Binding Site Structure of Insect Odorant Receptors.

描述（由申请人提供）：许多昆虫种类通过作为疾病传播媒介或通过造成农业破坏对人类健康产生负面影响。化学感觉受体、气味受体（ORs）和味觉受体对昆虫发挥这些作用的能力至关重要。昆虫化学感觉受体是一类全新的受体，与任何已知的受体家族（包括哺乳动物化学感觉受体）都没有遗传或结构上的关系。因此，这些受体是开发新的昆虫防治化合物的有吸引力的目标。实现这一目标需要对昆虫化学感觉受体的结构和功能有详细的了解。然而，这些受体最显著的特征之一是我们对它们的结构知之甚少。在这个项目中，我们将识别昆虫嗅觉的结构特征，这些特征在气味结合和识别中很重要。我们利用非洲爪蟾卵母细胞表达系统和机器人电生理学建立了一个强大的昆虫气味受体功能分析。利用这个实验，我们提出了一个项目，将确定昆虫嗅觉识别的结构基础。在Aim 1中，我们将使用我们的检测方法表达和筛选一大组果蝇嗅觉受体（dors）。在目标2中，我们将通过使用半胱氨酸反应性甲乙硫磺酸试剂筛选受体面板，确定dors气味结合亚基上感兴趣的区域。该筛选将利用DmOR家族中半胱氨酸残基的自然分布。在目标3中，我们将使用取代半胱氨酸可及性方法（SCAM）来探索昆虫嗅觉结合亚基的关键功能区（在目标2中确定）。在Aim 4中，我们将使用位点定向诱变和功能分析来揭示昆虫气味受体识别气味的结构基础。本项目将在分子水平上对昆虫嗅觉配体的识别提供新的认识。这一项目对人类健康具有重要意义，因为昆虫的ORs是开发防治有害昆虫化合物的新靶点。

DESCRIPTION (provided by applicant): Many insect species exert a negative impact on human health through serving as disease propagation vectors or through causing agricultural damage. Chemosensory receptors, odorant receptors (ORs) and taste receptors, are critical to the ability of insects to function in these roles. Insect chemosensory receptors are an entirely novel receptor class, having no genetic or structural relationship to any known receptor family (including mammalian chemosensory receptors). Thus, these receptors are appealing targets for the development of new compounds for insect control. Realizing this goal requires development of a detailed understanding of insect chemosensory receptor structure and function. However, one of the most notable characteristics of these receptors is how little we know about their structure. In this project, we will identify structural features of insect ORs that are important in odorant binding and recognition. We have established a robust functional assay for insect odorant receptors using the Xenopus oocyte expression system and robotic electrophysiology. Using this assay, we are proposing a project that will determine the structural basis for odorant recognition by insect ORs. In Aim 1, we will express and screen a large panel of Drosophila melanogaster odorant receptors (DmORs) using our assay. In Aim 2, we will identify regions of interest on the odorant binding subunits of DmOrs by screening the receptor panel with cysteine-reactive methanethiosulfonate reagents. This screen will take advantage of the natural distribution of cysteine residues across the DmOR family. In Aim 3, we will use the Substituted Cysteine Accessibility Method (SCAM) to explore critical functional regions (identified in Aim 2) of the odorant binding subunits of insect ORs. In Aim 4, we will use site-directed mutagenesis and functional analysis to reveal the structural basis for odorant recognition by insect odorant receptors. This project will provide an understanding of odorant ligand recognition by insect ORs at the molecular level. This project is important to human health because insect ORs are a novel target for the development of compounds to control deleterious insect species. PUBLIC HEALTH RELEVANCE: Insects can have a negative impact on human health through serving as disease propagation vectors or through causing agricultural damage. Odorant receptors (ORs) are critical to the ability of insects to function in these roles. Insect ORs are an entirely novel receptor class, having no genetic or structural relationship to any known receptor family. In fact, insect ORs are not related to any proteins in humans and other mammals, making these receptors appealing targets for the development of compounds with higher selectivity and lower environmental toxicity than currently available insecticides. However, we currently know essentially nothing about the structure of these receptors. In this project, we will identify structural features of insect ORs that are important in odorant binding and recognition. This work will aid in the development of new compounds to control deleterious insect species.