Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide
Denis S. Kudryavtsev, Valentin М. Tabakmakher, Gleb S. Budylin, Natalia S. Egorova, Roman G. Efremov, Igor A. Ivanov, Svetlana Belukhina, Artjom V. Jegorov, Igor E. Kasheverov, Elena V. Kryukova, Irina V. Shelukhina, Evgeny A. Shirshin, Nadezhda G. Zhdanova, М. Н. Жмак, Victor I. Tsetlin
Abstract
Snake venom α-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 α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) 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 α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-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 αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.