of aspartate at the entrance (Asp542), three TRPV5 mutant structures, namely, serine mutant (D542S), threonine mutant (D542T) and tyrosine mutant (D542Y), were obtained.

In order to realize the manually-regulated channel opening, as shown in fig. 2 and fig. 3, the protein variant channel inlet residues are respectively phosphorylated on the basis of step 5, the action of attracting calcium ions into the channel protein TRPV5 is newly given, and the mutated TRPV5 protein is phosphorylated through CHARMM-GUI, so that a phosphorylated serine mutant (D542S-Phos), a phosphorylated threonine mutant (D542T-Phos) and a phosphorylated tyrosine mutant (D542Y-Phos) are obtained.

In order to investigate whether calcium ions can enter an ion channel, the coordinates of an ion channel inlet are determined by virtue of molecular visualization software such as VMD (virtual machine format), and the calcium ions are placed at a position which is about 3 angstroms from the inlet in a vertical manner by manually modifying a protein crystal structure (PDB) file. The obtained crystal structures of the six protein variants are respectively inserted into calcium ions to be ingested, so that preparation is provided for the subsequent construction of a cell membrane system.

The invention constructs an intact cell membrane system for the obtained crystal structure of the protein variant containing calcium ions in CHARMM-GUI. The system mainly comprises protein, calcium ions, phospholipid bilayers, chloride ions, sodium ions and a solvent. The solvent is water. The invention adopts the most common palmitoyl oleoyl phosphatidylcholine (POPC, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine) to build up a phospholipid bilayer, and the X-axis and Y-axis dimensions of the phospholipid bilayer are both 150 angstroms. The invention selects sodium ion and chloride ion (Na)⁺/Cl^-) The system is adjusted to maintain the system electrically neutral. As a plurality of unnatural contacts are inevitably generated in the process of constructing the simulation system, the potential energy of the system is reduced by a dynamic simulation means.

Hair brushObviously, in order to reduce the abnormal conditions of overhigh potential energy of the system, elimination of atom position overlapping and the like, before the start of the dynamic simulation, the system is subjected to restrictive energy minimization optimization. The optimization is carried out aiming at phospholipid molecules, protein hydrogen atoms and solvent molecules, the positions and the structures of the atoms are optimized, unreasonable phenomena of atom overlapping, structure distortion and the like in a system are eliminated, and the potential energy of the system is further reduced. The CHARMM36m force field was used for the system in the present invention. The energy minimization optimization is carried out by adopting a Pmemd module in an Amber16 dynamic simulation tool, and a steepest descent method and a conjugate gradient method are sequentially adopted to carry out energy minimization optimization on a system. Firstly, a 2500-step steepest descent method is adopted to optimize the system, so that serious and unreasonable atom contact in the system is eliminated, and then a 2500-step conjugate gradient method is adopted to further optimize the system, so that the potential energy of the system is further reduced. Optimization process adopts

The weight of (2) fixes the protein heavy atoms, using

Calcium ion as a weight ofThe weight of (a) fixes the phospholipid molecules.

After the structural energy is minimized and optimized, the structure is subjected to pre-balance simulation, so that the potential energy of the system is further reduced under the preset experimental environment temperature and pressure. The invention uses a pmemd. cuda module of Amber16 to carry out pre-equilibrium simulation, and the system carries out six-step pre-equilibrium simulation in the step, and gradually releases the position limitation on protein heavy atoms and phospholipid molecules in the pre-equilibrium simulation process, wherein the position limitation weight of the protein heavy atoms is sequentially

In addition toThe position limiting weight of the phospholipid molecule is sequentially

Calcium ion global position restriction weight set to

The temperature control is carried out on the whole simulation process by adopting a Langevin temperature coupling method, wherein NVT pre-balance simulation with the temperature of 303.15K is adopted in the first step and the second step, NPT pre-balance simulation is adopted in the last four steps, so that the system adapts to the periodic boundary condition of 1 atmosphere, and a Monte Carlo pressure control method is adopted for pressure control. The SHAKE algorithm was used to limit all H-linked bonds to length, the cutoff for non-bond interactions was set to 12 angstroms, and the Particle-Mesh-EWald (PME) method was used to handle long-range electrostatic interactions. This step totals about 225000 steps for a total of 375ps of pre-balancing. This step ultimately allows for a lower energy architecture in preparation for subsequent kinetic simulations.

The invention carries out 1ns dynamic simulation on a well balanced system, thereby obtaining the ion motion situation and the structure change which are as close to the real environment as possible. In order to fully explore the movement path of calcium ions in the process of kinetic simulation, the kinetic simulation is divided into 10 parts to be sequentially carried out, each part is subjected to the kinetic simulation of 0.1ns, and a result file is output to be observed by using a VMD at a later stage. The invention uses the pmemd. cuda module of Amber16 to perform dynamic simulation on the system, the pressure control adopts Monte Carlo pressure control method, the temperature control adopts Langevin method, the environment is set to be 303.15K, the NPT ensemble of one atmosphere, and the cutoff value of non-bond interaction is set to be 10 angstroms.

After the simulation calculation is finished, the ambdbb module of Amber16 is used for converting the result file finally generated in each step of the simulation calculation into a PDB file for subsequent observation.

The invention adopts VMD software to analyze the tested calcium ion position and channel entrance structure change, measures the channel entrance width and the distance between the calcium ion and the channel entrance and the key residue in the channel by means of the software ranging tool, and analyzes whether the structure meets the requirement. Experiments show that the structures of the protein variants have certain defects, wherein the serine mutant (D542S) cannot effectively block the entrance of ions due to the small steric hindrance of serine, and cannot meet the function of closing the entrance of a channel. And the phosphorylated tyrosine mutant (D542Y-Phos) completely blocks the channel inlet due to the excessive steric hindrance, so that calcium ions stop near the channel inlet and are difficult to enter.

The threonine mutant (D542T) and the phosphotyrosine mutant (D542Y-Phos) finally obtained by the invention meet the requirements of artificial regulation and control of channel switches, and are shown in figure 4 and figure 5. Then, calcium ions are gradually placed at positions (such as 6 angstroms, 9 angstroms, 12 angstroms, 20 angstroms and the like) farther away from the entrance of the threonine mutant (D542T) and the phosphotyrosine mutant (D542Y-Phos) channel, and the three steps of energy minimization optimization, pre-equilibrium simulation and kinetic simulation are repeated on the basis of the calcium ions and the same parameters. In the dynamic simulation process, the invention sets that the crystal structure coordinate information is recorded once every 5000 steps, and the information is recorded in a track file in a NetCDF format.

After the track file in the NetCDF format is obtained, the track file is analyzed by using an analysis module in molecular dynamics simulation software, and the ion motion state is directly observed by using VMD. The track file is analyzed by adopting a Cpptraj module in the Amber16, distance instructions in the Cpptraj module are used for acquiring distance change data of calcium ions and the center of the channel inlet, and the ion movement trend and the intake speed can be analyzed through specific numerical change. In addition, the result file of each intermediate step is converted into a PDB format by using an ambdbb module and is observed by using VMD software, so that the protein structure change and ion movement conditions of each step in the kinetic simulation process are analyzed more intuitively, and whether calcium ions can be normally ingested by threonine mutant (D542T) and phosphorylated tyrosine mutant (D542Y-Phos) is verified. Through verification experiments, the threonine mutant (D542T) and the phosphorylated tyrosine mutant (D542Y-Phos) thereof can meet the experimental expectation, and the calcium ion uptake capacity of the TRPV5 channel protein variant is controlled through phosphorylation and dephosphorylation, so that the TRPV5 channel protein variant is optimal.

The applicant declares that the above detailed description is a preferred embodiment described for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, i.e. it does not mean that the present invention must be implemented by means of the above embodiment. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

1. A method for obtaining a regulatable TRPV5 variant based on computer simulation, comprising the steps of:

2. The method of claim 1, wherein the critical position at S1 is aspartic acid at position 542 (Asp 542).

3. The method of claim 2, wherein the phosphorylation modification at S2 is: residue number 542 of said variant A1 was phosphorylated by CHARMM-GUI software.

4. The method of claim 1, wherein the cell membrane structure model of S2 is electrically neutral; the solute ions include chloride ions and sodium ions.

5. The method of claim 1, wherein the step S3 comprises determining coordinate information of mutant channel entrance by VMD software, and setting the position of calcium ion by modifying the PDB format of the mutant crystal structure file.

6. The method of claim 5, wherein the energy minimization optimization of S4 comprises optimizing the location and structure of phospholipid molecules, hydrogen atoms of proteins, and solution molecules; and (3) running a CHARMM36m force field in a Pmemd module in an Amber16 dynamics simulation tool, and successively optimizing the cell membrane structure model by adopting a steepest descent method and a conjugate gradient method for 5000-20000 steps together to obtain an energy optimized system.

7. The method of claim 5, wherein the energy minimization optimization process of S4 employs

The weight of (a) limits the protein heavy atoms; by using

The weight of (b) limits calcium ions; the phospholipid molecules have a position-limiting weight of

8. The method of claim 5, wherein the pre-balancing simulation of S5 is performed 375-450 ps using a pmemd. cuda module of Amber 16; the pre-equilibrium simulation is carried out in six steps, and the position limitation of protein heavy atoms and phospholipid molecules is gradually released at the ambient temperature of 303.15-330.5K.

9. The method of claim 5, wherein the pre-equilibrium simulated protein heavy atom position limit weight of S5 isThe phospholipid molecules have a position-limiting weight of

The calcium ion has a position-limiting weight of

10. The method as claimed in claim 5, wherein the molecular dynamics simulation pressure control of S6 is Monte Carlo pressure control method, the temperature control is Langevin method, the environment is set to 303.15-307.5K, one atmosphere NPT ensemble, and the cutoff value of non-bond interaction is set to 10 angstrom;

s8, storing the motion trail file as a NetCDF format file;

s9 adopts a Cpptraj module in Amber16 to analyze the motion track file in S8, and uses a distance instruction to acquire distance data between calcium ions and the center of the channel entrance.