Structural Similarity between Defense Peptide from Wheat and Scorpion Neurotoxin Permits Rational Functional Design

Background: Protein folds differ in size and complexity and hence in their utility for engineering purposes. Results: The three-dimensional structure of wheat antifungal peptide Tk-AMP-X2 was investigated, and a new functionality was engineered based on its -hairpin scaffold. Conclusion: -Hairpinins are an attractive simple structural template for functional engineering and drug design. Significance: The repertoire of available scaffolds for protein engineering is broadened. In this study, we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide was found to adopt a disulfide-stabilized -helical hairpin fold and therefore belongs to the -hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule, Tk-hefu, was engineered by incorporating the functionally important residues from -hefutoxin 1 onto the Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that although the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 approximate to 35 m) to -hefutoxin 1 (IC50 approximate to 40 m). We conclude that -hairpinins are attractive in their simplicity as structural templates, which may be used for functional engineering and drug design.