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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.

While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interest concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated. Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and further experimental designs (Lou et al., 2002. J. Mol. Recognit. 15: 175-179). However, if one only relies on such information, more advanced structure-functional interpretations for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unknown. In order to have better understanding of the molecular details regarding this issue, we have performed the docking simulation with the S3C sequence from the hanatoxin-insensitive K+-channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of the S3-S4 linker region, we suggested a significant movement of drk1 S3C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering with S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of the S3-S4 linker, a possible molecular mechanism to illustrate the hanatoxin binding-modified gating is proposed., (Copyright 2003 John Wiley & Sons, Ltd.)

The carboxyl terminus of S3 segment (S3(C)) in voltage-gated potassium channels was proposed to bear the binding site for gating modifier toxins like Hanatoxin and a helical secondary structural arrangement was suggested. Due to the lack of complete structure in high resolution for such a channel molecule, no further direct experimental data to elucidate the mechanism for their binding conformations could thus far be derived. In order to examine the putative three-dimensional structure of S3(C) and to illustrate the residues required for Hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of Hanatoxin and the structural information from lysine-scanning results for S3(C) fragment. From our results, it is indicated that both hydrophobic and electrostatic interactions are utilized to stabilize the toxin binding. Detailed docking residues and appropriate orientation for binding regarding hydrophobic/-philic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of S3 segment in voltage-gated potassium channels can therefore be further emphasized., (Copyright 2002 John Wiley & Sons, Ltd.)