|Year : 2019 | Volume
| Issue : 3 | Page : 223-231
Homology modeling and docking study of Shewanella-like protein phosphatase involved in the development of ookinetes in Plasmodium
Sandhini Singh, Ruchi Yadav
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus, Lucknow, India
|Date of Web Publication||9-Jul-2019|
Assistant Professor, Amity Institute of Biotechnology, Amity University Lucknow, Lucknow 226028, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Parasites of the genus Plasmodium cause a great deal of morbidity and mortality worldwide, largely in regions with limited access and indication to the tools necessary to control mosquito populations and to treat human infections of malaria. Five species of this class of eukaryotic pathogens cause different human diseases, with Plasmodium falciparum alone infecting approximately 500 million people per year and resulting in approximately one million deaths. Materials and Methods: The two genes encoding the Shewanella-like protein phosphatases of P. falciparum, SHLP1 and SHLP2, are conserved among members of Plasmodiidae family. SHLP is frequently found in asexual blood stages and expressed at all stages of the life cycle of parasite. SHLP deletion results in a reduction in microneme formation, ookinetes (zygote) development, and complete ablation of oocyst formation, thereby blocking transmission of parasite. Structure modeling of SHLP protein can be helpful in understanding the active site and binding site information and hence can be used for drug designing and for therapeutics against malaria. Study of SHLP and its variants was carried out using UniProtKB database. Homology modeling was performed using Schrödinger software, and the modeled structure was verified using Ramachandran plot. Ten antioxidants were searched in PubChem database for docking and comparative analysis. Docking was carried out against SHLP-modeled protein, and the ligand–protein interaction map was analyzed. Effective role of resveratrol was studied against SHLP protein using docking method to identify protein–ligand interaction scheme and bond formation. Results: SHLP protein was modeled and docking was carried out to identify the binding sites and interaction with the SHLP protein. Docking study suggested that resveratrol has a strong interaction with SHLP protein and can be used as a potential ligand for drug designing. Conclusion: SHLP plays a crucial role in ookinetes and microneme development in Plasmodium; hence ligand, which can interact and inhibit SHLP protein, can be a potential drug against malarial parasite development. We studied the binding of antioxidant, such as resveratrol, with this protein-using docking method and it was found that resveratrol as an antioxidant can bind with the target SHLP protein.
Keywords: Malaria, microneme, molecular docking, resveratrol, Shewanella-like protein phosphatase, Schrödinger software
|How to cite this article:|
Singh S, Yadav R. Homology modeling and docking study of Shewanella-like protein phosphatase involved in the development of ookinetes in Plasmodium. J Pharm Bioall Sci 2019;11:223-31
|How to cite this URL:|
Singh S, Yadav R. Homology modeling and docking study of Shewanella-like protein phosphatase involved in the development of ookinetes in Plasmodium. J Pharm Bioall Sci [serial online] 2019 [cited 2019 Aug 22];11:223-31. Available from: http://www.jpbsonline.org/text.asp?2019/11/3/223/262192
| Introduction|| |
The unicellular parasites of the genus Plasmodium are the causative agents of malaria, and result in annual fatalities close to 1.25 million. Malaria is caused by infection with the apicomplexan parasite Plasmodium, which is transmitted via the female Anopheles mosquito, and in 2012, it resulted in approximately 207 million clinical infections and over 600,000 deaths. The life cycle of Plasmodium progresses through different morphologically distinct stages of development, including asexual proliferation in hepatocytes, followed by clinically overt intraerythrocytic multiplication in the vertebrate host.
Ingestion of developmentally arrested gametocytes triggers the development of sexual stage of parasites in the mosquito. This leads to eventual migration of parasites to the salivary glands and transmission during process of feeding. During each stage, the parasite uses and controls a number of signal transduction mechanisms, including reversible protein phosphorylation catalyzed by protein kinases (PKs) and protein phosphatases (PPs). This signaling mechanism is a conserved, ubiquitous regulatory process for many prokaryotic and eukaryotic cellular pathways. Although PKs are well recognized as important therapeutic targets, PPs are only now emerging as targets for the purpose of clinical intervention.
Shewanella-like protein phosphatase
Mammalian Plasmodium development process initiates species proceeds via asexual exoerythrocytic proliferation and intraerythrocytic multiplication that exist in mammalian liver hepatocytes and erythrocytes, respectively, whereas sporogony and sexual development take place in the mosquito.Plasmodium is related to the phylum Apicomplexa, which is characterized and indicated by the presence of distinct apical organelles consisting of micronemes, dense granules, and rhoptries that are used by the parasite for gliding motility and host invasion. Of the three invasive stages (sporozoites, merozoites, and ookinetes), the ookinetes uniquely lack rhoptries and dense granules.
There are two nonconventional PPs, one of them containing an N-terminal β-propeller formed by kelch-like motifs and the other a Shewanella-like PP (SHLP1), which are required during ookinetes-to-oocyst transition and subsequent transmission of the malaria through P. berghei. Recently, researchers have shown that SHLP2 (second SHLP member of family) has function in dephosphorylation of the host proteins, which results in invasion of oocyst in the erythrocytes during the merozoites phase of parasite development. In eukaryotes other than in Apicomplexa, SHLPs have been found in Archaeplastida, some fungi, and some Chromalveolates and have been structurally related to a segment of bacterial phosphoprotein phosphatase, those which have been identified first in the psychrophilic bacteria Shewanella and Colwellia. Studies has reported that there are two genes present in the genome of P. berghei, that encodes for Shewanella-like PPs. Genes which has signal peptide and apicoplast targeting sequence are known as Shewanella-like PPs (SLPs) and other genes that lacks these signal sequences are termed as Shewanella-like PPs (SHLP1 and SHLP2).
The analysis of Plasmodium SHLPs explained that only the highly conserved serine/threonine PPs (STP) catalytic residues and binding sites of metal ions are largely conserved. The rest of the conserved STP residues, okadaic acid, and microcystin inhibitor–binding sites, as well as PP1 regulatory subunit-binding sites and PP1 substrate-binding sites are only partially or not conserved. SHLP1 plays an important (though not essential) role at an early stage in ookinetes development and differentiation. SHLP1 in Plasmodium is essential for parasite transmission via the mosquito and thus, it is a potential target for the development of transmission-blocking drugs. Ookinetes-to-oocyst transition represents one of the biggest bottlenecks in the life cycle of the malarial parasite, and SHLP1 plays a crucial role in this current process.
Potential inhibitors of Shewanella-like protein phosphatase
Reversible protein phosphorylation is of central importance in the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of PKs and PPs, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations.
An antioxidant is a molecule that inhibits the oxidation of other molecules. The term “antioxidant” is mainly used for two different groups of substances: first group of antioxidants are used by food industry, these industries use antioxidant as food preservatives, and these antioxidants are called as synthetic antioxidant, for example, like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), and second group of antioxidants are naturally occurring, found in foods and body tissue, for example, tocopherol and ascorbic acid; these antioxidants have beneficial health effects. Resveratrol, a natural product, is known to affect a broad range of intracellular mediators and also to possess some antioxidant activity.
Resveratrol (3, 5, 4′-trihydroxy-trans-stilbene) is a stilbenoid, a type of natural phenol, and a phytoalexin produced by several plants in response to injury or when the plant is under attack by pathogens such as bacteria or fungi. Sources of resveratrol in food include the skin of grapes, blueberries, raspberries, mulberries, lingonberry, and senna. Resveratrol is produced in plants by the action of the enzyme, resveratrol synthase.
| Materials and Methods|| |
Study of SHLP protein and its variants was carried out using UniProtKB database (http://www.uniprot.org/). Homology modeling was performed using Schrödinger software suite (version 10.4.018; Schrödinger Software, New York, NY), and the modeled structure was verified using Ramachandran plot.
Ten antioxidants were selected from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) for interaction analysis with the SHLP proteins. All modeled proteins were docked with the 10 ligands using Glide Dock program of Schrödinger software suite. Docking results were analyzed and protein–ligand interaction map was studied to identify the best antioxidant against SHLP protein.
SHLP protein was searched in UniProt database, and two types of SHLP proteins were found, SHLP1 and SHLP2. [Table 1] lists the details of SHLP protein, UniProt ID, gene name, and source organism.
|Table 1: List of SHLP protein identified from UniProt database with UniProt ID|
Click here to view
Ligands for docking
Ligands were retrieved from PubChem database for interaction analysis with modeled SHLP protein. [Table 2] lists the details of antioxidant ligands used for docking with the homology modeled protein (modeled protein A, B, C, and D).
|Table 2: List of antioxidants used as ligands retrieved from PubChem database. These ligands were used for docking|
Click here to view
| Results and Discussion|| |
Homology modeling using Schrödinger software
SHLP1 is an ancient bacterial protein also found in fungi, protists, and plants. SHLP1 protein in malarial parasite Plasmodium is involved in ookinetes (zygote) development and microneme formation, and it is found abundantly in asexual stages of parasitic life cycle. In this paper, we have predicted the structure of SHLP protein by homology modeling. Structure conservation of SHLP protein was also studied in different species of Plasmodium.
Two variants of SHLP protein (SHLP1 and SHLP2) were found in four different species of Plasmodium (Plasmodium malariae, P. ovale, P. reichenowi, and P. berghei), which were retrieved through UniProt database. Sequence of these proteins was retrieved from UniProt database and shown in [Table 3].
Homology modeling of all the four proteins was carried out using Schrödinger software suite. [Table 3] shows the list of SHLP protein. Nomenclature A, B, C, and D were used for the four SHLP proteins for easy discussion and result representation. Two templates with Protein Data Bank (PDB) IDs 1V73 and 2Z72 were identified to model the four proteins of SHLP (protein A–D). Template protein used for homology modeling was of the protein—tyrosine phosphatase—of a psychrophile Shewanella. Four SHLP protein structures were modeled using these templates, and they are shown in [Figure 1], [Figure 3], [Figure 5], [Figure 7], which are of proteins A, B, C, and D, respectively., , ,
Structure verification of these proteins was carried out using Ramachandran plot analysis, and are shown in [Figure 2], [Figure 4], [Figure 6], [Figure 8], which are of proteins A, B, C, and D, respectively. Ramachandran plot analysis of all the four modeled proteins (A–D) shows that approximately 79%–96% residue lies in the favored region, which can be further used for docking analysis., , ,
Modeled structure of protein A (SHLP2, UniProt ID: A0A1C3KEC2)
Modeled structure of protein B (SHLP2, UniProt ID: A0A1D3TM00)
Modeled structure of protein C (SHLP2, UniProt ID: A0A060RV45
Modeled structure of protein D (SHLP1, UniProt ID: A0A1C6X2Z2)
[Table 4] shows the Ramachandran plot analysis of all the modeled proteins. Comparative analysis shows that modeled proteins A and C have 96% and 94% residues, respectively, in the favored region and are best models of the SHLP protein. Modeled proteins B and D have 75.2% and 79% residues, respectively, in the favored region and only 0.4% and 2.1% residues, respectively, in the disallowed region, this signifies that these modeled structures can also be used for the docking and interaction prediction, as only few residues belongs to the disallowed region.
Structural detail of all the four homology modeled proteins (A–D) shows that SHLP protein structure is highly conserved among different species of Plasmodium. Structural analysis of all the four SHLP proteins signifies that these proteins are mostly helices protein with 60% helices, 20% beta sheets, and 20% belongs to loops and turns secondary structures. These homology modeled structures were used for docking studies to identify the potential inhibitor against SHLP protein.
For docking, we have used resveratrol compound, which is a potential antioxidant, to study its interaction with the SHLP protein. Resveratrol ligands were retrieved from PubChem database and two-dimensional structures were used for docking. Resveratrol is a polyphenolic compound, which has been derived from plants. It activates sirtuin1 (an enzyme, which deacetylates the proteins that contribute to the regulation of cells), which further binds to peroxisome proliferator–activated receptor-γ coactivator-1 alpha (a transcriptional coactivator that takes care of biogenesis and functioning of mitochondria) and activates it by the process of deacetylation.
First step in docking was to identify binding sites in the target protein for grid map generation. Binding site was predicted in all the four homology-modeled structures (proteins A–D) by using SiteMap tool of Schrödinger software suite, and applying default parameters. Five binding sites were predicted for each protein, and the binding site with maximum score was used for grid generation. Grid file was generated using grid generation program of Schrödinger software and further used for Glide docking. Docking generates the pose-viewer file; this file was used for visualization of interaction between ligand and protein.
Docking results were validated using LigPlot tool of Schrödinger software and interaction map was studied to identify the nature of interactions such as hydrogen bonds, pi–pi interaction, side chain bond, and backbone hydrogen bonds. Ligand–protein interaction map was also used to study the position and the interacting amino acids with the target protein.
[Table 5] shows the docking result of best ligands with the four SHLP proteins. SHLP protein A (SHLP2 [A0A1C3KEC2]) shows the best interaction with ligand Pinostilbine, with the GlideScore of –6.80. Protein–ligand interaction map shows that Pinostilbine makes four hydrogen bond at positions Val 74, Leu 72, Ser 285, and Gln 288. Protein B (SHLP2 [A0A1D3TM00]) has the best interaction with the ligand resveratrol tripalmitate with the Glide energy of –7.72. Resveratrol tripalmitate makes two hydrogen bond at positions Val 309 and Thr 76 along with one pi–pi interaction at position Phe 190 with protein B.
|Table 5: Docking result of SHLP proteins with the ligands. Ligand–protein interaction map of the best ligand with each SHLP protein|
Click here to view
Protein C (SHLP2 [A0A060RV45]) also shows best interaction with the resveratrol tripalmitate with the Glide energy of –6.308. This ligand shows four hydrogen bond at positions, Asn 127, Tyr 130, Asp 87, and Ile 16. Protein D (SHLP1 [A0A1C6X2Z2]) also shows the same result as protein B and C, it has the best interaction with the resveratrol tripalmitate, with the GlideScore of –7.24. Protein–ligand interaction map of resveratrol tripalmitate shows that protein D makes two hydrogen bonds at positions, Tyr 156 and Lys 235, and it also makes one pi–pi interaction at position Trp 250.
Docking result shows that resveratrol tripalmitate has a strong binding to SHLP protein. Ligand–protein interaction map as shown in [Table 5] shows that resveratrol tripalmitate makes hydrogen bonds with SHLP protein. Hence, resveratrol can be a potential drug against malaria, which is caused by the development and proliferation of ookinetes in Plasmodium.
| Conclusion|| |
In most organisms, differentiation and development processes involved in cell cycle are regulated by proteins of reversible phosphorylation. Although kinases are well identified as basic targets for drugs, the SHLP’s study reports have only recently begun to identify them as therapeutic targets of potential activity. Variants of SHLP from different Plasmodium species were studied and their structures were modeled using Schrödinger software suite. Template of tyrosine phosphatase of a psychrophile Shewanella was used for homology modeling of all the four SHLP proteins. Researches have shown that SHLP plays a crucial role in ookinetes and microneme development in Plasmodium; hence, any ligand, which can play the role of antagonist for these proteins, can be regarded as a potential drug against malarial parasite development. We established the binding of antioxidant, such as resveratrol, with SHLP protein through in silico–based docking method. Docking result signifies that many of the antioxidants were able to bind with the target protein.
We also analyzed the binding interaction and the types of bonds formed with the modeled SHLP protein. Docking result states that resveratrol and its derivatives such as resveratrol tripalmitate can bind with the target SHLP protein. This study can be helpful in designing drugs against SHLP protein by the use of resveratrol as potential ligand against malaria disease. This study also established that resveratrol and its other derivatives can be significant lead molecule that can be used for drug development process.
We acknowledge Amity University Uttar Pradesh, Lucknow campus, Lucknow, India, for providing the Schrödinger Software in Bioinformatics Lab, to perform the docking studies included in this paper. We also thank other bioinformatics databases and tools, which are used to conduct this research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]