VrD1

It was shown previously that a bacterially expressed mungbean defensin VrCRP exhibited both antifungal and insecticidal activities. To isolate this protein in a large quantity for its characterization, the defensin cDNA was expressed in Pichia pastoris and the recombinant defensin (rVrD1) was purified. The recombinant VrD1 was shown to inhibit the growth of fungi such as Fusarium oxysporum, Pyricularia oryza, Rhizoctonia solani, and Trichophyton rubrum and development of bruchid larva. The protein also inhibits in vitro protein synthesis. These biological activities are similar to that of the bacterially expressed defensin. Functional expression of VrD1 in Pichia pastoris provides a highly feasible system to study the structure-function relationship of VrD1 using the mutagenesis approach.

Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity. We report herein the nuclear magnetic resonance solution structure of VrD1 and the implication on its insecticidal activity. The root-mean-square deviation values are 0.51 +/- 0.35 and 1.23 +/- 0.29 A for backbone and all heavy atoms, respectively. The VrD1 structure comprises a triple-stranded antiparallel beta-sheet, an alpha-helix, and a 3(10) helix stabilized by four disulfide bonds, forming a typical cysteine-stabilized alphabeta motif. Among plant defensins of known structure, VrD1 is the first to contain a 3(10) helix. Glu26 is highly conserved among defensins; VrD1 contains an arginine at this position, which may induce a shift in the orientation of Trp10, thereby promoting the formation of this 3(10) helix. Moreover, VrD1 inhibits Tenebrio molitor alpha-amylase. Alpha-amylase has an essential role in the digestion of plant starch in the insect gut, and expression of the common bean alpha-amylase inhibitor 1 in transgenic pea imparts complete resistance against bruchids. These results imply that VrD1 insecticidal activity has its basis in the inhibition of a polysaccharide hydrolase. Sequence and structural comparisons between two groups of plant defensins having different specificity toward insect alpha-amylase reveal that the loop between beta2 and beta3 is the probable binding site for the alpha-amylase. Computational docking experiments were used to study VrD1-alpha-amylase interactions, and these results provide information that may be used to improve the insecticidal activity of VrD1.

A variety of evolutionarily related defensin molecules is found in plants and animals. Plant gamma-thionins and scorpion neurotoxins, for instance, may be categorized in this functional group, although each class recognizes a distinct receptor binding site. Such molecules are also categorized into the superfamily of cysteine-rich proteins. Plant defensins were generally believed to be involved in antimicrobial or antifungal mechanisms and, unlike scorpion toxins, little is known about whether these molecules are also endowed with the function of insect resistance. We have previously reported the isolation of a cDNA encoding a small cysteine-rich protein designated VrD1 (VrCRP) from a bruchid-resistant mungbean, which is apparently the first discovered plant defensin exhibiting in vitro and in vivo both insecticidal and antifungal activities. Our previous data also successfully demonstrated that VrD1 is toxic to E. coli and able to completely arrest the growth of Sf-21 insect cells at low concentration. However, the molecular and structural basis of this unique insecticidal activity of VrD1 is not clear. Therefore, in the present study, we use structural approach and phylogenic analysis to investigate the evolutionary and functional relations for such unique insecticidal activity. From our results, it is suggested that VrD1, in addition to gamma-thionins and several amylase inhibitors, is highly homologous to scorpion toxins, especially the short toxins. Moreover, based on the observation from our homology structures, VrD1 may utilize a newly found cluster of basic residues to achieve its insecticidal function, whereas all the other plant gamma-thionins were known to use a previously identified basic cluster conserved for gamma-thionins. Considering the general feature of short scorpion toxins to act on insect cell membranes with K(+)- or Cl(-)-channels as molecular targets, our analysis of interaction and recognition modes provides reasonable correlations between this newly found basic cluster and the insecticidal activity of VrD1, which is also comprehended as a possible link for "homoplasy evolution" between plant and animal defensin molecules.