Structural similarity between defense peptide from wheat and scorpion neurotoxin permits rational functional design

Abstract

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.

Keywords: Antimicrobial Peptides; Hairpin; Hefutoxin; Plant Defense; Potassium Channels; Protein Design; Protein Engineering; Protein Folding.

FIGURE 1.

Production of recombinant Tk-AMP-X2. A, expression and purification of Trx-Tk-AMP-X2 fusion protein as followed by SDS-PAGE (10%). Lane 1, whole-cell lysate of E. coli BL21(DE3) cells carrying the plasmid pET-32b-Tk-AMP-X2 before isopropyl β-d-thiogalactopyranoside treatment; lane 2, induced with 0.2 mm isopropyl β-d-thiogalactopyranoside; lane 3, affinity column flow-through; lane 4, purified fusion protein; lane 5, molecular mass markers (LMW-SDS Marker kit from GE Healthcare); the corresponding molecular mass values are labeled in kDa. The desired product is labeled by an arrow. B, reversed-phase HPLC of the fusion protein Trx-Tk-AMP-X2 cleaved with CNBr on a Jupiter C₅ column. The faction corresponding to Tk-AMP-X2 is indicated with an arrow.

FIGURE 2.

Overview of NMR data. Data determining the secondary structure of Tk-AMP-X2 are shown. ³J_HαHβ2 and ³J_HαHβ3, ³J_NHβ2 and ³J_NHβ3, and ³J_HNHα coupling constants; temperature coefficients of H_N chemical shifts (TempGrad); and NOE connectivities are shown versus the Tk-AMP-X2 sequence. ³J_HαHβ are marked by closed squares if <5 Hz and by open squares if >10 Hz, ³J_NHβ are marked by closed squares if <2 Hz and by open squares if >3 Hz, and ³J_HNHα are marked by closed squares if <5 Hz. Closed triangles correspond to residues, H_N protons of which demonstrate temperature gradients higher than −4.5 ppb/K. Elements of secondary structure are shown on a separate line; helices are shown by rectangles: α-helices, closed, 3₁₀ helix, open.

FIGURE 3.

Three-dimensional structure of Tk-AMP-X2. A, ribbon representation of spatial structure of Tk-AMP-X2. Disulfide bonds (yellow) and positively charged (blue) and negatively charged (red) side chains are shown as sticks. B and C, surface of Tk-AMP-X2 colored with respect to molecular hydrophobicity potential (37) (B) and electrostatic surface potential (C).

FIGURE 4.

Comparison of Tk-AMP-X2 and κ-hefutoxin 1 structures. Tk-AMP-X2 is shown in light brown (left), and κ-hefutoxin 1 is shown in light cyan (middle). Cysteine residues are colored yellow. The functional dyad of κ-hefutoxin 1 (Tyr-5 and Lys-19) is colored violet, and corresponding residues of Tk-AMP-X2 (Glu-6 and Met-22) are colored red. The three-dimensional structure alignment is shown at right.

FIGURE 5.

Activity profile of Tk-hefu on several Kv channel isoforms. Representative whole-cell current traces in control and toxin conditions are shown. The dotted line indicates the zero current level. The asterisk (*) marks the steady-state current traces after application of 40 μm toxin. Traces shown are representatives of at least three independent experiments (n ≥ 3).

FIGURE 6.

Concentration and voltage dependence of the Tk-hefu-induced decrease of potassium currents (I). A, concentration-response curves for Tk-hefu (squares) and Tk-AMP-X2 (circles) on Kv1.3 channels. B, steady-state activation curves in control (squares) and after toxin application (40 μm Tk-hefu; circles). Error bars represent S.E.