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Exploring the molecular mechanism of the effect of puerarin on P2X3
Journal Pre-proofs Exploring the molecular mechanism of the effect of puerarin on P2X3 Shuangmei Liu, Mengke Wang, Na Wang, Shizhen Li, Rui Sun, Jingming Xing, Yueying Wang, Shicheng Yu, Lin Li, Guodong Li, Shangdong Liang PII: DOI: Reference:
S0141-8130(19)35089-5 https://doi.org/10.1016/j.ijbiomac.2019.09.120 BIOMAC 13365
To appear in: Received Date: Revised Date: Accepted Date:
4 July 2019 15 September 2019 16 September 2019
Please cite this article as: S. Liu, M. Wang, N. Wang, S. Li, R. Sun, J. Xing, Y. Wang, S. Yu, L. Li, G. Li, S. Liang, Exploring the molecular mechanism of the effect of puerarin on P2X3, (2019), doi: https://doi.org/ 10.1016/j.ijbiomac.2019.09.120
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Exploring the molecular mechanism of the effect of puerarin on P2X3 Shuangmei Liua, Mengke Wanga, Na Wangb, Shizhen Lib, Rui Sunc, Jingming Xingd, Yueying Wanga, Shicheng Yu a, Lin Lia, Guodong Lia, Shangdong Lianga* a
Department of Physiology, Basic Medical School of Nanchang University,
Nanchang, Jiangxi, P.R. China b
Undergraduate student of Second Clinical Department, Medical School of
Nanchang University, Nanchang, Jiangxi, P.R. China c
Undergraduate student of Anesthesiology Department, Medical School of
Nanchang University, Nanchang, Jiangxi, P.R. China d
Undergraduate student of Basic Medical Science Department, Medical School
of Nanchang University, Nanchang, Jiangxi, P.R. China Corresponding author: Shangdong Liang, PhD Department of Physiology, Basic Medical School of Nanchang University Nanchang, 330006 Jiangxi, China Phone: 0791-86363885 Email:[email protected] Abbreviations: ALA, Alanine; ATP, Adenosine triphosphate; CADD, computer aided drug design; DRG, dorsal root ganglia; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GFP, Green fluorescent protein; GLN, Glutamine; HEK, human embryonic kidney cell; HP, holding potential; PCR, Polymerase Chain Reaction; Pue, puerarin; SBDD, structure-based drug design; SDM, Site-directed mutagenesis; THR, Threonine; VAL, Valine; WT, wild-type; GLY, Glycine.
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Abstract P2X3 is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+. Adenosine triphosphate (ATP) activation of the P2X3 on primary sensory ganglion neurons is involved in nociceptive transmission. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen. Puerarin inhibits nociceptive signal transmission by inhibiting the P2X3 in the dorsal root ganglia (DRG) and sympathetic ganglia, but its molecular mechanism is unclear. The aim of this study was to explore the molecular mechanism of puerarin on the P2X3. Here, molecular docking results revealed that puerarin binds well to the human P2X3 protein in the vicinity of the ATP binding pocket. Protein-ligand docking showed that the V64A mutation reduced the effect of puerarin but had little effect on ATP. V64A site-directed mutagenesis of P2X3 was performed using an overlap extension PCR technique. The wild-type and V64A mutant pEGFP-C1-P2X3 recombinant plasmids were transfected into HEK 293 cells. The electrophysiology results demonstrated that puerarin exerted an obvious inhibitory effect on ATP-activated currents in HEK 293 cells transfected with the wild-type P2X3, while little inhibition was observed in HEK 293 cells transfected with the mutant P2X3. These studies suggest that puerarin inhibits the P2X3 by binding to V64A.
Key words: P2X3;
Puerarin;
Site-directed mutagenesis
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1. Introduction Purinergic receptors include the P1 and P2 subtypes. Adenosine and its analogues activate P1 receptors, and adenosine triphosphate (ATP) and its analogues act on P2 receptors[1-4]. P2 receptors are divided into P2X receptors and P2Y receptors[1-4]. P2X3 is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+[3-5]. Studies have shown that nociceptive stimulation causes cell damage and cell stress, resulting in injured cells and the release of inflammatory substances, such as ATP, from nerve endings. ATP-mediated activation of the P2X3 on primary sensory ganglion neurons is involved in nociceptive signaling transmission[3, 4, 6, 7]. Studies have demonstrated that pain sensitivity in P2X3 knockout mice is decreased compared to wild-type mice, and P2X3 antagonists inhibit neuropathic pain in rats[2-4, 8]. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen (Radix Puerariae, RP). Puerarin exhibits a wide range of pharmacological effects in China and is traditionally used to treat cardiovascular and cerebrovascular diseases[9, 10, 11]. Puerarin has also been reported to inhibit inflammatory effects, oxidative damage and to prevent peripheral neuropathic pain[12, 13]. In our previous studies, we observed that puerarin inhibits nociceptive signal transmission by decreasing P2X3 expression in DRG and sympathetic ganglia[9，14， 15], but the molecular mechanism by which this occurs is not clear. Molecular simulation can be used to study the interaction between biological macromolecules and small molecules with respect to biological activity. Molecular docking is an important method of molecular simulation and involves simulating the interaction between two or more molecules by calculation[16]. Homology modeling is a method that combines bioinformatics and computer technology[17, 18]. The crystal structure of the P2X4 receptor in zebrafish has been elucidated[19]. The homology modeling method was used to construct the structure of P2X3. This structure can be used as a model for virtual screening and can identify relationships between docking scoring and activity from the view of the receptor, providing a theoretical basis for virtual screening[20-23]. We used homology modeling to obtain a P2X3 model and to simulate molecular docking with puerarin. By molecular dynamics simulation, the receptor structure was optimized and the binding sites of puerarin and P2X3 were 3 / 28
observed. Through virtual screening of specific drug targets and experimental verification, we can avoid the randomness of drug target screening. The binding site of puerarin on P2X3 was tentatively identified, which could help for exploring the molecular mechanism of puerarin acting on P2X3.. The three-dimensional structure of proteins provides an effective way to understand its molecular mechanism. On this basis, the experimental design will be more targeted, which is helpful for the research of point mutation and the design of specific inhibitors[24, 25]. Structure-based drug design (SBDD) is a type of computer aided drug design (CADD). Using this method, we can analyze the active sites between receptors and drugs and study the binding energy and relative positional relationship between them. Studying the mode of action of small molecule drugs in the active sites of proteins can provide bioactive conformation of small molecule ligands to intuitively understand their specific mode of action in the active binding pockets of proteins and surrounding residues. Site-directed mutagenesis (SDM) is the insertion, deletion, or point mutation of bases in specific sites of DNA fragments using PCR and other methods[26, 27]. SDM is an effective tool for studying the relationship between protein structure and function. Using site directed mutagenesis technology, P2X3 was mutated and used to study the molecular mechanism of the effect of puerarin on the P2X3[28, 29]. Thus, the purpose of this study was to explore the molecular mechanism of puerarin on P2X3.
2. Materials and Methods 2.1. Cells and reagents HEK 293 cells were purchased from the Shanghai cell bank of the Chinese Academy of Sciences. ATP as a disodium salt was purchased from Sigma Chemicals (St. Louis, MO, USA). ATP was dissolved in distilled water or recording buffer as a 1 mol/L stock solution and diluted into different concentrations of working solutions using extracellular fluid. Puerarin was purchased from the China Food and Drug Inspection Institute and was dissolved in DMSO as a 1 mol/l stock solution. EcoR I, BglⅡ, T4 DNA ligase, and T4 DNA ligase buffer were purchased from Takara Company. Plasmid extraction and gel recovery kits were purchased from TIANGEN 4 / 28
Company (Beijing, China) and Promega, respectively. cDNA of the wild-type (WT) P2X3 (Changsha win run Biotechnology Co., Ltd.) was cloned into the pEGFP-C1 vector containing cDNA of enhanced green fluorescence protein to enable the identification of efficiently transfected cells under a fluorescence microscope. The pCR4-TOPO-P2X3 gene (Changsha ying run Biotechnology Co., Ltd.) was used as a template for production of plasmids containing the following described amino acid residue mutations.
2.2. Homology modeling and molecular docking Homology modeling based on the homology relationship between known target protein sequences and template protein sequences can predict and construct three-dimensional structure models of proteins[17]. When the sequence similarity between two proteins is greater than 30%, it indicates that these two proteins may be homologous. The higher the homology of the sequence of the unknown target protein and the known template protein, the higher the accuracy of the target protein constructed by homology modeling.. At present, three-dimensional structures of proteins can be obtained by X-ray diffraction, nuclear magnetic resonance and other methods. These structural documents are aggregated within the protein crystal database (Data Bank Protein, PDB) and are available to download. SWISS-PROT is a protein sequence database of the European Bioinformatics Institute (EBI). In this study, sequence information of human P2X3 was retrieved from the SWISS-PROT website (UniProtKB number: P56373). Through the sequence alignment function of SWISS-PROT, a known template protein crystal 3I5D (PDB database number) with high sequence similarity was matched. Based on the published crystal structure of the zebra fish P2X4 channel, the extracellular loop and transmembrane areas of the human P2X3 (hP2X3) receptor were modeled[19, 21]. Based on preliminary modeling, the established model was further optimized using the Chiron server and was assessed using the SVAES server to guarantee that amino acid residues in the activated region of the receptor protein were close to the native conformation. By alternating optimization and evaluation, a high quality model was obtained for the subsequent step of molecular docking studies[30-32]. In this study, we used the P2X3 model 5 / 28
established above to perform docking simulations with puerarin. Using Discovery Studio 3.5 software, the interaction between the docking protein (P2X3) and ligands (adenosine triphosphate or puerarin) was assessed to obtain the best docking score between them[31-34]. The mutation site of P2X3 was selected according to instructions of the Discovery Studio 3.5 software. Analyze Ligand Poses was used to establish the heatmap of hydrogen bonds (Fan Dehua collision). A heatmap can quickly display hydrogen bond maps and the number of close contacts to observe amino acid residues that make an important contribution to ligand binding. Application of the LibDock molecular docking module in Discovery Studio software defined wild-type and mutant P2X3 as receptor molecules and drugs (adenosine triphosphate or puerarin) as ligands for molecular docking[33-35]. By comparing and analyzing the amino acid sequence of P2X3 and its 3D structure, we determined the amino acid site in the activity center of P2X3 that would have an effect on its binding with puerarin.
2.3. Site-directed mutagenesis of the P2X3 gene by PCR Overlap extension PCR technique can be used to effectively perform gene recombination in vitro by PCR. The process of overlap extension PCR involved designing 2 pairs of primers, in this case, P2X3-Forward, P2X3-Reverse and V64A-Forward, V64A-Reverse. The P2X3-Forward and P2X3-Reverse primers were at the ends of the P2X3 gene, while the V64A-Reverse and V64A-Forward primers were in the middle of the P2X3 gene. The mutation point can be designed using V64A-Forward or V64A-Reverse or both primers. It should be noted that the mutation point was better designed at the 5 'end of the primer, avoiding the 3' end. The
primers
sequences
were
as
follows:
5'-GAAGATCTATGAACTGCATATCCGACTTCTTC-3', 5'-GGAATTCC
P2X3-Forward P2X3-Reverse
TAGTGGCCTATGGAGAAGGC-3',
V64A-Forward
5’-GTAACCAAGGCCAAGGGCTCC-3’,
V64A-Reverse
5’-GGAGCCCTTGGCCTTGGTTAC- 3’. The procedure of overlap extension PCR are
as
follows.
First,
we
used
two
pairs
of
primers,
primer
P2X3-Forward/V64A-Reverse and primer P2X3-Reverse/V64A-Forward to generate 6 / 28
two DNA fragments using the target gene as the template. The PCR products were gel-purified. These two DNA fragments were therefore able to anneal together with their complementary overhanging cohesive ends, which were then extended and amplified by PCR using the primer pair P2X3-Forward/ P2X3-Reverse to get a full length gene with the expected mutation.
2.4. Construction of a recombinant plasmid of mutant pEGFP-C1-P2X3 Amplification products of mutant P2X3 were cloned into the pEGFP-C1 vector. First, enzyme digestion of the PCR products and the pEGFP-C1 vector was performed. The enzyme digestion reaction solutions for the PCR products (PCR product, 6 µl; EcoR I, 1 µl; BglⅡ 1, µl; 10×H Buffer, 2 μl; ddH2O, 10 μl) and vector (vector, 2 μl; EcoR I, 1 µl; BglⅡ, 1 µl; 10×H Buffer, 2 μl; ddH2O, 14 μl) were prepared. Then, the solutions were placed in a 37ºC water bath overnight. After enzyme digestion, we assessed whether there was a P2X3 fragment (approximately 1.2 kb) or a linear pEGFP-C1 band (approximately 4.7bp) by agarose gel electrophoresis. Then, the gel bands were recycled. Third, the P2X3 fragment and linear pEGFP-C1 were connected. The reaction solutions included pEGFP-C1, 1 μl; P2X3 fragment, 5 μl; 10×ligation buffer, 1.5 μl; T4 DNA ligase, 1.5 μl; 10×BSA,1.5 μl; and ddH2O, 4.5 μl. The solutions were then placed at 16℃ overnight. The ligation products were transformed into Escherichia coli cells, and positive clones were sequenced by the Nanjing Genscript Company to ensure the success of the construction of the recombinant plasmid. After the recombinant plasmid was extracted and digested, agarose gel electrophoresis of the ligation products was used to determine the success of the cloning. If 1.2-kb and 4.7-kb bands were observed, it preliminarily indicated that the P2X3 gene fragment was successfully cloned into the pEGFP-C1 vector.
2.5. HEK 293 cell culture and transfection HEK 293 cells were grown in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were transiently transfected with the wild-type and mutant hP2X3 plasmids using Lipofectamine 2000 7 / 28
reagent (Invitrogen) according to the manufacturer’s instructions. When HEK 293 cells were 70–80% confluent, the cell culture media was replaced with OptiMEM 2 hours before transfection. The transfection medium was prepared as follows: (a) 4 µg plasmid DNA was diluted into a 250 µl final volume of OptiMEM; (b) 10 µl lipofectamine 2000 was diluted into a 250 µl final volume of OptiMEM; and (c) the lipofectamine-containing solution was mixed with the plasmid-containing solutions and incubated at RT for 20 min. Subsequently, 500 µl of the cDNA/lipofectamine solution was added to a 35 mm well. Cells were incubated for 6 h at 37°C, 5% CO2. After incubation, cells were washed in MEM containing 10% FBS and incubated for 24-48 h. GFP fluorescence was assessed as a reporter to determine the transfection efficiency, which was detected under a fluorescence microscope by calculating the percentage of positive cells. Whole-cell patch clamp recordings were performed 1-2 days after transfection.
2.6. Whole-cell patch clamp recording Electrophysiological recording was performed using a patch/whole cell clamp amplifier (Axopatch 200B). HEK 293 cells expressing green fluorescence were subjected to electrophysiological recording. Microelectrodes were filled with an internal solution (in mM) composed of K gluconate 145, EGTA 0.75, HEPES 10, CaCl2 0.1, MgATP 2, Na3GTP 0.3. The bath was continuously perfused with an extracellular solution containing (in mM) NaCl, 126; KCl, 2.5; glucose, 10; MgCl2, 1.2; CaCl2, 2.4; and NaHCO3 18. The osmolarity of the extracellular and internal solutions was adjusted to 340 mOsm using sucrose, the pH of the extracellular solution was adjusted to 7.4 using NaOH and the pH of the internal solution was adjusted to 7.3 using KOH. The resistance of the recording electrodes was 2-6 MΩ. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1-10 GΩ), and then, a more negative pressure was applied to rupture it. The holding potential (HP) was set to -60 mv. ATP and puerarin were diluted in the external solution and rapidly applied through a manifold comprising 10 capillaries made of fused silica coated with polyimide with a 200 μm internal diameter. The distance from the tubule mouth to the cell examined was approximately 100 μm. The solutions were 8 / 28
delivered by gravity flow from independent reservoirs. One barrel was used to apply a drug-free external solution to enable rapid termination of drug application. Data were low-pass filtered at 2 Hz, digitized at 5 kHz and stored on a laboratory computer using the Digidata 1200 interface and pClamp10.0 software (Axon instruments). A concentration response curve was constructed by Sigmaplot 12.0 software. Traces were acquired using pClamp software and plotted using Origin 8 (Microcal, Northampton, MA, USA).
2.7. Data analysis Statistical analysis of the data was performed using SPSS 11.5 software. Statistical significance was determined by an unpaired t-test between two groups in the electrophysiological experiments. P<0.05 was considered statistically significant.
3. Results 3.1. Molecular docking of puerarin and P2X3 The interaction between puerarin and the hP2X3 protein was analyzed by Autodock 4.2 (molecular docking software). The three dimensional structure files of ATP (CID5957) and puerarin (CID 5281807) were downloaded from the PubChem database, and Utra ChemBioDraw 11 and Chimera1.6.1 software was used to convert the format. The Lamarckian genetic algorithm (Lamarckian genetic algorithm selection, LGA) was used for molecular docking. The flexible sites of the hP2X3 protein were Lys-63, Lys-65, Phe171, Thr172, Asn-279, Arg-281, Lys-299. The molecular docking results are shown in Figure 1. A structure of puerarin was as Fig. 1A. Puerarin binds well with the hP2X3 protein in the vicinity of the ATP binding pocket and its surrounding residues and forms hydrogen bonds (Fig. 1 B, C and D). Score MOE software analysis showed that the optimal docking energy of puerarin with hP2X3 was -6.1829 kcal/mol, greater than the -3.81kcal/mol of ATP with hP2X3. It is speculated that puerarin binding to hP2X3 prevents ATP from binding with hP2X3, increasing the concentration of free ATP and inhibiting the activity of hP2X3 protein channels.
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3.2. Protein-ligand docking score of the selected P2X3 mutation site The ligand poses process of the Discovery Studio 3.5 software was used to analyze the interaction between receptor (such as P2X3) and ligand (such as ATP or puerarin). We chose P2X3 mutation site according to the close contact (Van Edward force collision) heatmap. Using the molecular docking module of Studio LibDock's Discovery, the interaction between P2X3 molecule (before and after the mutation) and ligand (ATP or puerarin) was scored. GLY130, VAL64, THR82, and GLN85 P2X3 mutants were constructed and docked with ATP or puerarin. Figure 2 shows that the VAL64 mutation had the maximum effect on the molecular docking results. The docking scores of the V64A mutation with ATP (Figure 2A) and puerarin (Figure 2B) were 154.924 and 122.257, respectively. The results indicate that the V64A mutation reduces the effects of P2X3 and puerarin and has little effect on the interaction between P2X3 and ATP.
3.3. Results of recombinant plasmid construction and the transfection efficiency of HEK 293 cells The P2X3 fragment of the PCR product was detected using agarose gel electrophoresis, and a band of approximately 1.2 kb was observed in the GIS gel system (Figure 3A). The length of the band was consistent with the P2X3 fragment. The P2X3 gene fragment was then cloned into the pEGFP-C1 vector. pEGFP-C1-P2X3 positive clones were identified by EcoR I and Bgl II, cutting out vector bands of approximately 4.7 kb and target fragment bands of approximately 1.2 kb (Fig. 3B). It was initially speculated that the P2X3 was cloned into the pEGFP-C1 vector. The experimental results are shown in Figure 3B. Positive clones were sequenced, and the results demonstrated that the pEGFP-C1-P2X3 was correctly constructed (P2X3 gene sequence number NM_002559.3) (Fig. 4). The efficiency of HEK 293 transfection with the wild-type and mutant P2X3 plasmids was greater than 80% (Fig. 5).
3.4. Effects of puerarin on the ATP-activated current in HEK 293 cells transfected with the wild type or mutant P2X3 10 / 28
Electrophysiological experiments were recorded 24 hours after transfection. Figure 6A shows that ATP-activated currents in HEK 293 cells transfected with the wild-type (WT) P2X3 and mutant P2X3 exhibited no differences. When applied with puerarin and ATP, ATP-activated currents were significantly decreased compared to only application of ATP in HEK 293 cells transfected with wild-type P2X3. In HEK 293 cells transfected with the mutant P2X3 plasmid, the inhibitory effect of puerarin on the ATP-activated current was not obvious. Figure 6B shows that the inhibitory effect of puerarin on the ATP-activated current in the wild-type and mutant P2X3 was increased in response to the upregulation of the puerarin concentration. The inhibition rate of the mutant was reduced compared to the wild type. These results suggest that the mutation site of the P2X3 plasmid is the active site of puerarin and P2X3.
4. Discussion The crystal structure of the zebrafish P2X4 receptor is known and was used as a template to construct P2X3 structure using homology modeling[20-23]. When the sequence similarity between the target protein and the template protein is greater than 30%, the two proteins may be homologous. The length of the P2X subunit was between 379 to 595 amino acids, and 35%-54% of the sequence was homologous. Therefore, it is feasible to use the zebrafish P2X4 receptor to construct a model of the human P2X3. The structure of P2X3 was solved in our laboratory using homology modeling. Molecular simulation can be used to study the interactions between receptor proteins and small active molecules. Molecular docking is an important method of molecular simulation, involving simulation of the interaction between two or more molecules using bioinformatics methods[16]. Virtual screening of specific drug targets can provide insight for further experimental validation, avoiding the randomness of drug target screening. In this study, we established a model of P2X3 protein structure that was subsequently used in molecular docking simulations with puerarin. The results revealed that there was an interaction between puerarin and hP2X3. Puerarin bound well with the hP2X3 protein in the vicinity of the ATP binding pocket and its surrounding residues. The optimal docking energy of puerarin with the hP2X3 protein was greater than that of ATP with the hP2X3 protein, suggesting that 11 / 28
puerarin prevents ATP from binding with P2X3. Therefore, the molecular docking method can be used to determine the binding site of puerarin and P2X3. This method is helpful for exploring the molecular mechanism of P2X3 inhitbited by puerarin. Studying the role of small molecules derived from natural drugs in the active sites of receptor proteins is helpful to understand the specific role of small molecule ligands in the protein binding pocket and surrounding residues[24, 25]. Using Discovery Studio 3.5 software, we analyzed the interactions between P2X3 protein and ATP or puerarin[33-35] and subsequently selected the P2X3 mutation sites. By comparing and analyzing the amino acid sequence of P2X3 and its 3D structure, we determined the possible amino acid sites that exerted an effect on puerarin binding in the active center of P2X3. In this experiment, the Studio LibDock's Discovery molecular docking module was used to determine the docking score of the wild-type and mutant P2X3and ligands (ATP or puerarin)[33-35]. Mutants of GLY130, VAL64, THR82, and GLN85 in P2X3 were constructed and docked with ATP or puerarin. The results demonstrated that the V64A mutation had the maximum effect on molecular docking results. The docking scores of the V64A mutation with ATP and puerarin were 154.924 and 122.257, respectively, suggesting that the V64A mutation reduces the effect of puerarin on P2X3 and has little effect on the interaction between ATP and P2X3. Hence, the V64A mutation of P2X3 was selected for the next experiment in which a fixed point mutation of P2X3 was designated by overlapping extension PCR[26, 27, 29], and the sequence of the point mutation was identified. Compared to the one-step PCR method, the shortcomings of overlapping extension PCR are that obtaining mutant proteins requires two pairs of primers for two PCR reactions. However, its advantage is that mutants obtained by two PCR reactions can be entirely separated from the original template, and the mutation rate is nearly 100%. This method reduces the workload of the screening process after mutation[26, 27, 36]. So overlapping extension PCR is a simple and effective method for site-directed mutagenesis. SDM can be used for insertion, deletion or point mutation of specific sites of target DNA to affect the traits and characteristics of the target protein[15, 26, 27]. SDM is an effective means to study the relationship between protein structure and 12 / 28
function. Using SDM technology to accomplish site-directed mutation of P2X3, we observed the interaction between puerarin and P2X3[28, 29]. Wild-type and mutant P2X3
plasmids were
successfully transfected into HEK 293 cells.
The
electrophysiological experimental results demonstrated that the ATP-activated current in both groups of HEK 293 transfected cells was not significantly different. Puerarin significantly reduced ATP-activated currents in HEK 293 cells transfected with the wild-type P2X3 plasmid but not in HEK 293 cells transfected with the mutant P2X3 plasmid. At the same time, we observed that inhibition of the ATP-activated current in the wild-type and mutant P2X3 by puerarin was increased with the increasing puerarin concentration, but the inhibitory rate in the mutant P2X3 was significantly lower than in the wild type. These experimental results indicate that the site-directed mutation of V64A significantly affects ATP-activated currents in HEK 293 cells transfected with mutant P2X3. Therefore, the V64A mutation of P2X3 is important for puerarin binding to P2X3, providing an experimental model to further explore the molecular mechanisms of the inhibition of P2X3 by puerarin.
5. Conclusions We used molecular docking to identify the V64A mutation and performed cell transfection and whole cell patch clamp experiments to verify the role of the mutation site. The point mutant V64A reduced the interaction between puerarin and the P2X3 but had little effect on the interaction between ATP and the P2X3. Therefore, position 64 (as shown by the V64A mutation) is an important site for the inhibition of P2X3 by puerarin.
Author’s contributions Participated in research design: Shangdong Liang Conducted experiments: Shuangmei Liu, Mengke Wang, Na Wang, Lin Li Performed data analysis: Shuangmei Liu, Shizhen Li, Rui Sun, Jingming Xing Wrote or contributed to writing of the manuscript: Shuangmei Liu, Yueying Wang, Shangdong Liang
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Conflicts of interest The all authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by grants (№s: 81701114, 81870574, 81570735, 81970749, 31560276) from the National Natural Science Foundation of China, and grant (№: 8181101216) from International Cooperation project between NSFC and DFG.
Data availability The data used to support the findings of this study are included within the article. Additional data can be requested by e-mail: [email protected]
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Figure Legends. Figure 1. Molecular docking of puerarin and hP2X3. A shows the chemical structure of puerarin. Simulation of puerarin docking with hP2X3 was performed by a computer. ATP-binding sites were located at opposite sites of the same subunit and were therefore only able to form a binding pocket at the interface of two adjacent subunits (B, C). Puerarin interacts with the hP2X3 protein near the ATP-binding pocket and forms hydrogen bonds with Thr155, Trp152 and Arg281 (C, D). 20 / 28
Figure 2. Docking score of the V64A mutation of P2X3 with ATP and puerarin. The scores of P2X3-ATP (A) and P2X3-puerarin (B) are 154.924 and 122.257, respectively.
Figure 3. Enzyme identification results of pEGFP-C1-P2X3. A. Gly electrophoresis image of P2X3 PCR product. M: DNA Marker IV; 1: PCR product. B. Identification chart of the recombinant pEGFP-C1-P2X3 plasmid digested by double enzymes. M: DNA Marker IV; 1: bands of pEGFP-C1-P2X3 after double enzymatic digestion.
Figure 4. Comparison results of the gene sequence of the positive clone of pEGFP-C1-P2X3. The pEGFP-C1-P2X3 plasmid was correctly constructed.
Figure 5. Detection of the transfection efficiency in HEK 293 cells transfected with the P2X3 plasmid. The left image shows all cells. The right image shows cells expressing green fluorescent protein that were successfully transfected with the pEGFP-P2X3 plasmid. The percent of green cells (right) of all cells (left) is the transfection efficiency. The efficiency of HEK 293 transfection with the wild-type and mutant P2X3 plasmids was greater than 80%.
Figure 6. Inhibitory effects of puerarin (Pue) on ATP-induced currents in HEK 293 cells transfected with the wild-type or the mutant hP2X3. (A) Original tracings showing that puerarin (10 µM) mostly inhibited ATP (100 µM)-induced currents in the wild-type hP2X3 but had little effect on the mutant receptor. (B) Graph showing the dose–response curve of ATP-activated currents in the wild-type and mutant hP2X3 in response to different concentrations of puerarin. The inhibitory effects of puerarin on ATP-induced currents in HEK 293 cells transfected with the wild-type hP2X3 was stronger than those transfected with the mutant receptor at the same concentration of puerarin. *P < 0.05, **P < 0.01, compared to the wild-type hP2X3 using the unpaired t-test.
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Highlights
• Molecular docking revealed puerarin binds well to hP2X3 protein. • Puerarin interacts with hP2X3 protein near ATP-binding pocket. • V64A mutation reduces the effects of the P2X3 receptor and puerarin. • Puerarin inhibits P2X3 receptor by binding to V64A.
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S0141-8130(19)35089-5 https://doi.org/10.1016/j.ijbiomac.2019.09.120 BIOMAC 13365
To appear in: Received Date: Revised Date: Accepted Date:
4 July 2019 15 September 2019 16 September 2019
Please cite this article as: S. Liu, M. Wang, N. Wang, S. Li, R. Sun, J. Xing, Y. Wang, S. Yu, L. Li, G. Li, S. Liang, Exploring the molecular mechanism of the effect of puerarin on P2X3, (2019), doi: https://doi.org/ 10.1016/j.ijbiomac.2019.09.120
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© 2019 Published by Elsevier B.V.
Exploring the molecular mechanism of the effect of puerarin on P2X3 Shuangmei Liua, Mengke Wanga, Na Wangb, Shizhen Lib, Rui Sunc, Jingming Xingd, Yueying Wanga, Shicheng Yu a, Lin Lia, Guodong Lia, Shangdong Lianga* a
Department of Physiology, Basic Medical School of Nanchang University,
Nanchang, Jiangxi, P.R. China b
Undergraduate student of Second Clinical Department, Medical School of
Nanchang University, Nanchang, Jiangxi, P.R. China c
Undergraduate student of Anesthesiology Department, Medical School of
Nanchang University, Nanchang, Jiangxi, P.R. China d
Undergraduate student of Basic Medical Science Department, Medical School
of Nanchang University, Nanchang, Jiangxi, P.R. China Corresponding author: Shangdong Liang, PhD Department of Physiology, Basic Medical School of Nanchang University Nanchang, 330006 Jiangxi, China Phone: 0791-86363885 Email:[email protected] Abbreviations: ALA, Alanine; ATP, Adenosine triphosphate; CADD, computer aided drug design; DRG, dorsal root ganglia; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GFP, Green fluorescent protein; GLN, Glutamine; HEK, human embryonic kidney cell; HP, holding potential; PCR, Polymerase Chain Reaction; Pue, puerarin; SBDD, structure-based drug design; SDM, Site-directed mutagenesis; THR, Threonine; VAL, Valine; WT, wild-type; GLY, Glycine.
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Abstract P2X3 is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+. Adenosine triphosphate (ATP) activation of the P2X3 on primary sensory ganglion neurons is involved in nociceptive transmission. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen. Puerarin inhibits nociceptive signal transmission by inhibiting the P2X3 in the dorsal root ganglia (DRG) and sympathetic ganglia, but its molecular mechanism is unclear. The aim of this study was to explore the molecular mechanism of puerarin on the P2X3. Here, molecular docking results revealed that puerarin binds well to the human P2X3 protein in the vicinity of the ATP binding pocket. Protein-ligand docking showed that the V64A mutation reduced the effect of puerarin but had little effect on ATP. V64A site-directed mutagenesis of P2X3 was performed using an overlap extension PCR technique. The wild-type and V64A mutant pEGFP-C1-P2X3 recombinant plasmids were transfected into HEK 293 cells. The electrophysiology results demonstrated that puerarin exerted an obvious inhibitory effect on ATP-activated currents in HEK 293 cells transfected with the wild-type P2X3, while little inhibition was observed in HEK 293 cells transfected with the mutant P2X3. These studies suggest that puerarin inhibits the P2X3 by binding to V64A.
Key words: P2X3;
Puerarin;
Site-directed mutagenesis
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1. Introduction Purinergic receptors include the P1 and P2 subtypes. Adenosine and its analogues activate P1 receptors, and adenosine triphosphate (ATP) and its analogues act on P2 receptors[1-4]. P2 receptors are divided into P2X receptors and P2Y receptors[1-4]. P2X3 is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+[3-5]. Studies have shown that nociceptive stimulation causes cell damage and cell stress, resulting in injured cells and the release of inflammatory substances, such as ATP, from nerve endings. ATP-mediated activation of the P2X3 on primary sensory ganglion neurons is involved in nociceptive signaling transmission[3, 4, 6, 7]. Studies have demonstrated that pain sensitivity in P2X3 knockout mice is decreased compared to wild-type mice, and P2X3 antagonists inhibit neuropathic pain in rats[2-4, 8]. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen (Radix Puerariae, RP). Puerarin exhibits a wide range of pharmacological effects in China and is traditionally used to treat cardiovascular and cerebrovascular diseases[9, 10, 11]. Puerarin has also been reported to inhibit inflammatory effects, oxidative damage and to prevent peripheral neuropathic pain[12, 13]. In our previous studies, we observed that puerarin inhibits nociceptive signal transmission by decreasing P2X3 expression in DRG and sympathetic ganglia[9，14， 15], but the molecular mechanism by which this occurs is not clear. Molecular simulation can be used to study the interaction between biological macromolecules and small molecules with respect to biological activity. Molecular docking is an important method of molecular simulation and involves simulating the interaction between two or more molecules by calculation[16]. Homology modeling is a method that combines bioinformatics and computer technology[17, 18]. The crystal structure of the P2X4 receptor in zebrafish has been elucidated[19]. The homology modeling method was used to construct the structure of P2X3. This structure can be used as a model for virtual screening and can identify relationships between docking scoring and activity from the view of the receptor, providing a theoretical basis for virtual screening[20-23]. We used homology modeling to obtain a P2X3 model and to simulate molecular docking with puerarin. By molecular dynamics simulation, the receptor structure was optimized and the binding sites of puerarin and P2X3 were 3 / 28
observed. Through virtual screening of specific drug targets and experimental verification, we can avoid the randomness of drug target screening. The binding site of puerarin on P2X3 was tentatively identified, which could help for exploring the molecular mechanism of puerarin acting on P2X3.. The three-dimensional structure of proteins provides an effective way to understand its molecular mechanism. On this basis, the experimental design will be more targeted, which is helpful for the research of point mutation and the design of specific inhibitors[24, 25]. Structure-based drug design (SBDD) is a type of computer aided drug design (CADD). Using this method, we can analyze the active sites between receptors and drugs and study the binding energy and relative positional relationship between them. Studying the mode of action of small molecule drugs in the active sites of proteins can provide bioactive conformation of small molecule ligands to intuitively understand their specific mode of action in the active binding pockets of proteins and surrounding residues. Site-directed mutagenesis (SDM) is the insertion, deletion, or point mutation of bases in specific sites of DNA fragments using PCR and other methods[26, 27]. SDM is an effective tool for studying the relationship between protein structure and function. Using site directed mutagenesis technology, P2X3 was mutated and used to study the molecular mechanism of the effect of puerarin on the P2X3[28, 29]. Thus, the purpose of this study was to explore the molecular mechanism of puerarin on P2X3.
2. Materials and Methods 2.1. Cells and reagents HEK 293 cells were purchased from the Shanghai cell bank of the Chinese Academy of Sciences. ATP as a disodium salt was purchased from Sigma Chemicals (St. Louis, MO, USA). ATP was dissolved in distilled water or recording buffer as a 1 mol/L stock solution and diluted into different concentrations of working solutions using extracellular fluid. Puerarin was purchased from the China Food and Drug Inspection Institute and was dissolved in DMSO as a 1 mol/l stock solution. EcoR I, BglⅡ, T4 DNA ligase, and T4 DNA ligase buffer were purchased from Takara Company. Plasmid extraction and gel recovery kits were purchased from TIANGEN 4 / 28
Company (Beijing, China) and Promega, respectively. cDNA of the wild-type (WT) P2X3 (Changsha win run Biotechnology Co., Ltd.) was cloned into the pEGFP-C1 vector containing cDNA of enhanced green fluorescence protein to enable the identification of efficiently transfected cells under a fluorescence microscope. The pCR4-TOPO-P2X3 gene (Changsha ying run Biotechnology Co., Ltd.) was used as a template for production of plasmids containing the following described amino acid residue mutations.
2.2. Homology modeling and molecular docking Homology modeling based on the homology relationship between known target protein sequences and template protein sequences can predict and construct three-dimensional structure models of proteins[17]. When the sequence similarity between two proteins is greater than 30%, it indicates that these two proteins may be homologous. The higher the homology of the sequence of the unknown target protein and the known template protein, the higher the accuracy of the target protein constructed by homology modeling.. At present, three-dimensional structures of proteins can be obtained by X-ray diffraction, nuclear magnetic resonance and other methods. These structural documents are aggregated within the protein crystal database (Data Bank Protein, PDB) and are available to download. SWISS-PROT is a protein sequence database of the European Bioinformatics Institute (EBI). In this study, sequence information of human P2X3 was retrieved from the SWISS-PROT website (UniProtKB number: P56373). Through the sequence alignment function of SWISS-PROT, a known template protein crystal 3I5D (PDB database number) with high sequence similarity was matched. Based on the published crystal structure of the zebra fish P2X4 channel, the extracellular loop and transmembrane areas of the human P2X3 (hP2X3) receptor were modeled[19, 21]. Based on preliminary modeling, the established model was further optimized using the Chiron server and was assessed using the SVAES server to guarantee that amino acid residues in the activated region of the receptor protein were close to the native conformation. By alternating optimization and evaluation, a high quality model was obtained for the subsequent step of molecular docking studies[30-32]. In this study, we used the P2X3 model 5 / 28
established above to perform docking simulations with puerarin. Using Discovery Studio 3.5 software, the interaction between the docking protein (P2X3) and ligands (adenosine triphosphate or puerarin) was assessed to obtain the best docking score between them[31-34]. The mutation site of P2X3 was selected according to instructions of the Discovery Studio 3.5 software. Analyze Ligand Poses was used to establish the heatmap of hydrogen bonds (Fan Dehua collision). A heatmap can quickly display hydrogen bond maps and the number of close contacts to observe amino acid residues that make an important contribution to ligand binding. Application of the LibDock molecular docking module in Discovery Studio software defined wild-type and mutant P2X3 as receptor molecules and drugs (adenosine triphosphate or puerarin) as ligands for molecular docking[33-35]. By comparing and analyzing the amino acid sequence of P2X3 and its 3D structure, we determined the amino acid site in the activity center of P2X3 that would have an effect on its binding with puerarin.
2.3. Site-directed mutagenesis of the P2X3 gene by PCR Overlap extension PCR technique can be used to effectively perform gene recombination in vitro by PCR. The process of overlap extension PCR involved designing 2 pairs of primers, in this case, P2X3-Forward, P2X3-Reverse and V64A-Forward, V64A-Reverse. The P2X3-Forward and P2X3-Reverse primers were at the ends of the P2X3 gene, while the V64A-Reverse and V64A-Forward primers were in the middle of the P2X3 gene. The mutation point can be designed using V64A-Forward or V64A-Reverse or both primers. It should be noted that the mutation point was better designed at the 5 'end of the primer, avoiding the 3' end. The
primers
sequences
were
as
follows:
5'-GAAGATCTATGAACTGCATATCCGACTTCTTC-3', 5'-GGAATTCC
P2X3-Forward P2X3-Reverse
TAGTGGCCTATGGAGAAGGC-3',
V64A-Forward
5’-GTAACCAAGGCCAAGGGCTCC-3’,
V64A-Reverse
5’-GGAGCCCTTGGCCTTGGTTAC- 3’. The procedure of overlap extension PCR are
as
follows.
First,
we
used
two
pairs
of
primers,
primer
P2X3-Forward/V64A-Reverse and primer P2X3-Reverse/V64A-Forward to generate 6 / 28
two DNA fragments using the target gene as the template. The PCR products were gel-purified. These two DNA fragments were therefore able to anneal together with their complementary overhanging cohesive ends, which were then extended and amplified by PCR using the primer pair P2X3-Forward/ P2X3-Reverse to get a full length gene with the expected mutation.
2.4. Construction of a recombinant plasmid of mutant pEGFP-C1-P2X3 Amplification products of mutant P2X3 were cloned into the pEGFP-C1 vector. First, enzyme digestion of the PCR products and the pEGFP-C1 vector was performed. The enzyme digestion reaction solutions for the PCR products (PCR product, 6 µl; EcoR I, 1 µl; BglⅡ 1, µl; 10×H Buffer, 2 μl; ddH2O, 10 μl) and vector (vector, 2 μl; EcoR I, 1 µl; BglⅡ, 1 µl; 10×H Buffer, 2 μl; ddH2O, 14 μl) were prepared. Then, the solutions were placed in a 37ºC water bath overnight. After enzyme digestion, we assessed whether there was a P2X3 fragment (approximately 1.2 kb) or a linear pEGFP-C1 band (approximately 4.7bp) by agarose gel electrophoresis. Then, the gel bands were recycled. Third, the P2X3 fragment and linear pEGFP-C1 were connected. The reaction solutions included pEGFP-C1, 1 μl; P2X3 fragment, 5 μl; 10×ligation buffer, 1.5 μl; T4 DNA ligase, 1.5 μl; 10×BSA,1.5 μl; and ddH2O, 4.5 μl. The solutions were then placed at 16℃ overnight. The ligation products were transformed into Escherichia coli cells, and positive clones were sequenced by the Nanjing Genscript Company to ensure the success of the construction of the recombinant plasmid. After the recombinant plasmid was extracted and digested, agarose gel electrophoresis of the ligation products was used to determine the success of the cloning. If 1.2-kb and 4.7-kb bands were observed, it preliminarily indicated that the P2X3 gene fragment was successfully cloned into the pEGFP-C1 vector.
2.5. HEK 293 cell culture and transfection HEK 293 cells were grown in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were transiently transfected with the wild-type and mutant hP2X3 plasmids using Lipofectamine 2000 7 / 28
reagent (Invitrogen) according to the manufacturer’s instructions. When HEK 293 cells were 70–80% confluent, the cell culture media was replaced with OptiMEM 2 hours before transfection. The transfection medium was prepared as follows: (a) 4 µg plasmid DNA was diluted into a 250 µl final volume of OptiMEM; (b) 10 µl lipofectamine 2000 was diluted into a 250 µl final volume of OptiMEM; and (c) the lipofectamine-containing solution was mixed with the plasmid-containing solutions and incubated at RT for 20 min. Subsequently, 500 µl of the cDNA/lipofectamine solution was added to a 35 mm well. Cells were incubated for 6 h at 37°C, 5% CO2. After incubation, cells were washed in MEM containing 10% FBS and incubated for 24-48 h. GFP fluorescence was assessed as a reporter to determine the transfection efficiency, which was detected under a fluorescence microscope by calculating the percentage of positive cells. Whole-cell patch clamp recordings were performed 1-2 days after transfection.
2.6. Whole-cell patch clamp recording Electrophysiological recording was performed using a patch/whole cell clamp amplifier (Axopatch 200B). HEK 293 cells expressing green fluorescence were subjected to electrophysiological recording. Microelectrodes were filled with an internal solution (in mM) composed of K gluconate 145, EGTA 0.75, HEPES 10, CaCl2 0.1, MgATP 2, Na3GTP 0.3. The bath was continuously perfused with an extracellular solution containing (in mM) NaCl, 126; KCl, 2.5; glucose, 10; MgCl2, 1.2; CaCl2, 2.4; and NaHCO3 18. The osmolarity of the extracellular and internal solutions was adjusted to 340 mOsm using sucrose, the pH of the extracellular solution was adjusted to 7.4 using NaOH and the pH of the internal solution was adjusted to 7.3 using KOH. The resistance of the recording electrodes was 2-6 MΩ. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1-10 GΩ), and then, a more negative pressure was applied to rupture it. The holding potential (HP) was set to -60 mv. ATP and puerarin were diluted in the external solution and rapidly applied through a manifold comprising 10 capillaries made of fused silica coated with polyimide with a 200 μm internal diameter. The distance from the tubule mouth to the cell examined was approximately 100 μm. The solutions were 8 / 28
delivered by gravity flow from independent reservoirs. One barrel was used to apply a drug-free external solution to enable rapid termination of drug application. Data were low-pass filtered at 2 Hz, digitized at 5 kHz and stored on a laboratory computer using the Digidata 1200 interface and pClamp10.0 software (Axon instruments). A concentration response curve was constructed by Sigmaplot 12.0 software. Traces were acquired using pClamp software and plotted using Origin 8 (Microcal, Northampton, MA, USA).
2.7. Data analysis Statistical analysis of the data was performed using SPSS 11.5 software. Statistical significance was determined by an unpaired t-test between two groups in the electrophysiological experiments. P<0.05 was considered statistically significant.
3. Results 3.1. Molecular docking of puerarin and P2X3 The interaction between puerarin and the hP2X3 protein was analyzed by Autodock 4.2 (molecular docking software). The three dimensional structure files of ATP (CID5957) and puerarin (CID 5281807) were downloaded from the PubChem database, and Utra ChemBioDraw 11 and Chimera1.6.1 software was used to convert the format. The Lamarckian genetic algorithm (Lamarckian genetic algorithm selection, LGA) was used for molecular docking. The flexible sites of the hP2X3 protein were Lys-63, Lys-65, Phe171, Thr172, Asn-279, Arg-281, Lys-299. The molecular docking results are shown in Figure 1. A structure of puerarin was as Fig. 1A. Puerarin binds well with the hP2X3 protein in the vicinity of the ATP binding pocket and its surrounding residues and forms hydrogen bonds (Fig. 1 B, C and D). Score MOE software analysis showed that the optimal docking energy of puerarin with hP2X3 was -6.1829 kcal/mol, greater than the -3.81kcal/mol of ATP with hP2X3. It is speculated that puerarin binding to hP2X3 prevents ATP from binding with hP2X3, increasing the concentration of free ATP and inhibiting the activity of hP2X3 protein channels.
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3.2. Protein-ligand docking score of the selected P2X3 mutation site The ligand poses process of the Discovery Studio 3.5 software was used to analyze the interaction between receptor (such as P2X3) and ligand (such as ATP or puerarin). We chose P2X3 mutation site according to the close contact (Van Edward force collision) heatmap. Using the molecular docking module of Studio LibDock's Discovery, the interaction between P2X3 molecule (before and after the mutation) and ligand (ATP or puerarin) was scored. GLY130, VAL64, THR82, and GLN85 P2X3 mutants were constructed and docked with ATP or puerarin. Figure 2 shows that the VAL64 mutation had the maximum effect on the molecular docking results. The docking scores of the V64A mutation with ATP (Figure 2A) and puerarin (Figure 2B) were 154.924 and 122.257, respectively. The results indicate that the V64A mutation reduces the effects of P2X3 and puerarin and has little effect on the interaction between P2X3 and ATP.
3.3. Results of recombinant plasmid construction and the transfection efficiency of HEK 293 cells The P2X3 fragment of the PCR product was detected using agarose gel electrophoresis, and a band of approximately 1.2 kb was observed in the GIS gel system (Figure 3A). The length of the band was consistent with the P2X3 fragment. The P2X3 gene fragment was then cloned into the pEGFP-C1 vector. pEGFP-C1-P2X3 positive clones were identified by EcoR I and Bgl II, cutting out vector bands of approximately 4.7 kb and target fragment bands of approximately 1.2 kb (Fig. 3B). It was initially speculated that the P2X3 was cloned into the pEGFP-C1 vector. The experimental results are shown in Figure 3B. Positive clones were sequenced, and the results demonstrated that the pEGFP-C1-P2X3 was correctly constructed (P2X3 gene sequence number NM_002559.3) (Fig. 4). The efficiency of HEK 293 transfection with the wild-type and mutant P2X3 plasmids was greater than 80% (Fig. 5).
3.4. Effects of puerarin on the ATP-activated current in HEK 293 cells transfected with the wild type or mutant P2X3 10 / 28
Electrophysiological experiments were recorded 24 hours after transfection. Figure 6A shows that ATP-activated currents in HEK 293 cells transfected with the wild-type (WT) P2X3 and mutant P2X3 exhibited no differences. When applied with puerarin and ATP, ATP-activated currents were significantly decreased compared to only application of ATP in HEK 293 cells transfected with wild-type P2X3. In HEK 293 cells transfected with the mutant P2X3 plasmid, the inhibitory effect of puerarin on the ATP-activated current was not obvious. Figure 6B shows that the inhibitory effect of puerarin on the ATP-activated current in the wild-type and mutant P2X3 was increased in response to the upregulation of the puerarin concentration. The inhibition rate of the mutant was reduced compared to the wild type. These results suggest that the mutation site of the P2X3 plasmid is the active site of puerarin and P2X3.
4. Discussion The crystal structure of the zebrafish P2X4 receptor is known and was used as a template to construct P2X3 structure using homology modeling[20-23]. When the sequence similarity between the target protein and the template protein is greater than 30%, the two proteins may be homologous. The length of the P2X subunit was between 379 to 595 amino acids, and 35%-54% of the sequence was homologous. Therefore, it is feasible to use the zebrafish P2X4 receptor to construct a model of the human P2X3. The structure of P2X3 was solved in our laboratory using homology modeling. Molecular simulation can be used to study the interactions between receptor proteins and small active molecules. Molecular docking is an important method of molecular simulation, involving simulation of the interaction between two or more molecules using bioinformatics methods[16]. Virtual screening of specific drug targets can provide insight for further experimental validation, avoiding the randomness of drug target screening. In this study, we established a model of P2X3 protein structure that was subsequently used in molecular docking simulations with puerarin. The results revealed that there was an interaction between puerarin and hP2X3. Puerarin bound well with the hP2X3 protein in the vicinity of the ATP binding pocket and its surrounding residues. The optimal docking energy of puerarin with the hP2X3 protein was greater than that of ATP with the hP2X3 protein, suggesting that 11 / 28
puerarin prevents ATP from binding with P2X3. Therefore, the molecular docking method can be used to determine the binding site of puerarin and P2X3. This method is helpful for exploring the molecular mechanism of P2X3 inhitbited by puerarin. Studying the role of small molecules derived from natural drugs in the active sites of receptor proteins is helpful to understand the specific role of small molecule ligands in the protein binding pocket and surrounding residues[24, 25]. Using Discovery Studio 3.5 software, we analyzed the interactions between P2X3 protein and ATP or puerarin[33-35] and subsequently selected the P2X3 mutation sites. By comparing and analyzing the amino acid sequence of P2X3 and its 3D structure, we determined the possible amino acid sites that exerted an effect on puerarin binding in the active center of P2X3. In this experiment, the Studio LibDock's Discovery molecular docking module was used to determine the docking score of the wild-type and mutant P2X3and ligands (ATP or puerarin)[33-35]. Mutants of GLY130, VAL64, THR82, and GLN85 in P2X3 were constructed and docked with ATP or puerarin. The results demonstrated that the V64A mutation had the maximum effect on molecular docking results. The docking scores of the V64A mutation with ATP and puerarin were 154.924 and 122.257, respectively, suggesting that the V64A mutation reduces the effect of puerarin on P2X3 and has little effect on the interaction between ATP and P2X3. Hence, the V64A mutation of P2X3 was selected for the next experiment in which a fixed point mutation of P2X3 was designated by overlapping extension PCR[26, 27, 29], and the sequence of the point mutation was identified. Compared to the one-step PCR method, the shortcomings of overlapping extension PCR are that obtaining mutant proteins requires two pairs of primers for two PCR reactions. However, its advantage is that mutants obtained by two PCR reactions can be entirely separated from the original template, and the mutation rate is nearly 100%. This method reduces the workload of the screening process after mutation[26, 27, 36]. So overlapping extension PCR is a simple and effective method for site-directed mutagenesis. SDM can be used for insertion, deletion or point mutation of specific sites of target DNA to affect the traits and characteristics of the target protein[15, 26, 27]. SDM is an effective means to study the relationship between protein structure and 12 / 28
function. Using SDM technology to accomplish site-directed mutation of P2X3, we observed the interaction between puerarin and P2X3[28, 29]. Wild-type and mutant P2X3
plasmids were
successfully transfected into HEK 293 cells.
The
electrophysiological experimental results demonstrated that the ATP-activated current in both groups of HEK 293 transfected cells was not significantly different. Puerarin significantly reduced ATP-activated currents in HEK 293 cells transfected with the wild-type P2X3 plasmid but not in HEK 293 cells transfected with the mutant P2X3 plasmid. At the same time, we observed that inhibition of the ATP-activated current in the wild-type and mutant P2X3 by puerarin was increased with the increasing puerarin concentration, but the inhibitory rate in the mutant P2X3 was significantly lower than in the wild type. These experimental results indicate that the site-directed mutation of V64A significantly affects ATP-activated currents in HEK 293 cells transfected with mutant P2X3. Therefore, the V64A mutation of P2X3 is important for puerarin binding to P2X3, providing an experimental model to further explore the molecular mechanisms of the inhibition of P2X3 by puerarin.
5. Conclusions We used molecular docking to identify the V64A mutation and performed cell transfection and whole cell patch clamp experiments to verify the role of the mutation site. The point mutant V64A reduced the interaction between puerarin and the P2X3 but had little effect on the interaction between ATP and the P2X3. Therefore, position 64 (as shown by the V64A mutation) is an important site for the inhibition of P2X3 by puerarin.
Author’s contributions Participated in research design: Shangdong Liang Conducted experiments: Shuangmei Liu, Mengke Wang, Na Wang, Lin Li Performed data analysis: Shuangmei Liu, Shizhen Li, Rui Sun, Jingming Xing Wrote or contributed to writing of the manuscript: Shuangmei Liu, Yueying Wang, Shangdong Liang
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Conflicts of interest The all authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by grants (№s: 81701114, 81870574, 81570735, 81970749, 31560276) from the National Natural Science Foundation of China, and grant (№: 8181101216) from International Cooperation project between NSFC and DFG.
Data availability The data used to support the findings of this study are included within the article. Additional data can be requested by e-mail: [email protected]
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Figure Legends. Figure 1. Molecular docking of puerarin and hP2X3. A shows the chemical structure of puerarin. Simulation of puerarin docking with hP2X3 was performed by a computer. ATP-binding sites were located at opposite sites of the same subunit and were therefore only able to form a binding pocket at the interface of two adjacent subunits (B, C). Puerarin interacts with the hP2X3 protein near the ATP-binding pocket and forms hydrogen bonds with Thr155, Trp152 and Arg281 (C, D). 20 / 28
Figure 2. Docking score of the V64A mutation of P2X3 with ATP and puerarin. The scores of P2X3-ATP (A) and P2X3-puerarin (B) are 154.924 and 122.257, respectively.
Figure 3. Enzyme identification results of pEGFP-C1-P2X3. A. Gly electrophoresis image of P2X3 PCR product. M: DNA Marker IV; 1: PCR product. B. Identification chart of the recombinant pEGFP-C1-P2X3 plasmid digested by double enzymes. M: DNA Marker IV; 1: bands of pEGFP-C1-P2X3 after double enzymatic digestion.
Figure 4. Comparison results of the gene sequence of the positive clone of pEGFP-C1-P2X3. The pEGFP-C1-P2X3 plasmid was correctly constructed.
Figure 5. Detection of the transfection efficiency in HEK 293 cells transfected with the P2X3 plasmid. The left image shows all cells. The right image shows cells expressing green fluorescent protein that were successfully transfected with the pEGFP-P2X3 plasmid. The percent of green cells (right) of all cells (left) is the transfection efficiency. The efficiency of HEK 293 transfection with the wild-type and mutant P2X3 plasmids was greater than 80%.
Figure 6. Inhibitory effects of puerarin (Pue) on ATP-induced currents in HEK 293 cells transfected with the wild-type or the mutant hP2X3. (A) Original tracings showing that puerarin (10 µM) mostly inhibited ATP (100 µM)-induced currents in the wild-type hP2X3 but had little effect on the mutant receptor. (B) Graph showing the dose–response curve of ATP-activated currents in the wild-type and mutant hP2X3 in response to different concentrations of puerarin. The inhibitory effects of puerarin on ATP-induced currents in HEK 293 cells transfected with the wild-type hP2X3 was stronger than those transfected with the mutant receptor at the same concentration of puerarin. *P < 0.05, **P < 0.01, compared to the wild-type hP2X3 using the unpaired t-test.
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Highlights
• Molecular docking revealed puerarin binds well to hP2X3 protein. • Puerarin interacts with hP2X3 protein near ATP-binding pocket. • V64A mutation reduces the effects of the P2X3 receptor and puerarin. • Puerarin inhibits P2X3 receptor by binding to V64A.
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