Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Scientific reports 2020 Mar 02

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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.

Citation

Denis S Kudryavtsev, Valentin М Tabakmakher, Gleb S Budylin, Natalia S Egorova, Roman G Efremov, Igor A Ivanov, Svetlana Yu Belukhina, Artjom V Jegorov, Igor E Kasheverov, Elena V Kryukova, Irina V Shelukhina, Evgeny A Shirshin, Nadezhda G Zhdanova, Maxim N Zhmak, Victor I Tsetlin. Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide. Scientific reports. 2020 Mar 02;10(1):3861