CN112034184B - Auxiliary screening method of protein interaction blocking polypeptide - Google Patents

Abstract

The invention provides an auxiliary screening method of protein interaction blocking polypeptide, which is a screening method taking Chinese prawn protein PcRab7 and white spot syndrome virus envelope protein VP28 as targets for blocking polypeptide by interaction. The method of the invention is to construct a complex structure PcRab7-VP28 of a prawn Rab7 protein and a white spot syndrome virus WSSV envelope protein VP 28; determining the interaction interface of a complex structure PcRab7-VP 28; then, WSSV envelope protein VP28 is used as a receptor target protein, and an active pocket is set according to the active region of the receptor target protein; sequentially butting the polypeptide fragments to be screened with receptor target protein VP28 at the set active pocket position, and calculating the binding free energy of the polypeptide and VP 28; screening the blocking polypeptide according to the free energy. The screening method of protein interaction blocking polypeptide established by the invention is based on the blocking polypeptide screening of interaction, can make up for the traditional high-cost short plate which only depends on experimental screening, and can screen the polypeptide in multiple ranges and lengths.

Description

Auxiliary screening method of protein interaction blocking polypeptide

Technical Field

The invention belongs to the technical field of protein-polypeptide interaction, and particularly relates to an auxiliary screening method of protein interaction blocking polypeptide.

Background

The White Spot Syndrome of the prawn is a comprehensive disease caused by White Spot Syndrome Virus (WSSV), is one of main diseases of the fulminant epidemic disease of the prawn, has extremely high mortality, seriously harms the prawn breeding industry, and is an important aquatic animal infectious disease required to be reported by international veterinary institute (OIE) regulations.

The genome size of WSSV is around 300kb, and it is predicted to encode about 180 proteins, of which more than 50 virus-encoded proteins are identified as structural proteins. Among these structural proteins, the envelope protein is an important determinant of viral infection and virulence, and is considered to be the first molecule in direct contact with the host, and thus plays a key role in cell targeting and host defense. Although the precise mechanism of entry of WSSV viruses into prawn bodies is not known to date, relevant studies speculate that viral entry into host cells occurs through the interaction of viral envelope proteins with host cell receptors. For example, the envelope protein enters the prawn body through the interaction with beta-integrin protein, PmRab7 and the like to cause infection. Since the virus enters the host cell through the interaction between the virus envelope protein and the host cell receptor, the screening of the interaction blocker has important significance for the development of anti-white spot syndrome virus medicines in the future.

The screening of drugs is always one of the main approaches for the development of new drugs. The blocking agent can be obtained accurately through in vivo and in vitro experimental screening. The polypeptide fragment 2E6(VAVNNSY) obtained by a phage surface display method by using VP28 as a target can effectively inhibit virus infection. Although the phage surface display method can obtain active polypeptide fragments by a panning method, the method has the problems of single fragment length and high experimental cost in constructing a polypeptide library, so that the method has no way of exhausting all fragment lengths. So that the experimental method has certain limitation on the screening scale and range.

Disclosure of Invention

The invention provides an auxiliary screening method of protein interaction blocking polypeptide, in particular to a screening method taking Chinese prawn protein PcRab7 and white spot syndrome virus envelope protein VP28 as an interaction blocking polypeptide as a target, thereby making up the defects of the prior art.

The invention provides a screening method of Chinese prawn protein PcRab7 and white spot syndrome virus envelope protein VP28 interaction blocking polypeptide, which comprises the following steps:

1) constructing a complex structure PcRab7-VP28 of a Chinese prawn Rab7 protein and white spot syndrome virus WSSV envelope protein VP 28;

2) determining the interaction interface of a complex structure PcRab7-VP 28;

3) WSSV envelope protein VP28 is used as a receptor target protein, and an active pocket is set according to the active region of the receptor target protein;

4) and sequentially butting the polypeptide fragments to be screened with receptor target protein VP28 at the set active pocket position, calculating the binding free energy of the polypeptide and VP28, and screening the blocking polypeptide according to the free energy.

The construction method of the PcRab7-VP28 complex structure in the step 1) is as follows:

A. the construction of a PcRab7 tertiary structure is completed through protein structure prediction software I-TASSER;

B. generating a plurality of PcRab7-VP28 complex structures through a ZDock2 program, and sorting according to binding energy;

C. according to the docking result, the ligand conformation with the highest Auto Dock score is selected, and the complex structure of PcRab7-VP28 is determined.

The step 2) of determining the interaction interface of the complex structure PcRab7-VP28 comprises the following steps:

A. obtaining a molecular dynamics track through molecular dynamics simulation of a complex structure;

B. calculating the binding free energy between PcRab7-VP28 by using an MM-PBSA energy calculation method of a single molecular kinetic trajectory, and analyzing main factors driving reversible binding of PcRab7-VP 28;

C. the contribution of each residue of PcRab7 to binding self-energy was calculated using the MM-GBSA energy decomposition method, and the residue that is responsible for PcRab7-VP28 binding was determined.

The VP28 active pocket in the step 3) is set by selecting residues at an interaction interface of VP28 and PcRab7 as an interaction site and generating an interaction region through software Discovery studio 4.0;

sequentially docking the polypeptide fragment to be screened with receptor target protein VP28 in the step 4), and jointly completing the docking by using REDUCE, Autodock Tools and Autodock4.2 software; the Autodock docking parameters are set as follows: the lattice point spacing is a cube region formed by lattice points with a box of 50 multiplied by 50, hydrogen bonds and Gaussian charges are added to the VP28 and the polypeptide fragment, then a completely flexible butt joint method of the polypeptide and a VP28 active site region is adopted, and the rest parameter settings are default values; and extracting interaction energy in the docking result, and selecting an average value of the conformation binding free energy in the cluster as a final scoring result.

Furthermore, the invention also provides a binding mode of the screened polypeptide and VP28, and molecular dynamics simulation is carried out on a target protein-polypeptide fragment system by using molecular dynamics software Amber 16;

the molecular dynamics simulation of the target protein-polypeptide fragment (VP28-IP) system is described as follows: the complex protein VP28-IP was immobilized to balance water and lipids for 2 ns; then, selecting all flexible and active site region residues of the polypeptide fragment as flexible residues, fixing C alpha atoms of other residues except the flexible residues, and gradually reducing the force constant from 10 kcal/mol to 0.1 kcal/mol; in all four-step balancing, each step runs for 10 ns; after equilibration, an MD simulation of 400ns was performed at constant temperature (310K) and pressure (1 atm).

In order to avoid the influence of randomness, two independent kinetic simulations are respectively carried out at different initial atomic speeds; the resulting traces were analyzed using the cbptraj module of the amber16 software package.

The screening method of the protein interaction blocking polypeptide established by the invention has the following advantages:

1) the screening is efficient, the blocking polypeptide screening based on the interaction can make up for the traditional high-cost short plate which only depends on experimental screening, and the screening of the polypeptides in multiple ranges and lengths can be realized.

2) The method has wide applicability, can successfully screen the PcRab7-VP28 interaction blocking agent, and is also applicable to screening other system interaction blocking polypeptides.

Drawings

FIG. 1: the PcRab7 model structure and its rationality test chart, wherein (A) cartoon picture display of PcRab7 tertiary structure, the picture is generated by molecular graphics software pymol, the picture is displayed in rainbow color (N-end is blue, C-end is red). (B) The result of the Ramachandran Plot of Pcrab7, generated by DS4.2, where the red triangle indicates residues in the unacceptable range.

FIG. 2: the structure diagram of a complex formed by molecular docking of PcRab7 and VP28, wherein PcRab7 is displayed in cartoon form, and VP28 is displayed on a hydrophilic and hydrophobic surface.

FIG. 3: an interaction interface diagram of VP28 and PcRab7 is displayed in a hydrophobic surface mode;

FIG. 4: an RMSD track model diagram of VP28-PP1, the part in a box is a finally selected clustering structure;

FIG. 5: interaction diagram of VP28 and PP1, wherein the interaction diagram is generated by calculation of Discovery studuo4.0visualizer (DS 4.0).

Detailed Description

The envelope protein VP28 has obvious advantages as prawn subunit vaccine and is an important target for antiviral drug screening. The location of an immunoelectron microscope proves that VP28 is distributed on the outer surface of the virus and can be specifically combined with Rab7(PmRab7) protein in the penaeus monodon and PcRab7 in the penaeus monodon and VP 28. Therefore, the inhibition of any protein of VP28 and Rab7 can effectively reduce the risk of WSSV infection of prawns. The invention selects the interaction of Chinese prawn protein PcRab7 and WSSV envelope protein VP28 as the target to screen the interaction blocker.

The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.

Example 1: establishment of screening method

(1) Firstly, carrying out structure modeling on PcRab7, and carrying out butt joint simulation on the modeled structure and VP28 to obtain a PcRab7-VP28 complex structure;

(2) performing 300ns water-developing molecular dynamics simulation on the complex structure through an amber software package, and performing interaction analysis on the balanced structure to predict and determine the key residue region of the PcRab7-VP28 interaction as D76-M83;

(3) the method is characterized in that fragments which are contacted with each other on an interaction interface of PcRab7 and VP28 are intercepted independently, and VP28 is used as a screening target protein, so that the fragment (D76-M83, DFLTKEVM) which is contacted with VP28 in PcRab7 is selected as a 'mother fragment', a polypeptide fragment library is constructed, and the method mainly comprises the following steps:

firstly, truncating the parent fragment (D76-M83) from two ends, and selecting fragments with various lengths from pentapeptide to octapeptide to form a polypeptide library 1(PPD 1);

table 1: polypeptide fragment library 1(PPD1)

Pentapeptide	TKEVM	DFLTK	FLTKE	LTKEV
					Hexapeptides	LTKEVM	DFLTKE	FLTKEV
Heptapeptides	FLTKEVM	DFLTKEV
					Octapeptides	DFLTKEVM

② all residues in PPD1 are mutated into other same groups (classified into nonpolar group of residues: Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, polar group: Ser, Thr, Tyr, Asn, Gln, Cys, Gly, positively charged group: Lys, Arg, His and negatively charged group: Asp, Glu) residues, and randomly combined to form polypeptide library 2(PPD2)

3. Virtual screening of polypeptide fragments

Molecular butt joint: and (3) taking VP28 as a receptor and the polypeptide fragments as ligands, and carrying out molecular docking on all polypeptide fragments in the polypeptide fragment library and VP28 by utilizing Autodock 4.2. Taking (35.395, 93.984, 76.586) as a center, and taking a cube space formed by grid points of 60 multiplied by 60 as a box, carrying out grid point division, and determining a binding region;

analyzing the docking result: all the post-docking conformations of each polypeptide fragment are derived, and the conformation with the lowest binding energy is selected as the representative conformation of the polypeptide fragment to form a polypeptide-VP 28 complex (PP-VP28) complex with VP 28. Selecting the first 100 polypeptide fragments with smaller PP-VP28 binding energy Ei and binding strength Ki;

and thirdly, scoring the complex conformation after docking: complexes of 100 selected polypeptide fragments with VP28 were scored using score2.01, pKd values were calculated, and the top 10 fragments of pKd (table 2) were selected for further analysis.

Table 2: ten possible polypeptide fragments

These polypeptide fragments may serve as candidate polypeptide blockers of the PcRab7-VP28 interaction.

(4) For the selected possible fragments, molecular dynamics software Amber16 is used to carry out 400ns molecular dynamics simulation on two systems of VP28-PP1 and VP28-PP 2. Wherein, VP28 adopts AMBER99SB force field, and the polypeptide fragment adopts leaprc.ff03.r1 force field. Generating a topological file of the system by using a tleap program, wherein the center of mass of the system is positioned in the center of a cubic box, and adding a protein around the system

Cubic water box of (1), solvent waterThe molecular model used the TIP3P model. Meanwhile, 11 Na + were added to maintain the electrical neutrality of the system. In order to eliminate the collision between atoms, 5000-step energy optimization is carried out by adopting a steepest descent method, and then 5000-step energy optimization is carried out by adopting a simulated annealing method. After equilibration, an MD simulation of 400ns was performed at constant temperature (310K) and pressure (1 atm). The long-range electrostatic interaction is treated by applying a circumferential boundary condition PME (particle Mesh Ewald) method. Wherein the cutoff value for van der Waals and electrostatic interactions is 10. The simulation time step is 2fs, and track energy and structure topology files are saved once every 2ps for subsequent processing. To avoid the influence of randomness, two independent kinetic simulations were performed at different initial atomic velocities. The resulting traces were analyzed using the cbptraj module of the amber16 software package.

(5) The Root Mean Square Deviation (RMSD) of VP28-PP1 was calculated, respectively, as shown in FIG. 4, and the last 5ns of the traces were cluster analyzed with the mean value of RMSD as the cutoff. The central conformation of the largest cluster was selected and analyzed for binding pattern using DS4.0 and Pymol, fig. 5 shows: PP1 can form hydrogen bonds with Ser152 and Gly178 of VP28, and form hydrophobic interactions with Phe149, Val150, Pro180 and The184 to be fixed in The active pocket region of VP28, so as to inhibit VP28 activity.

The above examples, which describe in detail the methods of screening for protein interaction blocking polypeptides, are illustrative and not restrictive, and several examples are listed within the limits of the invention, and thus variations and modifications within the scope of the invention are intended to be covered by the present invention.

Claims (5)

1. A method for screening a blocking polypeptide of interaction between a Chinese prawn protein PcRab7 and a white spot syndrome virus envelope protein VP28 is characterized by comprising the following steps:

1) constructing a complex structure PcRab7-VP28 of a Chinese prawn Rab7 protein and white spot syndrome virus WSSV envelope protein VP 28; the construction method comprises the following steps:

firstly, constructing a PcRab7 tertiary structure through protein structure prediction software I-TASSER;

generating a plurality of PcRab7-VP28 complex structures through a ZDock2 program, and sequencing according to the binding energy;

thirdly, according to the docking result, selecting the ligand conformation with the highest Auto Dock score to determine the ligand conformation as a complex structure of PcRab7-VP 28;

2) determining the interaction interface of a complex structure PcRab7-VP 28; the method comprises the following steps:

firstly, obtaining a molecular dynamics track through molecular dynamics simulation of a complex structure;

calculating the free binding energy between PcRab7-VP28 by using an MM-PBSA energy calculation method of a single molecular dynamics track, and analyzing main factors driving reversible binding of PcRab7-VP 28;

thirdly, calculating the contribution value of each residue of PcRab7 to the binding energy by using an MM-GBSA energy decomposition method, and determining the residue which plays a role in determining the binding of PcRab7-VP 28;

3) WSSV envelope protein VP28 is used as a receptor target protein, and an active pocket is set according to the active region of the receptor target protein; the VP28 active pocket is set by selecting a fragment contacted with the interaction interface of VP28 and PcRab7 as a mother fragment and generating an interaction region through software Discovery studio4.0, and the method comprises the following steps:

firstly, truncating the parent segment from two ends, and selecting each length segment between pentapeptide and octapeptide to form a polypeptide library 1;

secondly, all residues in the polypeptide library 1 are mutated into other residues in the same group, wherein the residues comprise a nonpolar residue group: ala, Val, Leu, Ile, Pro, Phe, Trp, Met; polar residue group: ser, Thr, Tyr, Asn, Gln, Cys, Gly; positively charged group: lys, Arg, His and negatively charged groups: asp and Glu are randomly combined to form a polypeptide library 2;

4) the polypeptide fragment to be screened is sequentially butted with a receptor target protein VP28 at the position of the set active pocket, and the binding free energy of the polypeptide and VP28 is calculated.

2. The method of claim 1, wherein the step 4) of docking the polypeptide fragment to be screened with the receptor target protein VP28 sequentially is performed by using REDUCE, Autodock Tools and autodoc4.2 software; the Autodock docking parameters are set as follows: the lattice point spacing is a cube region formed by lattice points with a box of 50 multiplied by 50, hydrogen bonds and Gaussian charges are added to the VP28 and the polypeptide fragment, then a completely flexible butt joint method of the polypeptide and a VP28 active site region is adopted, and the rest parameter settings are default values; and extracting interaction energy in the docking result, and selecting an average value of the conformation binding free energy in the cluster as a final scoring result.

3. A method for determining the binding pattern of the blocking polypeptide to VP28, which is obtained by screening according to the method of claim 1, wherein the method comprises performing molecular dynamics simulation on the target protein-polypeptide fragment VP28-IP system by using molecular dynamics software Amber 16.

4. The method of claim 3, wherein the molecular dynamics of the target protein-polypeptide fragment VP28-IP system is simulated by immobilizing complex protein VP28-IP to balance water and lipid for 2 ns; then selecting all flexible and active site region residues of the polypeptide fragment as flexible residues, fixing C alpha atoms of other residues except the flexible residues, and gradually reducing the force constant from 10 kcal/mol to 0.1 kcal/mol; in all balances, each step was run for 10 ns; after equilibration, a 400ns MD simulation was performed at constant temperature and pressure.

5. The method of claim 4, wherein the molecular kinetic simulation is two separate kinetic simulations with different initial atomic velocities; the resulting traces were analyzed using the cbptraj module of the amber16 software package.

Images