STRUCTURAL AND FUNCTIONAL STUDIES OF PLANT PROTEINS

Abstract

Plants have been used as ornaments, decorative material, medicines and drugs since ancient times. Seed plants cover more than 80% of known plant species. Major seed proteins include 2S albumin, lectin, chitinase, globulins, protease inhibitors, β-1,3-glucanses, defensins, α- amylase, lipid-transfer and ribosome-inactivating proteins which help them during seedling growth, development and provide them protection against pathogens. These proteins play a major role in metabolic or structural processes of the plant; however, some of these groups of proteins are present in high amount in seeds to serve as plant’s nutrition. All these proteins exhibit a spectrum of biotechnologically exploitable applications. The aim of the work presented in this thesis, entitled “Structural and functional studies of plant proteins” is to characterize some of the important plant proteins, specifically, seed storage globulins both 11S and 7S, albumins and chitinase-like lectins. The present work was carried out to study structural-functional relationship of these proteins and to gain insight about their importance in seeds and for developing bio-molecules with enhanced functionality. Chapter1 describes the structural characteristics, evolutionary path and functional role of seed proteins. The chapter is sub-divided into seed storage proteins and inactive chitinases. Seed storage proteins (SSPs) accumulate in significant amount in the growing seed, and act as a storage reserve of nitrogen, carbon, and sulphur. The SSPs comprise of globulins, albumins, prolamins etc. Globulins are cupin superfamily proteins which form a major class of enzymatically inactive SSPs. They can be categorized into 11S legumin and 7S vicilin based on their sedimentation coefficients. Subunits from both classes share structural homology are thought to have evolved from either one-domain germin predecessor by duplication or by horizontal gene transfer of two-domain oxalate decarboxylase from bacteria to plants. Globulins are known to be involved in sucrose binding, desiccation, defense against microbes, hormone binding and oxidative stress, etc. Another category of SSPs, 2S albumin are low molecular weight heterodimeric proteins. These are multifunctional proteins that posses DNase, RNase, antifungal, anticancer and in-vitro translational inhibitory activities. SSPs form the major class of allergens because of their abundant presence and heat stability. Another class of protein that has been included in the thesis is Chitinase-like protein. Chitinases (EC 3.2.1.14) have specific binding affinity for chitin and can hydrolyze them to their monomer N-acetyl-d-Glucosamine. Plant genome contains a large number of catalyticallyinactive chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high II sequence and structural homology to chitinases of GH18 and GH19 families, they may lack the binding/catalytic activity. Molecular genetic analysis suggests that gene duplication events followed by mutation in the existing chitinase gene resulted in the loss of activity. Evidences of adaptive functional diversification of the CLPs have been achieved through alterations in the flexible regions than in the rigid structural elements. In some studies, it has been observed that physiological roles of CLPs are similar to chitinase; such mutations have led to pluri-functional enzymes. Initially our group reported preliminary results related to the density of an indole-like molecules in the cavity of an SSP, Wrightia tinctoria 11S globulin (WTG). Here in the chapter 2 of the thesis the identification of the bound ligand and structural details of the protein-ligand has been discussed. Firstly, the ligand bound to the Wrightia tinctoria 11S globulin was isolated and characterized using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-resolution mass spectrometry (HRMS). The small molecule was characterized as Indole-3-acetic acid (auxin). The detailed structural analysis of the WTG 11S globulin-auxin complex was carried out to identify the key members of auxin binding motif. The detailed analysis showed that ligand binds near the disordered region IV of the protein; and are stabilized by non-covalent interactions at the binding site, contributed by four chains in each cavity. In 11S SSP, the endopeptidase cleavage at the disordered region IV marks the hexamer formation. The hexamer assembles because of stacking interaction between the hydrophobic surfaces of two trimers, leaving space for the binding of ligands. Further, the auxin binding motif was analyzed in the available 11S globulin sequences and crystal structures. The 11S globulin for which structures were unavailable, the models were built to analyze the auxin binding pockets. Since the overall structure of globulins is well-conserved; also, the auxin binding site shows the presence of similar residues among many plant species. To analyze the presence of auxin in other 11S globulin, the Holarrhena antidysenterica 11S globulin was isolated and characterized. The N-terminal amino acid sequence of the HAG protein confirmed that the purified protein was 11S seed storage globulin. Crystallization of the protein yielded small crystals; however, in the absence of sequence, we could not proceed with sequence and structural analysis. The biological relevance of the study is that seed has adopted a mechanism to store auxin in isolated cavities of 11S globulin. The 11S globulins during endopeptidases action captures ligands and forms hexamer. As the process of germination proceeds, there is a change in the pH, which induces the dissociation of the hexamer and thus releases auxin. The compact hexameric assembly ensures the long-term, stable storage of the molecules (like hormones). III In chapter 3, the sequence, structural, biophysical and biochemical analysis of 7S globulin from Momordica charantia (Mc) seeds has been described. A seed storage protein of ~52 kDa was isolated from the seeds. MALDI-ESI-MS identified peptide fragments confirmed that isolated protein is 7S globulin (Mc7S). In the absence of protein or gene sequence, the contig-sequence was obtained from the Sequence Read Archive (SRA) database and aligned to deduced protein sequence. Crystal structure of Mc7S showed that the protein has a cupin fold and an extended helical arm in both N- and C-terminal domains. The 7S globulin exists as a trimer in biological state; the three monomers are arranged around a three-fold axis, and the extended helical arms from both terminals of each monomer interact in head-to-tail fashion with the neighboring monomers. The trimer is stabilized by hydrogen bonding and hydrophobic interactions. The Cterminal domain of Mc7S possesses an acetate binding site and a copper binding site. The pH and temperature largely affect the functional properties of the protein therefore; CD spectra studies were carried out under varying temperature and pH condition. The biochemical experiment was carried out to identify the additional functional features of the protein. Mc7S is a glycosylated protein exhibiting antioxidant activity. The 7S globulins are a class of allergens; therefore B-cell epitopes were predicted using the sequence of Mc7S protein. In-silico sequence analysis showed the presence of bioactive peptides. Thus, Mc7S is of high nutritional and functional value to the food industry and structure-function studies can help in modifying proteins for enhanced functionality. Chapter 4 describes purification, characterization and structural analysis of 2S albumin (WTA) from seeds of Wrightia tinctoria. Three step purification process involving ion exchange chromatography, ammonium sulfate precipitation and gel filtration chromatography were employed to get the pure form of the WTA in homogeneous condition. The WTA is a small heterodimer protein of ~16 kDa having a small subunit of ~5 kDa and a larger subunit of ~11 kDa bridged together through disulphide bonds. The protein has five helices, the first are part of the smaller subunit; and other three forms the larger subunit. The two subunits are separated upon peptide cleavage. The fourth and fifth helix is connected through a hyper-variable loop region. As per in-silico analysis this loop is an immunodominant B-cell epitope. The multiple sequence analysis showed that 2S albumin share very low sequence homology however the positions of eight cysteine residues are well conserved. These cysteine residues form intradomain and inter-domain disulphide bonds, which help in stabilization of the protein structure. The protein showed deoxyribonucleases activity against closed circular pBR322 plasmid DNA and linear BL21 genomic DNA. The presence of metal ion enhanced the DNase activity of the 2S albumin. Also, the WTA protein exhibited antibacterial activity against Gram negative IV pathogen. The surface of WTA protein has a large number of charged residues. The patch of positively charged surface forms the binding site for bacterial surface proteins. This study reinforces the hypothesis that 2S albumin may also play additional roles in defense against pathogens along with its seed storage and DNase activity. Chapter 5 of the thesis discusses the functional role for Tamarind Chitinase-like protein (TCLL) of GH18 class III proteins. TCLL has a triose-phosphate isomerase (TIM) barrel fold, typical fold of glycosyl hydrolase family 18 chitinases; however, the key catalytic residues are mutated with aliphatic hydrophobic residues. Family 18 chitinases have been known to play key roles in bacteria, fungus, plants and animals. Phylogenetic analysis reveals that plant chitinase-like proteins are closer to fungal chitinase than bacterial and mammalian chitinase. The TCLL protein was isolated, purified and crystallized in the presence of xanthine molecules. The structures helped in studying the cavities found on the surface of the plant chitinase molecule. The structural analysis suggests that sugar ligand GalNAc binding pocket of chitinase-like lectin is different from the xanthine binding pocket. Therefore, CLPs have a binding tendency to both GalNAc and hydrophobic (xanthine-like) molecule and the binding site for both the molecules are different.