Abstract:  【Aim】 To study the binding characteristics of the odorant binding protein AsinOBP1 from Anopheles sinensis with the mosiquito repellent N,N-diethyl-m-toluamide (DEET) in comparison with Aedes aegypti OBP1 (AaegOBP1) and Culex quinquefasciatus OBP1 (CquiOBP1), and to identify the key residues responsible for the binding to DEET by OBP1s from different mosquito species. 【Methods】 The recombinant protein AsinOBP1 was expressed using a prokaryotic expression system and then purified. The DEET binding properties of AsinOBP1 were evaluated by fluorescence competitive binding assays using N-phenyl-1-naphthylamine (1-NPN) as the fluorescence probe. The binding affinities of AsinOBP1, AaegOBP1 and CquiOBP1 to DEET were compared, and the key residues responsible for this binding were identified using molecular docking. 【Results】 AsinOBP1 was able to bind to DEET, with a dissociation constant of 29.55 μmol/L. Under the same experimental conditions, CquiOBP1 and AaegOBP1 were also able to bind to DEET, with their dissociation constants of 17.15 and 12.81 μmol/L, respectively. It was shown that AaegOBP1 had the strongest binding affinity to DEET, followed by CquiOBP1 and AsinOBP1. The molecular docking of AsinOBP1 revealed that one DEET molecule is bound to each subunit at a site located near the interface between the two monomers of AsinOBP1. The DEET binding pocket is formed by residues belonging to helices α4, α5 and α6 (Leu-92, Leu-95, His-96, Leu-99, Ala-107, Met-108, Met-110, Gly-110, Cys-114, Leu-115, Trp-133, Met-108′, Lys-112′ and Leu-115′). The residues interacting with the tolyl group and ketonic oxygen of DEET are identical in the three recombinant proteins. However, among the five key residues interacting with the diethyl group of DEET, one residue is different: in AaegOBP1 the residue is Leu, whereas in CquiOBP1 and AsinOBP1 it is Met. Given that the residue Leu is more hydrophobic than Met, it was speculated that this might contribute to a higher affinity of AaegOBP1 to DEET. 【Conclusion】 AsinOBP1, AaegOBP1 and CquiOBP1 are all able to bind to DEET, but differ in affinity. Further study should be focused on the causes of their difference in affinity so as to better understand the mechanisms by which OBP1s bind to DEET.

Key words: Anopheles sinensis; odorant binding protein, DEET, fluorescence competitive binding assay, molecular docking