Neonicotinoid insecticides selectively modulate insect nicotinic acetylcholine receptors (nAChRs). They have been used to date for crop protection and animal healthcare, but EU limited the use of some neonicotinoids due to possible risks to bees. Hence it is critical to understand mechanism of neonicotinoid selectivity in detail. It has been described that neonicotinoids bind to the canonical orthosteric site formed by six loops A–F at α and non-α subunit interfaces of nAChRs. To elucidate molecular mechanism of nAChR-neonicotinoid interactions, we co-crystallized the pond snail acetylcholine binding protein (AChBP), a surrogate that mimics the orthosteric site of nAChRs, with imidacloprid, clothianidin, thiacloprid, nitromethylene analog of imidacloprid (CH-IMI) and the denitrated metabolite of imidacloprid (DN-IMI). In the crystal structure of AChBP-imidacloprid complex, the pyridine nitrogen of imidacloprid formed hydrogen bond via water molecule with the backbone of loop E, while the imidazolidine ring stacked with Tyr185 of loop C and contacted by CH-π interactions with Trp143 in loop B. Notably, the nitro group formed a hydrogen bond with Gln55 in loop D, offering an explanation for selectivity of neonicotinoids to insect nAChRs because basic residues favorable for interactions with the nitro group are preserved at the corresponding position in loop D of insect non-α subunits. In accord with this, the guanidine tip of DN-IMI lacking the nitro group was directed away from loop D in AChBP. We also found that not only the nitro group of clothianidin and CH-IMI, but also the cyano group of thiacloprid interacted electrostatically with Lys34 on the β1 strand of the complementary side of AChBP. Interestingly, insect nAChR α subunits possess basic residues at positions corresponding to Lys34 of AChBP. Provided that such residues in loop G could underpin the interactions with nitro or cyano group of neonicotinoids, they would interact from the complementary side of a non-canonical orthosteric site formed by vicinal α subunits. Indeed, mutations in loop G affected concentration-response curves for neonicotinoids, supporting this hypothesis.

Canonical and non-canonical binding sites of neonicotinoids determining their selective actions on insect nicotinic acetylcholine receptors

254th National Meeting (2017)

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