Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain α-Neurotoxins Acting on Neuronal Nicotinic Receptors

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha 7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha-and kappa-neurotoxins is essential for their action on the alpha 7 and alpha 3 beta 2nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by H-1 and H-1-N-15 NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I block alpha 7 nAChRs with similar potency (IC50 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha 7 nAChR (IC50 126 nM) but did not convey activity at alpha 3 beta 2nAChRs. Docking of both chimeras to alpha 7 and alpha 3 beta 2nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha 3 beta 2nAChRs.