Complex approach for analysis of snake venom \u03b1-neurotoxins binding to HAP, the high-affinity peptide

Denis S Kudryavtsev,Igor A Ivanov,Evgeny A Shirshin,Nadezhda G Zhdanova,Roman G Efremov,Gleb S Budylin,Irina V Shelukhina,Svetlana Yu Belukhina,Igor E Kasheverov,Victor I Tsetlin,Artjom V Jegorov,Valentin М Tabakmakher,Natalia S Egorova,Maxim N Zhmak,Elena V Kryukova

doi:10.1038/s41598-020-60768-y

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.

Highlights

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor
Our attention was attracted to a 13-membered peptide high-affinity peptide (HAP) (“high affinity peptide”), which binds biotinylated αBgt with a high affinity (2–4 nM), characteristic for the αBgt binding to the whole-size nicotinic acetylcholine receptors (nAChRs)
A high affinity of HAP for αBgt was found in experiments which demonstrated that HAP pre-incubation with biotinylated αBgt diminished binding of the latter to the muscle-type nAChR and the respective concentration dependencies provided the binding parameters

Summary

Introduction

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. Α-neurotoxins for a long time were known to bind to synthetic fragments of nAChR α1 and α7 subunits encompassing their C-loops, as well as to the combinatorial peptides sharing a certain similarity with this loop[15] The affinity of such binding may be sufficiently high, and recently this property was utilized by introducing the respective sequences into the extracellular parts of diverse receptors, differing from the nAChRs. Fluorescent or biotinylated αBgt derivatives made possible detecting of such receptors in the cells and monitoring receptor assembly and cell internalization; among the successful examples were such ion channels as α-amino-3-hydroxy -5-methyl-4-isoxazolepropionic acid (AMPA) receptors, ionotropic γ-aminobutiryc acid (GABAA) receptors and voltage-gated potassium Kv4.2 channels, as well as such G-protein coupled receptors as γ-aminobutiryc acid receptors (GABAB)[16]. For αCtx we succeeded in only moderate increase in the affinity, we hope that the proposed approach will be helpful for analysis of various peptide-protein interactions

Methods

Results

Conclusion