Snake venom á-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of á1 and á7 nAChR subunits, binds biotinylated á-bungarotoxin (áBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of á-cobratoxin (áCtx), similar to áBgt in action on nAChRs. Using radioiodinated áBgt, we confirmed a high affinity of HAP for áBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with áCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-áBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with áCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of á-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in áCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.
Bungarotoxins Cobra Neurotoxin Proteins