摘要：
[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-l-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,His96,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 OBP1 s bind to DEET.%[目的]研究中华按蚊Anopheles sinensis气味结合蛋白1(AsinOBP1)与避蚊胺(N,N-diethylm-toluamide,DEET)的结合特性,并与埃及伊蚊Aedes aegypti AaegOBP1、致倦库蚊Culexquinquefasciatus CquiOBP1进行比较,分析与DEET互作的关键氨基酸残基.[方法]利用原核表达载体进行目的蛋白AsinOBP1的原核表达及纯化.以N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)作为荧光探针,通过荧光竞争结合实验对AsinOBP1与DEET的结合特性进行分析,并比较AsinOBP1,AaegOBP1和CquiOBP1与DEET的结合能力,通过分子对接鉴定其与DEET互作的氨基酸残基.[结果]AsinOBP1能与DEET结合,解离常数为29.55 μmol/L.在相同实验条件下,CquiOBP1和AaegOBP1都能结合DEET,解离常数分别为17.15和12.81 μmol/L.与AaegOBP1和CquiOBP1相比,AsinOBP1结合DEET的能力最弱,AaegOBP1最强.分子对接显示,DEET分子结合在AsinOBP1二聚体靠近交界面的结合口袋边缘,结合口袋由α4,α5和α6上的氨基酸残基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'和Leu-115'组成.比较分析发现3个蛋白中与DEET甲苯基团形成疏水性作用的氨基酸残基、与DEET羰基氧形成氢键的氨基酸残基是相同的,但与DEET二乙基侧链形成疏水性作用的氨基酸残基中,有一个位置存在差异,AaegOBP1是Leu,而AsinOBP1和CquiOBP1是Met残基.Leu的疏水性强于Met,可能是AaegOBP1与DEET的结合能力较强的原因之一.[结论]AsinOBP1能够结合DEET,不同蚊虫气味结合蛋白1与DEET的亲和力存在差异,进一步探索这些差异形成的原因对于阐明气味结合蛋白与DEET互作的模式具有重要的参考价值.
