G. Mourier et al., Chemical engineering of a three-fingered toxin with anti-alpha 7 neuronal acetylcholine receptor activity, PROTEIN ENG, 13(3), 2000, pp. 217-225
Though it possesses four disulfide bonds the three-fingered fold is amenabl
e to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a
three-fingered curaremimetic toxin from snake with high yield and showed th
at the synthetic and native toxins have the same structural and biological
properties. Both were characterized by the same 2D MMR spectra, identical h
igh binding affinity (Kd = 22 +/- 5 pM) for the muscular acetylcholine rece
ptor (AChR) and identical low affinity (K-d = 2.0 +/- 0.4 mu M) for alpha 7
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 pres
ent in longer snake toxins that bind with high affinity (K-d = 1-5 nM) to a
lpha 7 neuronal AchR. Not only did the chimera toxin still bind with the sa
me high affinity to the muscular AchR but also it displayed a 20-fold highe
r affinity (K-d = 100 nM) for the neuronal alpha 7 AchR, as compared with t
he parental short-chain toxin, This result demonstrates that the engineered
loop contributes, at least in part, to the high affinity of long-chain tox
ins for alpha 7 neuronal receptors. That three-fingered proteins with four
or five disulfide bonds are amenable to chemical synthesis opens new perspe
ctives for engineering new activities on this fold.