Chemical engineering of a three-fingered toxin with anti-α7 neuronal acetylcholine receptor activity

Abstract

Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 ± 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 ± 0.4 μM) for α7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to α7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal α7 AchR, as compared with the parental short-chain toxin, This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for α7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.