Characterization of the interaction between spider toxin Hv1a and acetylcholine binding protein

Neonicotinoid insecticides occupy the highest market share in the world. Their wide usage has led to the emergence of insect resistance and bee toxicity problem. Insect nicotinic acetylcholine receptors (nAChRs) are the molecular targets of these insecticides. Acetylcholine binding proteins (AChBPs) share a similar homologous pentameric structure with extramembrane ligand-binding domains of nAChRs. Therefore, AChBPs are used as an alternative tool for studying the ligand binding properties of nAChRs. Recently, the AChBP identified from Pardosa pseudoannulata, Pp-AChBP, was proven to be more similar to insect nAChRs in terms of the binding properties of known nAChR-targeting insecticides. Meanwhile, a spider peptides toxin, Hv1a (ω-hexatoxin-Hv1a), has been found to selectively bind to insect nAChRs, but the binding pocket is different from traditional neonicotinoid insecticides. The purpose of this study is to reveal the molecular mechanism of spider toxin Hv1a binding to Pp- AChBP. Hv1a gene was cloned and expressed in Escherichia coli. Pp-AChBP gene was cloned and expressed in Sf9 insect cells. We explored the strategies to achieve a high level of expression of both proteins. The pull-down assay result showed that Pp-AChBP do not bind with Hv1a, the possible reasons of which were discussed. This study provided a theoretical basis for the regulation mechanism of Hv1a.