CN106496301B - A method for screening food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch region - Google Patents

Abstract

The invention discloses a method for screening food-borne antioxidant oligopeptides by multi-site molecular docking in a Keap1Kelch area, which comprises the following steps: step one, constructing a food-borne oligopeptide molecular structure library; step two, searching for a PDB file which interacts with each other; step three, designing 3 binding sites; step four, carrying out molecular docking on 3 binding sites designed in step three; and step five, comprehensive evaluation, and preliminary screening of food-borne antioxidant oligopeptides. Has the advantages that: by designing multiple binding sites, the impact on alignment assays due to oligopeptides smaller than Nrf2 fragments comprising the ETGE sequence is reduced. In the subsequent test, the food-derived oligopeptides obtained by screening can be used as a blueprint, and the food-derived oligopeptides with the antioxidant function are successively screened out by utilizing an in vitro chemical test and a cell test, so that the test efficiency is effectively improved, and the test period is shortened.

Description

Screening of food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch region Methods

technical field

The invention relates to a method for screening food-derived antioxidant oligopeptides, in particular to a method for screening food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch region.

Background technique

At present, modern medicine generally believes that the oxidative stress caused by the increase of free radicals will cause tissue cell damage, which will lead to the aging of the body, and even induce malignant tumors in severe cases. Therefore, improving the body's antioxidant capacity has become an important means to maintain good health. Compared with other methods to improve the body's antioxidant capacity, eating foods containing natural antioxidants in the daily diet is a low-cost, effective and cost-effective method. Among natural antioxidants, food-derived antioxidant peptides play an important role.

Food-derived peptides are the degradation products of food proteins, and can be divided into food-derived oligopeptides (containing 2-10 amino acid residues) and food-derived polypeptides (containing more than 10 amino acid residues) according to their size. In general, food-derived oligopeptides have higher antioxidant activity than food-derived peptides. As one of the important biological activities of food-derived oligopeptides, antioxidant activity has been studied for a long time.

Usually our method for studying food-derived antioxidant oligopeptides consists of the following five steps: (1) enzymatic hydrolysis of food-derived proteins; (2) separation of enzymatically hydrolyzed crude peptides according to the molecular weight of the peptides; (3) using in vitro chemical tests, Cell tests and animal experiments to verify its antioxidant activity; (4) Mass spectrometry analysis of crude peptides with antioxidant activity was performed to obtain the molecular structure of the oligopeptides; (5) In vitro chemical tests, cell tests and animal experiments were used to verify these Antioxidative properties of oligopeptides. This method is inefficient, expensive and time-consuming. At the same time, this method is not suitable for screening highly active antioxidant oligopeptides from a large number of oligopeptides. Therefore, it is imperative to develop an efficient, economical and rapid screening method for food-derived antioxidant oligopeptides.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for screening food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch region in order to develop an efficient, economical and rapid method for screening food-derived antioxidant oligopeptides by molecular docking .

The method for screening food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch region provided by the present invention is as follows:

Step 1. Obtain the protein primary structure of the food-derived protein from the Uniprot database, and based on this, construct a food-derived oligopeptide molecular structure library;

Step 2. Find the PDB file from the PDB database that can reflect the interaction between the Kelch region of the Keap1 protein and the Nrf2 fragment containing the ETGE sequence;

Step 3. Design three binding sites with reference to the three standards of the Nrf2-binding Keap1 binding site of the PDB file, all amino acid residues on Keap1 that are too close to the Nrf2 fragment, and the ETGE sequence of the key position of the Nrf2 fragment. ;

Step 4. Use all oligopeptides in the oligopeptide molecular structure library as ligands, and use the Keap1 protein Kelch region in the PDB file as the receptor, and perform molecular docking on the three binding sites designed in step 3;

Step 5. Evaluate the docking results with the binding ability as an index, compare and analyze the interaction between the oligopeptide ligand with the highest binding ability and the active center and the interaction between the original Keap1 protein Kelch region in the PDB file and the Nrf2 fragment containing the ETGE sequence. Evaluation, preliminary screening of food-derived antioxidant oligopeptides.

Step 3: By designing 3 binding sites, the impact on the docking experiment caused by the oligopeptide being smaller than the Nrf2 fragment containing the ETGE sequence in the PDB file is reduced.

The mechanism of the present invention is as follows:

The Keap1-Nrf2 signaling pathway is one of the important cellular defense mechanisms of the body against oxidative stress. Under normal physiological conditions, Nrf2 is continuously produced in the body, and the Keap1 protein forms a homodimer through its BTB region and binds to the actin of the cell, and binds separately through the two Kelch regions of the dimer. The DLG and ETGE sequences in the Neh2 region of the Nrf2 protein (wherein the binding ability between the Kelch region and the ETGE sequence is 100 times the binding ability between the Kelch region and the DLG sequence), and then the Nrf2 protein is ubiquitinated and degraded. When the body is under oxidative stress, the external oxidants will change the conformation of cysteine residues in the BTB and IVR domains of Keap1 protein, thereby changing the conformation of the Keap1 dimer. This change may interfere with Keap1 and low-affinity DLG. Sequence interacts, but does not affect interaction with ETGE sequences. In turn, Nrf2 bound to Keap1 cannot be ubiquitinated and then degraded, resulting in the accumulation of intracellular Nrf2. This part of Nrf2 enters the nucleus and activates the expression of a series of genes related to phase II detoxification enzymes (including HO-1, NQO1, SOD, and CAT) with antioxidant and cytoprotective effects, thereby improving the body's antioxidant capacity.

Therefore, if foreign antioxidant active substances can affect the Keap1-Nrf2 interaction, thereby increasing the intracellular Nrf2 content, the pathway can be activated and the body's antioxidant capacity can be improved. Antioxidative active substances that affect the Keap1-Nrf2 interaction in this way are called Keap1-Nrf2 interaction inhibitors. Keap1-Nrf2 interaction inhibitors can be divided into two categories: indirect inhibitors and direct inhibitors. The indirect inhibitor is to change the conformation of key cysteine residues in the BTB and IVR domains of Keap1 protein, thereby inhibiting the Keap1-Nrf2 interaction and activating the pathway. The direct inhibitor directly binds to the Kelch region of the Keap1 protein and occupies the position where Keap1 binds to Nrf2, thereby inhibiting the Keap1-Nrf2 interaction and activating the pathway. Compared with indirect inhibitors, direct inhibitors are more specific and do not have the effect of indirect inhibitors due to their ability to change the conformation of cysteine residues. The normal physiology of other proteins containing key cysteine residues in vivo functional side effects. Therefore, direct inhibitors are more suitable for research than indirect inhibitors.

In the Keap1-Nrf2 interaction, the binding of the Keap1 protein Kelch region to the Nrf2 protein ETGE sequence is particularly critical. If foreign antioxidants can occupy the binding site between the Keap1 protein Kelch region and the Nrf2 protein ETGE sequence, it is very likely It will directly inhibit the Keap1-Nrf2 interaction, thereby activating the Keap1-Nrf2 signaling pathway and improving the body's antioxidant capacity.

Based on the above mechanism, in the present invention, the protein primary structure of the food-derived protein is firstly found from the Uniprot database, and based on this, a food-derived oligopeptide molecular structure library is constructed; secondly, the PDB database can reflect the Keap1 protein Kelch The PDB file that interacts with the Nrf2 fragment containing the ETGE sequence is the receptor file, and the binding site of the Nrf2 fragment containing the ETGE sequence in the receptor file and Keap1, and all amino acid residues on Keap1 that are too close to the Nrf2 fragment are respectively used in the receptor file. Base and Nrf2 fragment key position ETGE sequence as a reference, three new binding sites were designed; then, using molecular docking software, the receptor and oligopeptide molecular structure library were respectively performed at the selected three sites. Molecular docking was used to preliminarily select food-derived oligopeptides with antioxidant effects; finally, in vitro chemical tests were used to screen again to verify their antioxidant properties.

Beneficial effects of the present invention:

Through molecular docking screening based on the Kelch region of the Keap1 protein, the invention can efficiently and rapidly screen out dozens of food-derived oligopeptides with strong antioxidant activity from thousands or even tens of thousands of food-derived oligopeptides. At the same time, by designing multiple binding sites, the impact on the docking assay due to the oligopeptide being smaller than the Nrf2 fragment containing the ETGE sequence was reduced. Subsequent tests can also be based on the screened food-derived oligopeptides, and use in vitro chemical tests and cell tests to screen out food-derived oligopeptides with antioxidant functions, thereby effectively improving the efficiency of the test and reducing the test cycle.

Description of drawings

Fig. 1 is a schematic diagram of the interaction between the Kelch region of Keap1 protein of the PDB file (PDB ID: 2FLU) and the Nrf2 fragment comprising the ETGE sequence in the present invention.

Figure 2 is a schematic diagram of the interaction between Asp-Lys-Lys and the Kelch region of Keap1 protein in the present invention.

FIG. 3 is a schematic diagram of the results of the fluorescence polarization test of Asp-Lys-Lys in the present invention.

Detailed ways

Example 1 A multi-site molecular docking screening of egg white-derived antioxidant tripeptides based on the Keap1 Kelch region.

(1) The steps of molecular docking screening are as follows:

1. The protein primary structure of egg white protein was obtained from the Uniprot database, and based on this, an egg white source tripeptide molecular structure library was constructed;

Using the Uniprot protein database, the egg white protein was obtained, and its protein primary structure was obtained. Based on this, an egg white source tripeptide molecular structure library was established.

2. From the PDB database, search for the PDB file (PDB ID: 2FLU) that can reflect the interaction between the Kelch region of the Keap1 protein and the Nrf2 fragment containing the ETGE sequence;

Using the PDB database, all PDB files containing human Keap1 were searched, and the PDB files (PDB ID: 2FLU) that could reflect the Keap1-Nrf2 interaction were screened and downloaded.

3. Design 3 binding sites with reference to the three standards of the Nrf2 binding Keap1 binding site of the PDB file, all amino acid residues on Keap1 that are too close to the Nrf2 fragment, and the ETGE sequence of the key position of the Nrf2 fragment;

)处结合；其次，由于该Nrf2片段由16个氨基酸残基组成，远远大于由3个氨基酸残基组成的三肽，为了减少这种差异所造成对对接试验的影响，分别以该位点、距离Nrf2片段过近接触的Keap1上所有氨基酸残基和Nrf2片段关键位置ETGE序列三个标准作为设计新结合位点构建的依据，经过调整，最终得到3个结合位点：结合位点1(中心坐标：x：-4，y：6，z：0；半径：

)、结合位点2(中心坐标： x：5，y：9，z：1；半径：

)和结合位点3(中心坐标：x：7.36，y：8.33， z：1.77；半径：

)。In this step, first analyze the binding of the Kelch region of the Keap1 protein to the Nrf2 fragment containing the ETGE sequence in the PDB file (PDB ID: 2FLU) and find out its original binding site. In this PDB file, the Kelch region of the Keap1 protein is associated with Nrf2 fragment (containing ETGE sequence, consisting of 16 amino acid residues) at site (x: 2.28, y: 6.23, z: 1.63; radius:

); secondly, since the Nrf2 fragment is composed of 16 amino acid residues, which is much larger than the tripeptide composed of 3 amino acid residues, in order to reduce the impact on the docking test caused by this difference, the site , All amino acid residues on Keap1 that are too close to the Nrf2 fragment and the ETGE sequence of the key position of the Nrf2 fragment are three criteria as the basis for designing new binding sites. After adjustment, three binding sites are finally obtained: binding site 1 ( Center coordinates: x: -4, y: 6, z: 0; radius:

), binding site 2 (center coordinates: x: 5, y: 9, z: 1; radius:

) and binding site 3 (center coordinates: x: 7.36, y: 8.33, z: 1.77; radius:

).

4. Using all tripeptides in the egg white-derived tripeptide molecular structure library as ligands, and using the Keap1 protein Kelch region in the PDB file as the receptor, molecular docking is performed on the three binding sites designed in step 3. The main steps are as follows:

(1) Add a MMFF force field to all tripeptide ligands in the molecular structure library constructed in step 3, and perform energy minimization until the ligand molecular conformation does not change.

(2) Remove water molecules and Nrf2 fragments from the 2FLU file obtained in step 1, add hydrogen, and process to obtain an acceptor file.

(3) Add CHARMm force field to the receptor file obtained in step (1).

(4) On the receptor file with the CHARMm force field attached, set the three binding sites obtained in step 2 respectively.

(5) Using the receptor obtained in step (3) as a receptor, and using the tripeptide obtained in step (1) as a ligand at three selected sites respectively, perform molecular docking screening.

5. Using the binding ability as an index to evaluate the docking results, comprehensive evaluation, and preliminary screening of egg white-derived antioxidant tripeptides.

The docking results obtained by the screening in step 4 were sorted, and the egg white-derived tripeptide with antioxidant effect was preliminarily selected. In order to further improve the accuracy of the screening results, according to the ranking of the docking results, compare and analyze the interaction between the top-ranked tripeptide ligand and the active center and the interaction between the Kelch region of the original Keap1 protein in the PDB file and the Nrf2 fragment containing the ETGE sequence (As shown in Figure 1), egg white-derived tripeptides with antioxidant effects were selected.

After the above screening, the top ten tripeptides were screened in the three sites respectively. They bind to some amino acid residues in the Kelch region of the Keap1 protein, resulting in interactions. These amino acid residues also play a role in the interaction of the Kelch region of the Keap1 protein with the Nrf2 fragment containing the ETGE sequence. This further demonstrated that its binding to the Kelch region of the Keap1 protein could regulate the pathway, thereby exerting its antioxidant effect.

(2) Using fluorescence polarization test to screen egg white-derived tripeptide with antioxidant function.

Through the above-mentioned molecular docking screening, a series of tripeptides that can bind to keap1 at three newly set sites in its Kelch region were screened from the egg white-derived tripeptide ligand library. Then the fluorescence polarization test was carried out to verify its oxidation resistance.

1. We randomly selected a compound from the egg white-derived tripeptide obtained by molecular docking screening: Asp-Lys-Lys, whose molecular structure is shown in the following formula. Figure 2 shows its interaction with the Kelch region of Keap1 protein. The compound was selected for the following fluorescence polarization test.

The well plate used for fluorescence polarization was a black 384-well plate with no binding ability, and 40 μl of the test solution was added to each well. The test solution in the test group consisted of 10ul PBS, 10ul 4mM Asp-Lys-Lys, 10ul gradient Keap1 protein Kelch region and 10ul 200nM probe. The test solution in the control group consisted of 20ul PBS, 10ul gradient Keap1 protein Kelch region and 10ul 200nM probe. The probe is a FITC-labeled Nrf2 fragment (containing the ETGE sequence, consisting of 9 amino acid residues). After the hole is added, cover the hole and shake it for 30 minutes at room temperature in the dark. The Fp value is measured. The Kd value of the Nrf2 fragment of the sequence is the most evaluation index. The results are shown in Figure 3, the Kd value of the experimental group added with Asp-Lys-Lys is much higher than that of the control group, which indicates that Asp-Lys-Lys can inhibit the interaction of keap1-Nrf2. Conclusion: It is proved that Asp-Lys-Lys has the ability to bind keap1 in in vitro chemical test, and can be further used in cell test.

2. The egg white-derived tripeptide obtained by molecular docking screening can be subjected to fluorescence polarization test according to the Asp-Lys-Lys method to determine whether it has the ability to bind keap1 in the in vitro chemical test, so as to determine its antioxidant capacity sex.

Example 2 A multi-site molecular docking screening of soybean-derived antioxidant tetrapeptides based on the Keap1 Kelch region.

The method is the same as that of Example 1, except that the molecular structure library is established; the protein primary structure of soybean protein is found from the Uniprot database, and a soybean-derived tetrapeptide molecular structure library is established based on this.

SEQUENCE LISTING

<110> Jilin University

<120> A method for screening food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch region

<130> 2016

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 624

<212> PRT

<213> human

<400> 1

Met Gln Pro Asp Pro Arg Pro Ser Gly Ala Gly Ala Cys Cys Arg Phe

1 5 10 15

Leu Pro Leu Gln Ser Gln Cys Pro Glu Gly Ala Gly Asp Ala Val Met

20 25 30

Tyr Ala Ser Thr Glu Cys Lys Ala Glu Val Thr Pro Ser Gln His Gly

35 40 45

Asn Arg Thr Phe Ser Tyr Thr Leu Glu Asp His Thr Lys Gln Ala Phe

50 55 60

Gly Ile Met Asn Glu Leu Arg Leu Ser Gln Gln Leu Cys Asp Val Thr

65 70 75 80

Leu Gln Val Lys Tyr Gln Asp Ala Pro Ala Ala Gln Phe Met Ala His

85 90 95

Lys Val Val Leu Ala Ser Ser Ser Pro Val Phe Lys Ala Met Phe Thr

100 105 110

Asn Gly Leu Arg Glu Gln Gly Met Glu Val Val Ser Ile Glu Gly Ile

115 120 125

His Pro Lys Val Met Glu Arg Leu Ile Glu Phe Ala Tyr Thr Ala Ser

130 135 140

Ile Ser Met Gly Glu Lys Cys Val Leu His Val Met Asn Gly Ala Val

145 150 155 160

Met Tyr Gln Ile Asp Ser Val Val Arg Ala Cys Ser Asp Phe Leu Val

165 170 175

Gln Gln Leu Asp Pro Ser Asn Ala Ile Gly Ile Ala Asn Phe Ala Glu

180 185 190

Gln Ile Gly Cys Val Glu Leu His Gln Arg Ala Arg Glu Tyr Ile Tyr

195 200 205

Met His Phe Gly Glu Val Ala Lys Gln Glu Glu Phe Phe Asn Leu Ser

210 215 220

His Cys Gln Leu Val Thr Leu Ile Ser Arg Asp Asp Leu Asn Val Arg

225 230 235 240

Cys Glu Ser Glu Val Phe His Ala Cys Ile Asn Trp Val Lys Tyr Asp

245 250 255

Cys Glu Gln Arg Arg Phe Tyr Val Gln Ala Leu Leu Arg Ala Val Arg

260 265 270

Cys His Ser Leu Thr Pro Asn Phe Leu Gln Met Gln Leu Gln Lys Cys

275 280 285

Glu Ile Leu Gln Ser Asp Ser Arg Cys Lys Asp Tyr Leu Val Lys Ile

290 295 300

Phe Glu Glu Leu Thr Leu His Lys Pro Thr Gln Val Met Pro Cys Arg

305 310 315 320

Ala Pro Lys Val Gly Arg Leu Ile Tyr Thr Ala Gly Gly Tyr Phe Arg

325 330 335

Gln Ser Leu Ser Tyr Leu Glu Ala Tyr Asn Pro Ser Asp Gly Thr Trp

340 345 350

Leu Arg Leu Ala Asp Leu Gln Val Pro Arg Ser Gly Leu Ala Gly Cys

355 360 365

Val Val Gly Gly Leu Leu Tyr Ala Val Gly Gly Arg Asn Asn Ser Pro

370 375 380

Asp Gly Asn Thr Asp Ser Ser Ala Leu Asp Cys Tyr Asn Pro Met Thr

385 390 395 400

Asn Gln Trp Ser Pro Cys Ala Pro Met Ser Val Pro Arg Asn Arg Ile

405 410 415

Gly Val Gly Val Ile Asp Gly His Ile Tyr Ala Val Gly Gly Ser His

420 425 430

Gly Cys Ile His His Asn Ser Val Glu Arg Tyr Glu Pro Glu Arg Asp

435 440 445

Glu Trp His Leu Val Ala Pro Met Leu Thr Arg Arg Ile Gly Val Gly

450 455 460

Val Ala Val Leu Asn Arg Leu Leu Tyr Ala Val Gly Gly Phe Asp Gly

465 470 475 480

Thr Asn Arg Leu Asn Ser Ala Glu Cys Tyr Tyr Pro Glu Arg Asn Glu

485 490 495

Trp Arg Met Ile Thr Ala Met Asn Thr Ile Arg Ser Gly Ala Gly Val

500 505 510

Cys Val Leu His Asn Cys Ile Tyr Ala Ala Gly Gly Tyr Asp Gly Gln

515 520 525

Asp Gln Leu Asn Ser Val Glu Arg Tyr Asp Val Glu Thr Glu Thr Trp

530 535 540

Thr Phe Val Ala Pro Met Lys His Arg Arg Ser Ala Leu Gly Ile Thr

545 550 555 560

Val His Gln Gly Arg Ile Tyr Val Leu Gly Gly Tyr Asp Gly His Thr

565 570 575

Phe Leu Asp Ser Val Glu Cys Tyr Asp Pro Asp Thr Asp Thr Trp Ser

580 585 590

Glu Val Thr Arg Met Thr Ser Gly Arg Ser Gly Val Gly Val Ala Val

595 600 605

Thr Met Glu Pro Cys Arg Lys Gln Ile Asp Gln Gln Asn Cys Thr Cys

610 615 620

Claims (1)

1. a method for screening food-derived antioxidant oligopeptides by multi-site molecular docking in the Keap1 Kelch district, characterized in that: its method is as follows: Step 1. Obtain the protein primary structure of the food-derived protein from the Uniprot database, and based on this, construct a food-derived oligopeptide molecular structure library; Step 2. Find the PDB file that can reflect the interaction between the Kelch region of the Keap1 protein and the Nrf2 fragment containing the ETGE sequence from the PDB database, and the PDB file is ID: 2FLU; Step 3. Design three binding sites with reference to the three standards of the Nrf2-binding Keap1 binding site of the PDB file, all amino acid residues on Keap1 that are too close to the Nrf2 fragment, and the ETGE sequence of the key position of the Nrf2 fragment. ; center coordinates of binding site 1: x: -4, y: 6, z: 0; radius:

Center coordinates of binding site 2: x: 5, y: 9, z: 1; radius:

Center coordinates of binding site 3: x: 7.36, y: 8.33, z: 1.77; radius:

Step 4. Use all oligopeptides in the oligopeptide molecular structure library as ligands, and use the Keap1 protein Kelch region in the PDB file as the receptor, and perform molecular docking on the three binding sites designed in step 3; Step 5. Evaluate the docking results with the binding ability as an indicator, compare and analyze the interaction between the oligopeptide ligand with the highest binding ability and the active center and the original Keap1 protein Kelch region in the PDB file and the Nrf2 fragment containing the ETGE sequence. Evaluation, preliminary screening of food-derived antioxidant oligopeptides.

Images