An in silico approach for structural and functional analysis of heavy metal associated (HMA) proteins in Brassica oleracea

Jasmin Sutkovic, Mujo Kekić, Maida Ljubijankić, Petar Glamočlija


Heavy metal ATPases (HMAs) are the most important proteins involved in heavy metal accumulation process. Brassica oleracea has 5 HMA (1-5) homologues whose 3D structure has been predicted and validated in this study by different bioinformatics tools. Phylogenetic and multiple sequence alignment analyses showed high relationship between HMA2 and HMA4, while two same domains were identified in all five HMA proteins: E1-E2 ATPase and haloacid dehydrogenase (HAD) domain. Four HMA (2-5) proteins were identified to be localized in the plasma membrane, while HMA1 localization is predicted to be in plastid. Interactome analysis revealed high interaction of all HMA (1-5) proteins with many metal ion binding proteins and chaperones. Among these, interesting and strong interaction is observed between all HMA (1-5) proteins and ATX1, while HMA1, HMA2 and HMA4 have been found to strongly interact with FP3 (farnesylated protein 3) and FP6 (farnesylated protein 6) proteins. Docking site predictions and electrostatic potentials between HMA2/HMA4 and the interactome proteins were explained and discussed in this study.


Keywords: Protein structure prediction, Heavy metals; accumulation; transport; interactome; docking site

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A.M. Gardner, A. F. Brown, J. A. Juvik, “QTL analysis for the identification of candidate genes controlling phenolic compound accumulation in broccoli (Brassica oleracea L. var. italica),” Mol Breeding, vol. 36:81, 2014.

M. P. Mourato, I. N. Moreira, L. Leitão et al., “Effect of Heavy Metals in Plants of the Genus Brassica,” International Journal of Molecular Sciences, ISSN 1422-0067, pp 1-24, 2015.

H. J. Korry, J. W. Finley, A. Sigrid-Keck, R. J. Robbins,“ The Journal of Nutrition, vol. 135, no. 5, 2005.

L. Fengjuan, W.Yongsheng, F. Xiaoling, J. Yucui, W. Zhaojia, H. William, P. Carolyn, L. Lei et al., “A Novel Mechanism of Indole-3-Carbinol Effects on Breast Carcinogenesis Involves Induction of Cdc25A Degradation,” Cancer Prevention Research, vol. 3, no. 7, 2010.

M. W. Farnham and D.A. Kopsell, “Importance of Genotype on Carotenoid and Chlorophyll Levels in Broccoli Heads,” HortScience, vol. 44, no. 5, 2009.

B. Srilakshmi, Nutrition Science. New Age International. ISBN 978-81-224-1633-6, pp. 186–7, 2006.

S. Christensen, R. von Bothmer, P.Gert et al., “AFLP analyses of genetic diversity in leafy kale (Brassica oleracea L. convar. acephala (DC.) alef) landraces, cultivars ad wild population in Europe,’’ Genet Reseour Crop Evol, vol. 58, 2010.

G.A. Boamponsem, M. Kumi and I. Debrah, “Heavy Metals Accumulation In Cabbage, Lettuce And Carrot Irrigated With Wastewater From Nagodi Mining Site In Ghana,” International Journal of Scientific and Technology Research, vol. 1, no. 11, pp. 124-129, 2012.

S. Mohammad, A. Khan, Z.R. Goel and J. Musarrat, “Biomanagement of Metal-Contaminated Soils,” In: Environmental Pollution. Springer Netherlands (online), 2011.

L. Subasic, H. Gavranovic, I. Muhovic and A. Memon, “Heavy metal induced gene expression in Brassicaceae,” Department of Genetics and Bioengineering, Faculty of Engineering and Information Technologies, International Burch University, BiH, 2012.

M. Cho, C.N. Agnes and D. Karl-Josef, “Differential heavy metal tolerance of Arabidopsis halleri and Arabidopsis thaliana: a leaf slice test,“ The New Phytologist, vol. 158, no. 2, pp. 287-293, 2002.

V. Shanmugam, Lo, Jing-Chi and Kuo-Chen. Yeh, “Control of Zn uptake in Arabidopsis halleri: a balance between Zn and Fe,” Frontiers in Plant science, vol. 4, pp. 281, 2013.

X. Sun, G.Yu, Jing-Tao. Li et al., “A Heavy Metal-Associated Protein (AcHMA1) from the Halophyte, Atriplex canescens (Pursh) Nutt., Confers Tolerance to Iron and Other Abiotic Stresses When Expressed in Saccharomyces cerevisiae,” Int J Mol Sci., vol. 15, no. 8, pp. 14891–14906, 2014.

J. M. Argüello, E. Eren, M.González-Guerrero, “The structure and function of heavy metal transport P1B-ATPases,” M. Biometals, vol. 20, 2007.

E. Eren and José M. Argüello, “Arabidopsis HMA2, a Divalent Heavy Metal-Transporting PIB-Type ATPase, Is Involved in Cytoplasmic Zn2+ Homeostasis,” Plant Physiol., vol. 136, no. 3, pp. 3712–3723, 2004.

P. Ashton and K. Leon, “Identification of Thlaspi caerulescens Genes That May Be Involved in Heavy Metal Hyperaccumulation and Tolerance. Characterization of a Novel Heavy Metal Transporting ATPase, “ Plant Physiology, vol. 136, no. 3, pp. 3814-3823, 2004.

K. Ute, I. N. Talke and M. Hanikenne, “Transition metal transport,“ FEBS Letters, vol. 581, no. 12, pp. 2263–2272, 2007.

O. Siemianowski, A. Barabasz, M. Kendziorek, E. Bulska, L. E Williams and D.M. Antosiewicz, “HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier,“ J Exp Bot., vol. 65, no. 4, 2014.

A. Gravot, A. Lieutaud, F. Verret, P. Auroy, A. Vavasseur and P. Richaud, “AtHMA3, a plant P1B-ATPase, functions as a Cd/Pb transporter in yeast,” FEBS Lett., vol. 561, 2004.

Y. Kobayashi, K. Kuroda, K. Kimura et al., “Amino acid polymorphisms in strictly conserved domains of a P-type ATPase HMA5 are involved in the mechanism of copper tolerance variation in Arabidopsis,” Plant Physiol., vol. 148, no. 2, 2008.

A. S. Colás, V. S. Rodríguez-Navarro et al., “The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots,” The Plant Journal, vol. 45, no. 2, pp. 225–236, 2005.

N. Grotz and M. L. Guerinot, “Molecular aspects of Cu, Fe and Zn homeostasis in plants,” Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, vol. 1763, no. 7, pp. 595–608, 2006.

E.W. Sayers, T. Barrett, S.H. Benson, “Database resources of the National Center for Biotechnology Information,” Nucleic Acids Res., vol. 37, D5-15, 2009.

M. Goujon, H. McWilliam, W. Li et al., “A new bioinformatics analysis tools framework at EMBL-EBI,” Nucleic acids research, vol. 38. Suppl: W695-9, 2010.

A. M. Lesk, Introduction to Bioinformatics. Oxford University Press Inc., New York, USA, 2002.

M.A. Larkin, B. G. Blackshields, N.P. Brown et al., “ClustalW and ClustalX version 2,” Bioinformatics, vol. 23, no. 21, pp. 2947-2948, 2007.

L.A. Kelley and M.J.E. Sternberg, “Protein structure prediction on the web: a case study using the Phyre server,” Nature Protocols, vol. 4, pp. 363 – 371, 2009.

P. Benkert, M. Biasini and T. Schwede, “Toward the estimation of the absolute quality of individual protein structure models,” Bioinformatics, vol. 27, no. 3, 2011.

D. Eisenberg, R. Lüthy and J.U. Bowie, “Assessment of protein models with three-dimensional profiles,” Nature, vol. 356, no. 6364, 1992.

R.A. Laskowski, M.W. MacArthur, D.S. Moss and J.M. Thornton, “PROCHECK - a program to check the stereochemical quality of protein structures,” J. App. Cryst., vol. 2, pp. 283-291, 1993.

L. Liu, Z. Zhang, Q. Mei and M. Chen, “PSI: A Comprehensive and Integrative Approach for Accurate Plant Subcellular Localization Prediction,” PLoS ONE, vol. 8, no.10, 2013.

J. Schultz, F. Milpetz, P. Bork and C.P. Ponting, “SMART, a simple modular architecture research tool: Identification of signaling domains,” Proc. Natl. Acad. Sci., vol. 95, no. 11, pp. 5857-5864, 1998.

D. Szklarczyk, A. Franceschini, S. Wyder et al., “STRING v10: proteinprotein interaction networks, integrated over the tree of life,” Nucleic Acids Res., vol. 43, 2015.

D. Kozakov, D. Beglov, T. Bohnuud et al., “How good is automated protein docking?” Proteins, vol. 81, no. 12, pp. 2159-2166, 2013.

R.L. Cross and V. Muller, “The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H+/ATP coupling ratio,” FEBS Lett., vol. 576, pp. 1-4, 2004.

K. B. Axelsen and M.G. Palmgren, “Evolution of substrate specificities in the Ptype ATPase superfamily,” J Mol Evol., vol. 46, no. 1, pp. 84-101, 1998.

E.V. Koonin and R.L. Tatusov, “Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity. Application of an iterative approach to database search,” J Mol Biol, vol. 244, no. 1, 1994.

I. Letunic, T. Doerks and P. Bork, “SMART: recent updates, new developments and status in 2015,” Nucleic Acids Res., vol. 43, 2015.

H. Zhou and Y. Zhou, “Distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction,” Protein Sci., vol. 11, pp. 2714-2726, 2002.

K. Viehweger, “How plants cope with heavy metals,” Botanical Studies, vol. 55, no. 35, 2014.

H. D. Haydon, M. J. Wang et al., “P-Type ATPase Heavy Metal Transporters with Roles in Essential Zinc Homeostasis in Arabidopsis,” Plant Cell, vol. 16, no. 5, pp. 1327–1339, 2004.

L. A. Kelley et al., “Phyre2 Protein Fold Recognition Server,” Nature Protocols, vol. 10, pp. 845-858, 2015.

P. Benkert, M. Biasini and T. Schwede, “Toward the estimation of the absolute quality of individual protein structure models,” Bioinformatics, vol. 27, no. 3, 2011.

K. B. Axelsen and M.G. Palmgren, “Evolution of substrate specificities in the Ptype ATPase superfamily,” J Mol Evol., vol. 46, no. 1, pp. 84-101, 1998.

B. Olaf, S. Vogt, R. Uhlemann, Z. Wiebke and H. Klaus, “Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29,” Plant Mol Biology, vol. 69, pp. 213–226, 2009.

Braga de Abreu-Neto, A. C. Joao, L.F.V. Turchetto-Zolet et al., “Heavy metal-associated isoprenylated plant protein (HIPP): characterization of a family of proteins exclusive to plants,” The FEBS Journal, vol. 280, no. 7, 2013.

G, Wei, Y.L. Hong, X. Shi and Mee-Len Chye, “Protein interactors of acyl-CoA-binding protein ACBP2 mediate cadmium tolerance in Arabidopsis,” Plant Signal Behav., vol. 5, no. 8, pp. 1025–1027, 2010.

E. Dykema, R. Sipes, A. Marie et al., “A new class of proteins capable of binding transition metals,” Plant Mol Biol., vol. 41, no. 1, 1999.

E. Himelblau, H. Mira, S. Lin et al., “Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis,” Plant Physiol., vol. 117, no. 4, pp. 1227–1234, 1998.

N. J. Robinson and Dennis R. Winge, “Copper Metallochaperones,” Annu Rev Biochem., vol. 79, pp. 537–562, 2010.

S. Lung-Jiun, L. Jing-Chi and Y. Kuo-Chen, “Copper Chaperone Antioxidant Protein1 Is Essential for Copper Homeostasis,” Plant Physiol., vol. 159, no. 3, pp. 1099–1110, 2012.

K.C. Wong, E. Jarvis, S. Renée et al., “Functional analysis of the heavy metal binding domains of the Zn/Cd-transporting ATPase, HMA2, in Arabidopsis thaliana,” New Phytologist Journal, vol. 181, no. 1, pp. 79–88, 2009

A. Heifetz, E. Katchalski-Katzir and M. Eisenstein, “Electrostatics in protein–protein docking,” Protein Sci., vol. 11, no. 3, pp. 571–587, 2002.



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Digital Object Identifier DOI: 10.21533/pen

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