Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests

Plant cystatins show great poten- tial as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtec- tus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in mid- guts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna un- guiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from mid- guts of both A. obtectus and Z. subfasciatus bruch- ids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabiliza- tion of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The applica- tion of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed. Proteins 2006;63:662- 670.