Structural and biochemical characterization of the OprD membrane protein family

描述（由申请人提供）：革兰氏阴性菌的外膜（OM）可作为防止小分子渗透的有效屏障，从而保护细胞免受环境中遇到的有毒化合物的影响。为了获得细胞生长和功能所需的分子和离子，OM内存在转运通道。在大多数革兰氏阴性菌中，如E.在大肠杆菌中，大部分小分子被孔蛋白如OmpF和OmpC摄取。孔蛋白是非特异性通道，不结合其底物。它们在OM内形成充满水的孔隙，小分子在浓度梯度的驱动下通过这些孔隙扩散。革兰氏阴性菌铜绿假单胞菌是一种机会性人类病原体，最常见的与囊性纤维化患者的肺部感染有关。在治疗感染铜绿假单胞菌的患者中的严重问题是该细菌的高内在抗生素抗性。高抗生素耐药性的一个重要原因是OM的低渗透性，由于缺乏孔蛋白。在不存在孔蛋白的情况下，属于OprD家族的底物特异性通道负责铜绿假单胞菌中大部分低分子量化合物的摄取。OprD家族成员在序列上密切相关，但转化非常不同的底物。该家族的原型OprD用作碱性氨基酸和碳青霉烯类抗生素的摄取通道。没有任何OprD家族成员的结构信息可用，并且OprD家族内底物选择性和特异性的基础未知。在这个建议中，我们将确定一些OprD家族通道的X射线晶体结构，并使用一系列计算，生物化学和生物物理实验来表征其底物特异性。具体而言，我们将追求以下目标：1.铜绿假单胞菌OprD的底物结合和转运机制。2.使用X射线晶体学以高分辨率测定OprD家族成员的结构。3.具有确定结构的OprD成员的生物化学和生物物理表征（目的2）。OprD家族为研究密切相关的OM通道蛋白的底物特异性的结构和生化基础提供了极好的机会。这样的分析将是膜蛋白的第一次，除了基础知识外，还将提供可用于设计更有效的抗铜绿假单胞菌药物的信息。细菌细胞被膜包围，保护它们免受来自外部世界的有害和有毒化合物的侵害。然而，为了使细菌生长，小的营养分子必须穿过膜进入细胞。为了完成这项任务，细菌在细胞膜上配备了专门的蛋白质，这些蛋白质起着通道和转运蛋白的作用，将小分子从细胞外运送到细胞内。对于必须运输的不同类型的分子，细菌具有不同的通道蛋白家族。OprD家族就是这样一个家族，其成员存在于许多人类致病细菌中。OprD家族的成员如何执行运输任务是一个悬而未决的问题。该提案旨在通过X射线晶体学在原子水平上确定这些蛋白质的结构来回答这个问题。我们还将通过计算、生物化学和生物物理实验来确定OprD家族成员可以转运什么样的分子。总之，这些信息应该使我们能够了解OprD家族成员如何在细菌膜中执行任务。除了从基本的角度来看很重要之外，这些知识还可以被制药公司用来设计更好的药物来对抗致病细菌。

DESCRIPTION (provided by applicant): The outer membrane (OM) of gram-negative bacteria serves as an effective barrier against the permeation of small molecules, thereby protecting the cell from noxious compounds encountered in the environment. In order to obtain the necessary molecules and ions required for growth and function of cell, transport channels are present within the OM. In most gram-negative bacteria such as E. coli, the majority of small molecules are taken up by porins, such as OmpF and OmpC. Porins are non-specific channels and do not bind their substrates. They form water-filled pores within the OM, through which small molecules diffuse, driven by their concentration gradient. The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen and most prevalently associated with lung infections in cystic fibrosis patients. A severe problem in the treatment of patients infected with P. aeruginosa is the high intrinsic antibiotics resistance of this bacterium. An important reason for the high antibiotics resistance is the low permeability of the OM, due to the absence of porins. In the absence of porins, substrate specific channels belonging to the OprD family are responsible for uptake of the majority of low molecular weight compounds in P. aeruginosa. OprD family members are closely related in sequence, yet transform very different substrates. The archetype of the family, OprD, functions as an uptake channel for basic amino acids and carbapenem antibiotics. No structural information is available for any OprD family member, and the basis for substrate selectivity and specificity within the OprD family is unknown. In this proposal we will determine the X-ray crystal structures of a number of OprD-family channels and characterize their substrate specificity using a range of computational, biochemical and biophysical experiments. More specifically, we will pursue the following aims: 1. Mechanism of substrate binding and transport of P. aeruginosa OprD. 2. Structure determination of a number of OprD family members at high resolution using X-ray crystallography. 3. Biochemical and biophysical characterization of OprD members with determined structures (aim 2). The OprD family provides an excellent opportunity to investigate the structural and biochemical basis for substrate specificity of closely related OM channel proteins. Such an analysis will be the first of its kind for membrane proteins and will provide, besides fundamental knowledge, information that can be used to design more effective drugs against P. aeruginosa. Project narrative Bacterial cells are surrounded by membranes that protect them from harmful and toxic compounds from the outside world. In order for the bacteria to grow, however, small nutrient molecules have to be transported across the membrane into the cell. For this task the bacteria are equipped with specialized proteins in the cell membrane that function as channels and transporters, and which ferry small molecules from the outside of the cell to the inside. For different types of molecules that have to be transported, the bacteria have different families of channel proteins. One such family is the OprD family, members of which occur in a number of human-disease causing bacteria. How members of the OprD family perform their transport tasks is an unanswered question. This proposal aims to answer that question by determining the structures of these proteins at an atomic level by X-ray crystallography. We will also determine what kind of molecules can be transported by members of the OprD family by computational, biochemical and biophysical experiments. Together, this information should enable us to understand how OprD family members perform their tasks in the bacterial membrane. Besides being important from a fundamental point of view, such knowledge could also be used by pharmaceutical companies to design better drugs against disease-causing bacteria.