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In silico structural and interactional study of wheat proteins gliadin and

glutenin with comparison to wheat toxin alpha-purothionin

Article · December 2022

DOI: 10.57219/crrls.2022.1.1.0032

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In silico structural and interactional study of wheat proteins gliadin and glutenin with
comparison to wheat toxin alpha-purothionin
Sana Fatima 1, *, Aiman Nayab 1, Abida Bibi 1, Shaista Naz 1, Amjad Ullah Khan 1, Mehak Hayat1, Kalsoom
Begum 1, Ayesha Urooj 2, Muhammad Umer Farooq Qureshi 3 and Saqib Ali Rustam 4
1 Gomal Centre of Biochemistry and Biotechnology, Gomal University, Dera Ismail Khan 29050, Khyber Pakhtunkhwa,
Pakistan.
2 Department of Zoology, Kohat University of Science and Technology, Kohat 26000, Khyber Pakhtunkhwa, Pakistan.
3 Institute of Microbiology, Gomal University, Dera Ismail Khan 29050, Khyber Pakhtunkhwa, Pakistan.
4 Faculty of Veterinary & Animal Sciences, Gomal University, Dera Ismail Khan 29050, Khyber Pakhtunkhwa, Pakistan.

Comprehensive Research and Reviews in Life Sciences, 2022, 01(01), 007–013

Publication history: Received on 05 August 2022; revised on 09 September 2022; accepted on 11 September 2022

Article DOI: https://doi.org/10.57219/crrls.2022.1.1.0032

Abstract
Wheat is most important component of food throughout the world. Wheat is full of different proteins but most abundant
and most studied proteins are gliadin and glutenin. Interaction of gliadin and glutenin is important in biological
properties of the wheat flour. Wheat also produce a toxin known as purothionin, which is used by the plant to show
defense against different types of pathogen attack. Current in silico study was done to investigate the interaction
between gliadin and glutenin proteins against wheat toxin alpha-purothionin solely. Methodology that was used in the
current study consists of RaptorX tool for protein modeling, PROCHECK for predicted 3D model verification. CASTp 3.0
online tool for active site prediction. Online tool Cluspro and offline tool ligplus+v2.1 for protein-protein docking and
visualization of docked complex respectively. Results showed high interaction between gliadin and glutenin, which
supported the results of previous studies. While interaction between gliadin and purothionin proteins was high as
compared to interaction between glutenin and alpha-purothionin proteins. Different helices were noted in the 3D
structure of gliadin and glutenin proteins, where most of the time Gln amino acid was involved in making the helices.
Form the results it has been concluded that greater interaction between gliadin and alpha-purothionin was due to
similar sequence of gliadin with viral protein so the interaction of toxin purothionin against pathogens enhance the
interaction of gliadin with alpha-purothionin. Current in silico study will enhance the understanding about the
interaction of important wheat proteins.

Keywords: Wheat; Gliadin; Glutenin; Alpha-purothionin

1. Introduction
Wheat is a small plant and was originated in Tigris, Asia, and Euphrates river valley (Landriscina et al., 2017). Bread
wheat also known as Triticum aestivum, produces one-seeded fruits, called as grains (Langridge, 2017). The pleasant
and unique flavor and gluten-forming features of wheat products like bread, chapatti, pasta, etc. make them very
attractive among other cereals (Lemmens et al., 2019). Wheat has been grown in areas with moderate temperature and
is primarily used as food since ancient times (Li et al., 2014). Therefore, it is not an exaggeration to say that in the past,
human life in many parts of the world depended on wheat and wheat-based foods (IWGSC, 2018). Both quality and
quantity of wheat flour proteins being important in bread making quality.

* Corresponding author: Sana Fatima

Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
 Comprehensive Research and Reviews in Life Sciences, 2022, 01(01), 007–013

Main wheat storage proteins (endosperms) consists of gluten proteins. The gluten proteins consists of two main
proteins i.e. gliadin and glutenin, which are studied intensively due to their bread making quality. Gluten was identified
by Italian chemist, Beccari long ago, on the basis of wheat’s unique dough-forming properties. Gluten is among the one
of the protein, which was isolated in pure form from wheat endosperms (Lamacchia et al., 2018). Bread wheat, group
Triticeae has five genera namely, Triticum, Elymus, Aegilops, Secale, and Hordeum, in it. There are 7 chromosomes in
Triticum (Bush and Hefle, 1996).

Plants contain different bioactive compounds that are important for their protection against pathogens i.e. fungi and
bacteria. Among these bioactive compounds are purothionins. Purotuionins are small and mainly basic globular
proteins mostly found in the wheat epidermis (Triticum aestivum) (Duponte et al., 2007).

Major feature of purothionins is their harmfulness against many microorganism i.e. yeast, bacteria, and fungi
(Constantin et al., 2008), different animals (Palosuo et al., 2001), cultured cells (mammalian) (Stec et al., 2004) and
larvae of different insects (Sampson et al., 2006). Different in vitro studies have been documented to check the biological
activities of purothionins, but the important biological purpose of purothionins seems to be involved in the defense of
plants against their microscopic predators (Pahr et al., 2012).

Most of the documented biological properties result from the interaction of purothionins with the target cell membrane
(Pahr et al., 2012). This is supported by the statement that some thionins interact with phospholipid bilayers (Hughes
et al., 2000).

Current in silico study was done to investigate the interaction between the two important wheat proteins i.e. gliadin and
glutenin and also to check the interaction of gliadin and glutenin with alpha-purothionin protein solely.

2. Methodology
In current in silico study protein 3D structure of gliadin, glutenin and purothionin was done using RaptorX online tools.
RaptorX is a template-based protein structure modeling server. It requires protein sequence in FASTA format and
provide 3D structure in PDB format (Källberg et al., 2012). Predicted 3D models were verified using PROCHEK tool
(Laskowski et al., 1993), which provide results in the form of Ramachandran plot. 3D structures were visualized using
Chimera1.13.1 (Goddard et al., 2007).

CASTP 3.0 tool was used to predict the active site pockets of all the gliadin, glutenin and purothionin proteins
(Binkowski et al., 2003). Top three active site pockets in the gliadin, glutenin and purothionin proteins were noted. For
protein- protein interaction, another online tool Cluspro (Kozakov et al.,2017) and offline java based software LigPlot+
v2.1 (Laskowski et al.,2011) were used.

3. Results

3.1 Structural studies

Wheat gliadin protein was 290 amino acids long and its sequence was obtained from uniprot database to design its 3D
model. Wheat Alpha-gliadin protein 3D model, consists of coils and helices while no beta-sheets were noted in its 3D
structure.7 seven helices of different lengths were present in the 3D sequence of Wheat Alpha-gliadin protein i.e. Leu25-
Gln29, Gln111-Ser153, Gln164-Gln180, Glu183-Gln211, Pro240-Gln243 and Gln250-Cys269. Most of the time amino
acid Gln was involved in making helices in the wheat gliadin protein. Three Active site pockets were also predicated.
Largest pocket consists of 20 residues, 2 nd largest pocket consists of 17 residues and 3rd largest pocket consists of 12
residues (Table 1). 3D model, active site pockets and verification of 3D structure of gliadin protein through
Ramachandran plot are shown in figure 1.

Wheat Glutenin protein was 660 amino acids long and its sequence was obtained from uniprot database to design its
3D model. Wheat glutenin protein 3D model, also consists of coils and helices, while no beta-sheets were noted in its 3D
structure.17 seven helices of different lengths were present in the 3D sequence of glutenin protein i.e. Ala2-Glu24,
Gln28-Ser38, Leu40-Ala52, Leu55-Trp57, Thr59-Asp71, Ala74-Gln87, Ser121-Val124, Pro224-Gln226,Glu237-Gly239,
Gly272-Gln274, Ala354-Gln356, Pro414-Gln416, Gln473-Gln475, Pro490-Gln492, Ala625-Ala629, Ser631-Ala637 and
Pro640-Gln643,while rest of the amino acids were involved in making coils. Most of the time amino acid Gln was
involved in making helices in the glutenin protein. Three Active site pockets were also predicated. Largest pocket
consists of 29 residues, 2nd largest pocket consists of 20 residues and 3rd largest pocket consists of 36 residues (Table

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1). 3D model, active site pockets and verification of 3D structure of wheat glutenin protein through Ramachandran plot
are shown in figure 2. Both glutenin and gliadin proteins were rich in Gln amino acid.

Figure 1 (a) 3D model of gliadin protein predicted through RaptorX tool. (b) 3D model of gliadin protein showing top
three active site pockets (c) Predicted 3D model verified through Ramachandran plot

Table 1 Active site pockets of Gliadin, Glutenin and Alpha-Purothionin predicted through CASTp tool

Pocket Active site pockets of Gliadin

Size
1st 67Leu,68Pro,69Tyr,70Pro,71Gln,158Ser,159Ser,160Gln,163Gln,205Gln,206Gln,208Gln,209Gln,212Gln,
214Leu,215Ser,247Gln,249Gln,250Gln,253Gln.
2nd 11Pro,13Asn,15Ser,16Gln,17Gln,18Gln,19Pro,20Gln,21Glu,44Tyr,46Gln,47Pro,48Gln, 49Pro, 60Gln,
61Pro, 63Pro.
3rd 126Gln,129Gln,130Gln,133Leu,183Glu,184Gln,185Ser,187Cys,188Gln,274Pro,
276Tyr, 277Cys.
Active site pockets of Glutenin
1st 565Ile,566Gly,568Val,570Gln,594Gln,597Gln,601Gly,618Asp,620Pro,621Tyr,
622His,623Val,627Gln,634Val,638Gln,642Thr,643Gln,644Leu,645Pro,648Cys,
649Arg,650Met,651Glu,654Asp,655Ala,656Leu,657Ala,658Ala,660Gln.
2nd 5Leu,8Phe,9Ala,12Val,15Thr,16Thr,19Gly,20Glu,25Leu,26Gln,27Cys,30Glu,
31Leu,34Ser,37Glu,38Ala,71Asp,72Val,73Ser,74Ala.
3rd 135Arg,136Gln,137Gly,139Tyr,155Pro,156Gly,157Lys,158Trp,160Glu,165Gln,
166Gln,168Tyr,169Pro,170Ser,172Ser,173Leu,174Gln,175Gln,176Pro,
178Gln,180Gln,181Gln,186Lys,189Tyr,191Pro,194Leu,196Gln,199Gln,201Gln,
208Gln,209Gly,212Pro,215His,217His,227Gly,229Gln.
Active site pockets of Alpha-Purothionin
1st 6Ser, 8Leu, 30Arg.
2nd 1Lys, 2Ser, 13Tyr, 22Gln, 33Ile.
3rd 5Arg, 6Ser, 9Gly, 29Cys, 30Arg.

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Figure 2 (a) 3D model of Glutenin protein predicted through RaptorX tool. (b) 3D model of Glutenin protein showing
top three active site pockets (c) Predicted 3D model verified through Ramachandran plot

Figure 3 (a) 3D model of Alpha-purothionin protein (PDB id 2plh) (b) 3D model of Alpha-purothionin protein
showing top three active site pockets (c) Predicted 3D model verified through Ramachandran plot

Alpha-purothionin protein was 45 amino acids long (PDB id 2PLH). Three Active site pockets were also predicated.
Largest pocket consists of 3 residues, 2nd largest pocket consists of 5 residues and 3 rd largest pocket also consists of 5
residues (Table 1). 3D model, active site pockets and verification of 3D structure of Alpha-purothionin through
Ramachandran plot are shown in figure 3.

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3.2 Interaction Studies

Figure 4 (a) Protein-protein interaction between alpha-purothionin and gliadin (b) Protein-protein interaction
between alpha-purothionin and glutenin (c) Protein-protein interaction between glutenin and gliadin

Table 2 Protein-protein interaction between Gliadin, Glutenin and Alpha-Purothionin

Interacting Interacting residues Interacting Interacting Number of

Proteins of Gliadin residues residues of bonds
of Glutenin Purothionin

Gliadin and Gln34,Tyr76, __ Asn11,Arg5, 8

Purothionin Gln150, Lys32,Arg30,
Ser153, Pro75, Gln211 Lys23
Glutenin and __ Gln30 Arg5 1
Purothionin
Gliadin and Tyr289,Asn259,Tyr69,Phe62,Gln58, Ser172,Thr91, ___ 8
Glutenin Gln16, Asn16 Gln67,Gly60,Ser38,
Glu38,Lys3,Val6

Protein-protein interaction between gliadin and glutenin, gliadin and alpha-purothionin and glutenin and alpha-
purothionin was done. Gliadin and glutenin were interacting through 8 hydrogen bonds. Residues that were involved
in making interaction present in gliadin protein were Tyr289, Asn259, Tyr69, Phe62, Gln58, Gln16 and Asn16 and
residues involved in making interactions that were present in glutenin protein were Ser172, Thr91, Gln67, Gly60, Ser38,
Glu38, Lys3 and Val6. Gliadin and alpha-purothionin were interacting through 8 hydrogen bonds. Gliadin protein
residues that were involved in making interaction were Gln34, Tyr76, Gln150, Ser153, Pro75, Gln211 and residues
involved in making interactions in alpha-purothionin protein were Asn11, Arg5, Lys32, Arg30 and Lys23. Glutenin and

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Purothionin were interacting through only 1 hydrogen bond. Residues that were involved in making interaction in
glutenin protein was Gln30 and residue involved in making interactions in alpha-purothionin protein was Arg5 (figure
4). Description of all the interacting residues in three proteins are summarized in table 2.

4. Discussion
Our findings showed high interaction between gliadin and glutenin proteins. Which supported the studty of Lagranin
and his co-workers (2008) that for initial RVA viscosity the interaction of gliadin and glutenin is important. Loss of
hydrogens bonds (break on high temperature) or other conformational changes in the gluten proteins can happens if
the RVA values decreases (Lagrain et al., 2008).

The viscosity rise in the RVA profile at temperatures exceeding 90 °C was caused by formation of large glutenin polymers with
the incorporation of gliadin through SS bonds impacting the rotation of the RVA paddle. The sudden decrease in apparent
viscosity during cooling was due to the protein polymers aggregating tightly and sticking to the paddle caused by the loss of
kinetic energy from heating (Lagrain et al., 2008). The balance and interaction between gliadins and glutenins is responsible
for important rheological properties such as viscosity and elasticity (Gomez et al., 2011).

In 3D models of gliadin and glutenin high ratio of glutamine (Gln) were present in forming helices, studies showed that
amino acids Glu, Gln, Ala, Met and Leu are most often found in helices formation (Krivoshapko and Ivanov, 2015).

Our in silico interaction study showed high interaction between gliadin and alpha-purothionin protein, which supported
the study of Kagnoff et al., 1984. Kangnoff et al., compared the amino acid sequence of adeno virus and alpha-gliadin
proteins and found significant similarity between E1b viral protein and some part of alpha-gliadin protein (Kagnoff et
al., 1984). So the possibility is that the sequence similarly between viral and alpha gliadin protein is responsible for high
interaction between gliadin and alpha-purothionin proteins because the major function of alpha-purothionin protein is
to act against different pathogens including bacteria, fungi and virus. So therefore gliadin and alpha-purothionin protein
showed high interaction as compared to glutenin and alpha-purothionin protein.

5. Conclusion
Current In silico study was done to find out the interaction between different wheat proteins. From the results it has
been concluded that the interaction between purothionin and gliadin protein was higher as compared to interaction
between glutenin and purothionin protein, we conclude that as the gliadin protein resembles with viral protein so the
purothionin, which is actually a toxin against different pathogens, shows highs interaction for gliadin protein.

Compliance with ethical standards

Acknowledgments
We are thankful to our lab fellows for helping us in this research.

Disclosure of conflict of interest

The authors declare that they have no financial as well as competing interests.

References
[1] Binkowski, T. A., Naghibzadeh, S., & Liang, J. (2003). CASTp: computed atlas of surface topography of proteins.
Nucleic acids research, 31(13), 3352-3355.
[2] Constantin, C., Quirce, S., Grote, M., Touraev, A., Swoboda, I., Stoecklinger, A., ... & Valenta, R. (2008). Molecular
and immunological characterization of a wheat serine proteinase inhibitor as a novel allergen in baker's asthma.
The Journal of Immunology, 180(11), 7451-7460..
[3] Dupont, F. M., Chan, R. and Lopez, R. (2007). Molar fractions of high-molecular-weight glutenin subunits are
stable when wheat is grown under various mineral nutrition and temperature regimens. Journal of Cereal Science
45, 134-139.
[4] Goddard, T. D., Huang, C. C., & Ferrin, T. E. (2007). Visualizing density maps with UCSF Chimera. Journal of
structural biology, 157(1), 281-287.

12
 Comprehensive Research and Reviews in Life Sciences, 2022, 01(01), 007–013

[5] Gomez, A., Ferrero, C., Calvelo, A., Anon, M.C., Puppo, M.C. (2011). Effect of mixing time on structural and
rheological properties of wheat flour dough for breadmaking. International Journal of Food Properties, 14, 583–
598.
[6] Bush, R. K., & Hefle, S. L. (1996). Food allergens. Critical Reviews in Food Science & Nutrition, 36(S1), 119-163.
[7] Hughes, P., E. Dennis, M. Whitecross, D. Llewellyn, and P. Gage. 2000. The cytotoxic plant protein, -purothionin,
forms ion channels in lipidmembranes. J. Biol. Chem. 275:823–827.
[8] International Wheat Genome Sequencing Consortium (IWGSC). (2018). Shifting the limits in wheat research and
breeding using a fully annotated reference genome. Science, 361(6403), eaar7191.
[9] Kagnoff, M. F., Austin, R. K., Hubert, J. J., Bernardin, J. E., & Kasarda, D. D. (1984). Possible role for a human
adenovirus in the pathogenesis of celiac disease. The Journal of experimental medicine, 160(5), 1544-1557.
[10] Källberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., & Xu, J. (2012). Template-based protein structure
modeling using the RaptorX web server. Nature protocols, 7(8), 1511-1522.
[11] Kozakov, D., Hall, D. R., Xia, B., Porter, K. A., Padhorny, D., Yueh, C., & Vajda, S. (2017). The ClusPro web server for
protein–protein docking. Nature protocols, 12(2), 255.
[12] Krivoshapko, S. N., & Ivanov, V. N. (2015). Encyclopedia of analytical surfaces. Springer.
[13] Lagrain, B., Thewissen, B. G., Brijs, K., & Delcour, J. A. (2008). Mechanism of gliadin–glutenin cross-linking during
hydrothermal treatment. Food Chemistry, 107(2), 753-760.
[14] Lamacchia, C., Musaico, D., Henderson, M. E., Bergillos-Meca, T., Roul, M., Landriscina, L. Costabile, A. (2018).
Temperature-treated gluten proteins in Gluten-FriendlyTM bread increase T mucus production and gut-barrier
function in human intestinal goblet cells. Journal of Functional Foods, 48, 507–514.
[15] Landriscina, L., D’Agnello, P., Bevilacqua, A., Corbo, M. R., Sinigaglia, M., & Lamacchia, C. (2017). Impact of gluten-
friendlyTM technology on wheat kernel endosperm and gluten protein structure in seeds by light and electron
microscopy. Food Chemistry, 221, 1258–1268.
[16] Langridge, P. (2017). Achieving sustainable cultivation of wheat: Breeding, quality traits, pests and diseases.
Cambridge, UK: Burleigh Dodds Science Publishing Limited.
[17] Laskowski, R. A., & Swindells, M. B. (2011). LigPlot+: multiple ligand–protein interaction diagrams for drug
discovery.
[18] Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK: a program to check the
stereochemical quality of protein structures. Journal of applied crystallography, 26(2), 283-291.
[19] Lemmens, E., Moroni, A. V., Pagand, J., Heirbaut, P., Ritala, A., Karlen, Y.,. . . Delcour, J. A. (2019). Impact of cereal
seed sprouting on its nutritional and technological properties: A critical review. Comprehensive Reviews in Food
Science and Food Safety, 18, 305–328.
[20] Li, M., Zhu, K., Guo, X., Brijs, K., & Zhou, H. (2014). Natural additives in wheat-based pasta and noodle products:
Opportunities for enhanced nutritional and functional properties. Comprehensive Reviews in Food Science and
Food Safety, 13, 347–357.
[21] Pahr, S., Constantin, C., Mari, A., Scheiblhofer, S., Thalhamer, J., Ebner, C., ... & Valenta, R. (2012). Molecular
characterization of wheat allergens specifically recognized by patients suffering from wheat‐induced respiratory
allergy. Clinical & Experimental Allergy, 42(4), 597-609..
[22] Palosuo, K., Varjonen, E., Kekki, O. M., Klemola, T., Kalkkinen, N., Alenius, H., & Reunala, T. (2001). Wheat ω-5
gliadin is a major allergen in children with immediate allergy to ingested wheat. Journal of allergy and clinical
immunology, 108(4), 634-638..
[23] Sampson, H. A., Muñoz-Furlong, A., Campbell, R. L., Adkinson Jr, N. F., Bock, S. A., Branum, A., ... & Decker, W. W.
(2006). Second symposium on the definition and management of anaphylaxis: summary report—Second
National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network symposium. Journal
of Allergy and Clinical Immunology, 117(2), 391-397.
[24] Stec, B., Markman, O., Rao, U., Heffron, G., Henderson, S., Vernon, L. P., ... & Teeter, M. M. (2004). Proposal for
molecular mechanism of thionins deduced from physico‐chemical studies of plant toxins. The Journal of peptide
research, 64(6), 210-224.

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