Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

Abstract

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-p interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.

Insight in nAChR subtype selectivity from AChBP crystal structures

“…The analysis of the co-crystal structure of LsAChBP with imidacloprid also indicates that S186 in loop C makes hydrogen bonds with E163 and Y164 in loop F [31]. The mutation of the Drosophila α 2 subunit proline (P243) to glutamate, equivalent to LsAChBP E163, was further found to decrease the sensitivity of the α 2 β 2 nAChR to imidacloprid [53].…”

“…Owing Page 7 of 44 A c c e p t e d M a n u s c r i p t 7 to this high degree of structural identity, AChBP is also currently being used as a tool to search for subtype-specific nicotinic receptor ligands through a combined approach involving in silico docking, assaying AChBP binding to potential ligands, and the structural characterization of AChBP-ligand complexes by X-ray crystallography [30]. Such structural studies have also contributed to better understand the factors, which lead to the selectivity of neonicotinoid insecticides such as thiacloprid or imidacloprid for invertebrate nAChRs [31,32].…”

“…Co-crystal structures of imidacloprid with LsAChBP LsAChBP M114, which corresponds to basic or polar residues in the α 1 , α 2 or α 3 Drosophila melanogaster nAChRs and to non-polar residues in vertebrate nAChR subunits, is positioned close to the nitroimine group of the ligand. This chemical group is likely to accept hydrogen bonds from residues equivalent to the LsAChBP M114 in the insect α nAChR subtypes [31].…”

“…Only the human β 4 nAChR subunit contains a lysine (K75) at that position. The potential sensitization caused by this residue is relieved by a glutamate two residues downstream, which might interfere with the interaction between the lysine and the nitro group of imidacloprid [31].…”

“…Taking these data into account, it has been suggested that in heteromeric vertebrate nAChRs, the presence of acidic residues in the position equivalent to LsAChBP S186 would lead to electrostatic repulsion of acidic residues in the loop F on the complementary face. Such acidic residues are not found in the C loop of α subunits of heteromeric insect nAChRs, hence the increased binding efficiency of insecticide neonicotinoids to these receptors [31].…”