Abstract
Cysteine-stabilized αβ (CSαβ) motif consists an α-helix and an antiparallel β-sheet, connected by three or four disulfide bonds which provide high stablity and make this motif as a suitable framework for molecular engineering. Due to structural similarity, we utilized Vigna radiata plant defensin 1 protein (VrD1) as a template containing CSαβ motif to engineer a modified protein bearing not only its original function but also the channel blocking activity like scorpion toxin by point mutagenesis method in this study. Sequence K-C4-X-N is the toxin signature of α-KTxs scorpion toxins, which specifically affect voltage-gated potassium (Kv1) channel. According to sequence alignment, we mutated the corresponding positions (N32C33K34G35) in VrD1 to the toxin signature (K-C4-X-N) of α-KTxs, and also constructed mutants VrD1-E8Q, VrD1-T39K, and VrD1-Y41K to increase positive charge on the binding interface to channel. Both the electrophysiological recording and Tenebrio molitor larvae α-amylase (TMA) inhibition assay were conducted to investigate the function and characteristics of modified VrD1 proteins. The results showed that Lysine substitution could not increase hKv channel blocking ability of VrD1, and the mutations on or near Loop 3 of VrD1 (G35N, T39K and Y41K) reduced the TMA inhibition. On the contray, VrD1 bearing toxin signature K-C-X-N increased the inhibition to sepecific Kv1 channels and retain the TMA inhibition. Here, we created a dual functional protein; however, the ability of using point mutation to develope nontoxic plant defensin into an efficient ion channel blocking toxin was limited. According to docking analysis, the N-terminal region seemed to be the obstacle in the interaction between VrD1 and Kv1 channel. This provided a feasible direction for molecular design of nontoxic CSαβ motif into the potent ion channel blocker.