In silico analysis of sequential, structural and functional diversity of wheat cystatins and its implication in plant defense

Abstract

Phytocystatins constitute a multigene family that regulates the activity of endogenous and/or exogenous cysteine proteinases. Cereal crops like wheat are continuously threatened by a multitude of pathogens, therefore cystatins offer to play a pivotal role in deciding the plant response. In order to study the need of having diverse specificities and activities of various cystatins, we conducted comparative analysis of six wheat cystatins (WCs) with twelve rice, seven barley, one sorghum and ten corn cystatin sequences employing different bioinformatics tools. The obtained results identified highly conserved signature sequences in all the cystatins considered. Several other motifs were also identified, based on which the sequences could be categorized into groups in congruence with the phylogenetic clustering. Homology modeling of WCs revealed 3D structural topology so well shared by other cystatins. Protein-protein interaction of WCs with papain supported the notion that functional diversity is a con-sequence of existing differences in amino acid residues in highly conserved as well as relatively less conserved motifs. Thus there is a significant conservation at the sequential and structural levels; however, concomitant variations maintain the functional diversity in this protein family, which constantly modulates itself to reciprocate the diversity while counteracting the cysteine proteinases.

Figure 1

ClustalW alignment of all the cystatin sequences from wheat, rice, barley, sorghum and corn. Few N-terminal and C-terminal amino acid residues are not shown for the clarity of the picture. The conserved signature sequences of the PhyCys are highlighted by enclosing in colored rectangles (Yellow: N-terminal G; Red: LARFAV; Black: QXVXG; Green: P/AW).

Figure 2

Unrooted phylogenetic tree of wheat, rice, maize, sorghum and barley cystatins constructed by the neighbor-joining method. Bootstrap values are indicated against each branch. Bootstrap similarity is >50% and the tree was built after 1,000 cycles.

Figure 3

Block diagram representation and distribution of different non-overlapping motifs predicted by MEME software in cystatins of wheat, rice and barley. The sequences of the motifs are given in Table S3.

Figure 4

3D structures of oryzacystatin (A), WC1 (B), WC2 (C), WC3 (D), WC4 (E), WC5 (F) and WCMD (G). Variation in helix is marked with a red circle. 3D structures were constructed by MOE software using oryzacystatin as the template.

Figure 5

A. Amino acid sequence alignment of oryzacystatin (OC1) and wheat cystatins (WCs). The positions of secondary structures of OC1 are indicated except the first β-strand, which is not shared by most of the WCs. An array of red ovals indicates an α-helix and block arrows indicate β-strands. α-helices of WCs are shown in yellow boxes and β-strands are in cyan boxes. The four signature sequences of PhyCys are shown enclosed in differently colored rectangles. B. Diagrammatic representation of secondary structures of different wheat cystatins. Structure of OC1 is shown for comparison. Structures are not up to scale.

Figure 6

Protein–protein interactions of different WCs with papain molecule by taking the stefin–papain complex (A) as template, modeled in Swiss-Pdb viewer. B. WC1–papain complex. C. WC2–papain complex. D. WC3–papain complex. E. WC4–papain complex. F. WC5–papain complex. G. WCMD–papain complex. Structural graphics are produced by using Rasmol software version 2.7.3.1.

Figure 7

Superimposition of WC1–papain complex on stefin B–papain complex (1stf). Graphics were generated by MOE software. Color scheme: papain (white), WC1 (red), stefin B (yellow).