Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease

Abstract

Background: Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation.

Methods: Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61-74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter.

Results: 1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis.

Major conclusions: The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx.

General significance: Jbtx represents a new type of insecticidal and membrane-active peptide.

Keywords: C-terminal domain of Jbtx; CD; Insect; Jbtx; Jbtx C-ter; Jbtx N-ter; Jbtx-2Ec; JbtxΔ-β; LUV; MD; Membrane-disturbing; Molecular modeling; N-terminal domain of Jbtx; RMSD; Site-directed mutagenesis; Urease-derived peptide; a version of Jbtx containing a V5 epitope; circular dichroism; jaburetox; large unilamellar vesicle; molecular dynamics; root mean square deviation; β-hairpin; β-hairpin deleted version of Jbtx.