Comparative study of 7S globulin from Corylus avellana and Solanum lycopersicum revealed importance of salicylic acid and Cu-binding loop in modulating their function

https://doi.org/10.1016/j.bbrc.2019.11.072Get rights and content

Highlights

  • SOD activity of vicilin from Solanum lycopersicum (SL80.1) is not conserved in vicilin from Corylus avellana (HZ.1).

  • SA binding is an important trigger for SOD activity in vicilins.

  • Difference in a critical residue in the co-factor binding site, hinders SA binding in the case of HZ.1 as compared to SL80.1.

  • Copper-binding loop helps in channeling of superoxide radicals to active site of vicilins.

Abstract

The plant seed proteins referred to as vicilins belong to a structurally common superfamily. While some of them are reported to exhibit superoxide dismutase activity, vicilins from other sources do not possess this activity. Vicilin from Corylus avellana (HZ.1) and Solanum lycopersicum (SL80.1) were purified and subjected to structure-function analysis. The superoxide dismutase activity assays were performed to understand the functional differences between them. While SL80.1 has the superoxide dismutase activity, HZ.1 was enzymatically inactive. Crystal structure followed by mass spectrometry analysis of both the proteins revealed that while SL80.1 has bound salicylic acid, HZ.1 does not. Comparison of C-terminal binding pocket of both the structures revealed that a point mutation at residue 321 in HZ.1 (Gly→Cys) leads to obstruction in binding of salicylic acid in the pocket. Similarly, copper-binding loop of HZ.1 was reportedly found to be intact and shorter than the loops reported in SL80.1. The copper-binding loop of SL80.1 is rich in polar residues and the absence of these residues in HZ.1 copper-binding loop possibly indicates deficiency in channeling of oxygen radicals to the active center of the enzyme. Difference in the enzymatic activity of vicilin from two evolutionarily distinct sources is due to mutations in its co-factor binding pocket and copper-binding loop.

Introduction

Globulins constitute a major part of the seed proteome and are classified into 7S globulins and 11S globulins on the basis of their sedimentation rates [1]. The 7S globulins are also termed as vicilins and are distributed widely in the plant kingdom. They were classically known to be an important source of nutrition during the growth and development of the seeds [1], but further studies revealed that they have diverse activities. Vicilins from legumes are reported to bind to sugars and chitin, thus inhibiting the growth of fungi and pests that infect plants [2]. Vicilin from Pisum sativum indicated its role in desiccation tolerance [3]. A more recent study of vicilin from bell pepper (Capsicum annuum) showed that it has superoxide dismutase (SOD) function and its role has been associated with managing oxidative stress [4].

Interestingly, not all vicilins share common function. Of course, structural similarity does not imply same function and various plant proteins (germins, germin like proteins, fatty acid multifunctional proteins, ferredoxin:sulfite reductase, etc.) are already known to exhibit different functions in different species [5]. Similar to these proteins, the ability of vicilins to bind to chitin has only been reported in leguminous plants [6] and the SOD activity of C. annuum vicilin was absent in vicilins isolated from okra (A. esculentus) and cowpea (V. unguiculata) [4]. However, it is not yet clear what makes these proteins functionally diverse. The differences in their function could be possibly because of small differences in their structures, sequences and the environments in which they act [7].

In the present study, we have carried out functional and structural analyses of vicilins from tomato (S. lycopersicum; SL80.1) and hazelnut (C. avellana; HZ.1) to understand the basis for their functional diversity. The SOD activities of HZ.1 and SL80.1 were assayed. It was realized that while SL80.1 exhibits the SOD activity, HZ.1 lacks the same [4]. Structural comparison was further done to understand the underlying reasons behind the differential activities of vicilins. HZ.1 and SL80.1 were purified, crystallized and their structures were determined. The detailed analyses of the three-dimensional structures accompanied with biophysical characterization of bound ligand were aimed to establish a meaningful correlation with their enzymatic activities. Our findings provide key insights about the possible role of salicylic acid (SA) and copper-binding loop in regulating the SOD activity of vicilins.

Section snippets

Fractionation of seed proteome

Seeds of S. lycopersicum and C. avellana were washed with 70% alcohol and were grounded to powder. This was followed by delipidification in petroleum ether. The dried delipidified powder was homogenized in 50 mM Tris-Cl (pH 8 and pH 7.5 for S. lycopersicum and C. avellana, respectively) containing 150 mM NaCl along with protease inhibitor cocktail (Sigma). The extract in homogenization buffer was subjected to salt fractionation over a concentration range of 0–95% (w/v) ammonium sulfate. This

Results

The ammonium sulfate fractionation yielded an array of proteins. The precipitated proteins in each fraction in both the cases of S. lycopersicum and C. avellana could be visualised in Supplementary Figs. S1 and S2. For targeting globulins from S. lycopersicum and C. avellana seeds, we characterized all the fractions of ammonium sulfate. Two major abundantly present proteins in ammonium sulfate fractions of S. lycopersicum were identified as 7S vicilin (SL80.1) and pathogenesis-related protein

Discussion

Vicilins are important plant proteins that structurally belong to cupin superfamily which include enzymatically diverse proteins from all kingdoms. Vicilins were considered as enzymatically inactive cupins, but a recent structural study of a vicilin from C. annuum revealed that it has SOD activity. Reactive oxygen species are generated in plants during various biotic and abiotic stress conditions such as drought, desiccation as well as bacterial and fungal infections. Vicilins continue to be of

Authors’ contribution

AJ, DMS and MS conceived and designed the experiments. AJ and MS solved the structure. MS performed the biochemical experiments. AJ, DMS and MS wrote the manuscript. The authors declare no competing interests.

Acknowledgements

We thank the EMBL staff and EMBL-DBT for providing access to the BM14 beamline at the ESRF. We thank mass spectrometry facility and staff at RCB and SCIEX. The financial support from the Department of Biotechnology, Government of India through core grants is gratefully acknowledged. MS thanks CSIR for fellowship. PDB: 6L4M and 6L4C.

References (16)

  • B. d Moore

    Bifunctional and moonlighting enzymes: lighting the way to regulatory control

    Trends Plant Sci.

    (2004)
  • A.L. Kriz

    7S globulins of cereals

  • F.T. Moura et al.

    Effects of a chitin-binding vicilin from Enterolobium contortisiliquum seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus) and phytopathogenic fungi (Fusarium solani and Colletrichum lindemuntianum)

    J. Agric. Food Chem.

    (2007)
  • A. Jain et al.

    Crystal structure of the vicilin from Solanum melongena reveals existence of different anionic ligands in structurally similar pockets

    Sci. Rep.

    (2016)
  • M. Shikhi et al.

    Structure-guided identification of function: role of C. annuum vicilin during oxidative stress

    Biochem. J.

    (2018)
  • M.P. Sales et al.

    Do legume storage proteins play a role in defending seeds against bruchids?

    Plant Physiol.

    (2000)
  • J.M. Dunwell et al.

    Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily

    Microbiol. Mol. Biol. Rev.

    (2000)
  • A. Shevchenko et al.

    In-gel digestion for mass spectrometric characterization of proteins and proteomes

    Nat. Protoc.

    (2006)
There are more references available in the full text version of this article.

Cited by (0)

1

Both the authors equally contributed to the work.

View full text