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

ABSTRACT: Snake venom ?-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 ?1 and ?7 nAChR subunits, binds biotinylated ?-bungarotoxin (?Bgt) 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 ?-cobratoxin (?Ctx), similar to ?Bgt in action on nAChRs. Using radioiodinated ?Bgt, we confirmed a high affinity of HAP for ?Bgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with ?Ctx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-?Bgt binding constant directly (29?nM). These methods also confirmed weak HAP interaction with ?Ctx (>10000?nM). We attempted to enhance it by modification of HAP structure relying on the known structures of ?-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 ?Ctx binding (7000?nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

SUBMITTER: Kudryavtsev DS

PROVIDER: S-EPMC7052197 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Kudryavtsev Denis S DS Tabakmakher Valentin М VМ Budylin Gleb S GS Egorova Natalia S NS Efremov Roman G RG Ivanov Igor A IA Belukhina Svetlana Yu SY Jegorov Artjom V AV Kasheverov Igor E IE Kryukova Elena V EV Shelukhina Irina V IV Shirshin Evgeny A EA Zhdanova Nadezhda G NG Zhmak Maxim N MN Tsetlin Victor I VI

Scientific reports 20200302 1

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 ...[more]

PMID: 32123252

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Project description:?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the ?1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the ?1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the ?1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the ?1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the ?1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human ?1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the ?1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Dataset Information

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

Publications

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

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