Molecular Recognition of Neonicotinoid Insecticides by Honeybee Nicotinic Receptors and ACHBP Homologues

Abstract

Neonicotinoids, sometimes called ‘neonics’, are a class of insecticides targeting neuronal nicotinic acetylcholine receptors, which belong to the family of pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors. The widespread application of these neurotoxic insecticides in agriculture is tied to the worldwide decline of bee populations, due to their sublethal effects on bee memory, behavior and reproduction. In 2018, the member states of the European Union, agreed on a ban of most neonicotinoids, with exemption for greenhouses. However, a new generation of sulfoximine-based insecticides, such as sulfoxaflor, has recently been introduced to the agricultural market claiming they are chemically distinct from neonicotinoids. Using the acetylcholine binding protein (AChBP) as a well-established tool for structural studies of nicotinic receptors, we engineered AChBP variants that mimic the neurotransmitter binding site of honeybee nicotinic receptors. In parallel, we pursued functional characterization of these receptors expressed in Xenopus oocytes and using electrophysiological techniques. Three-dimensional structures of sulfoxaflor-bound AChBPs reveal that the molecule is recognized through receptor interactions that are virtually identical to the neonicotinoid thiacloprid. Surprisingly, binding assays reveal a ∼100-fold difference in affinity between sulfoxaflor and thiacloprid, leaving open important questions with respect to the precise molecular mechanism of sulfoxaflor recognition. Our study will attempt to begin addressing these central questions.