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

• Published in: The Journal of biological chemistry 2014-05, Vol.289 (20), p.14331-14340
• Main Authors: Berkut, Antonina A., Usmanova, Dinara R., Peigneur, Steve, Oparin, Peter B., Mineev, Konstantin S., Odintsova, Tatyana I., Tytgat, Jan, Arseniev, Alexander S., Grishin, Eugene V., Vassilevski, Alexander A.
• Format: Article
• Language: English
• Subjects: Animals; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - genetics; Antimicrobial Cationic Peptides - pharmacology; Antimicrobial Peptides; Disulfides - chemistry; Electrophysiological Phenomena - drug effects; Hairpin; Hefutoxin; Models, Molecular; Neurotoxins - chemistry; Neurotoxins - genetics; Nuclear Magnetic Resonance, Biomolecular; Plant Defense; Potassium Channel Blockers - chemistry; Potassium Channel Blockers - pharmacology; Potassium Channels; Protein Design; Protein Engineering; Protein Folding; Protein Structure and Folding; Protein Structure, Secondary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacology; Scorpions - chemistry; Triticum - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.530477

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 α-helical hairpin fold and therefore belongs to the α-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 κ-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 ∼ 35 μm) to κ-hefutoxin 1 (IC50 ∼ 40 μm). We conclude that α-hairpinins are attractive in their simplicity as structural templates, which may be used for functional engineering and drug design. Protein folds differ in size and complexity and hence in their utility for engineering purposes. The three-dimensional structure of wheat antifungal peptide Tk-AMP-X2 was investigated, and a new functionality was engineered based on its α-hairpin scaffold. α-Hairpinins are an attractive simple structural template for functional engineering and drug design. The repertoire of available scaffolds for protein engineering is broadened.