Structure-Function Insights of Jaburetox and Soyuretox: Novel Intrinsically Disordered Polypeptides Derived from Plant Ureases

Abstract

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.

Keywords: antifungal activity; biopesticides; insecticidal activity; mechanism of action; transgenic crops.

Figure 1

Structural representation of entomotoxic peptides and Jack Bean Urease. (A) Graphical representation of the location of the peptides (red) in the protein structure of Jack Bean Urease (pdb: 3LA4). Each monomer of the JBU hexamer is represented with a different shade of grey. (B) Jaburetox, (C) Soyuretox and (D) comparison of the primary sequences.

Figure 2

Disorder profile of Jaburetox and Soyuretox. Algorithm of disorder prediction VL-XT PONDR^® was applied to compare Jaburetox and Soyuretox amino acid sequences. The amino acid sequences (including the 6-His tags) were submitted to http://www.pondr.com/ to generate the graphics. PONDR score above 0.5 indicates disorder. Jaburetox is slightly more disordered than Soyuretox, especially in its N-terminal region. Jaburetox structure is based on conformer 1 (PDB 2MM8). The modeled structure of Soyuretox was obtained with Modeller 9.19 using Jaburetox as a model. The primary sequences of the peptides appear below each 3D-structure, negatively charged amino acids are highlighted in blue and positively charged in red.

Figure 3

Tissue-specific effects of urease-derived peptides in Rhodnius prolixus. In the flowchart, the brown boxes are the organs or the cells affected by the urease-derived peptides while the grey boxes indicate no change. The green boxes represent an increase in the assessed effect while the red boxes indicate a decrease. Acronyms stand for: 5-HT, Serotonin; ANT MIDGUT, Anterior midgut; CNS, Central nervous system; ET’S, Extracellular traps; JBTX, Jaburetox; MALP TUBULES, Malpighian tubules; NOS, Nitric oxide synthase; ROS, Reactive oxygen species; SG, Salivary glands; SYTX, Soyuretox; UAP, UDP-N-acetylglucosamine pyrophosphorylase; WGA, Wheat germ agglutinin. The numbers between brackets indicate the corresponding references: (1) Fruttero et al., [78]; (2) unpublished results; (3) Moyetta et al., [77]; (4) Fruttero et al., [82]; (5) Coste Grahl et al., [83]; (6) Kappaun et al., [64]; (7) and (8) unpublished results; (9) Staniscuaski et al., [81].

Figure 4

Schematic representation of the antifungal effects of Jaburetox and Soyuretox against filamentous fungi and yeasts.