Bacterial outer membrane biogenesis through a novel periplasmic protein that recognizes and traffics phospholipids

该项目的总体目标是确定细菌如何识别和组织外膜中的脂质。《科学》杂志的编辑Bruce Alberts对我们面临的挑战做了最好的描述，他说：“我痛苦地意识到，即使是最简单的细胞，我们对它的理解仍然存在巨大的差距。以常见的大肠杆菌为例，在分子生物学的早期，它是一种主要的模式生物。令人警醒的是，50多年后，在其基因组编码的4000多种蛋白质中，有近四分之一的功能仍然未知。通过对这些蛋白质的研究，可能会发现一些对所有细胞都有共同功能的生物分子的新类别吗？“******我们将通过阐明一种名为YraP的大肠杆菌蛋白的机制来解决这一空白。在包括各种病原体在内的一系列细菌中发现了至少7000种与YraP相似的蛋白质，因此对传染病有影响。此外，YraP作为一种独特的基因产物，在应激反应中表达，一旦其结构、功能和筛选方法成熟，将为抗菌药物的设计提供机会。YraP的进化亲缘关系包括溶血素、机械敏感通道、膜孔形成蛋白分泌素和多种真核蛋白。我们认为它们具有基于脂质识别的共同功能。******我们的重点是了解YraP的细胞功能，分子相互作用和3D结构。我们将揭示这种脂蛋白是如何与所有革兰氏阴性细菌周围的膜中发现的磷脂结合的。外膜的作用是形成半透层，控制营养物质和包括药物分子在内的其他物质的进出。插入这层膜的蛋白质包括孔、通道和抗原，它们是免疫反应的目标。我们认为它们都依赖于YraP将特定的脂质传递到外膜。我们将研究YraP如何识别和组织这种脂质，以及它如何从细胞内运输脂质。我们将使用包括磁共振波谱在内的方法来可视化结构和结合界面的原子，以揭示其氨基酸残基和它们接触的脂质的特定作用。由此产生的配体识别和膜生物发生原理将为抑制剂、疫苗和抗菌剂的发现提供信息。该项目是合作的，涉及加拿大国家核磁共振设施和英国微生物学与感染研究所的研究人员对新靶标的详细结构-功能分析。我们将利用我们共同发现的YraP在脂质运输中的作用，以及一个新的核磁共振系统，旨在阐明这种蛋白质超家族如何识别脂质，塑造膜和保护细胞。

The overall aim of this project is to determine how bacteria recognize and organize lipids in their outer membrane. The challenge we are tackling here is best described by Bruce Alberts, Editor of Science, who stated "I am painfully aware of the huge gap that remains in our understanding of even the simplest cells. Consider, for example, the common bacterium E. coli, which served as a predominant model organism in the early years of molecular biology. It is very sobering to report that more than 50 years later, nearly a quarter of the more than 4000 proteins encoded by its genome have functions that remain unknown. Might some new functional classes of biological molecules, common to all cells, be discovered by a focus on such proteins?"******We will address this gap by elucidating the mechanism of an E. coli protein called YraP. There are at least 7000 proteins with similarity to YraP which have been found in a range of bacteria including various pathogens, and hence there are implications for infectious diseases. Moreover, as a unique gene product that is expressed in response to stress, YraP could offer opportunities for the design of antimicrobial agents once its structure, function and screening assays are available. The evolutionary relatives of YraP includes hemolysins, mechanosensitive channels, the membrane-pore forming protein Secretin, and a variety of eukaryotic proteins. We propose that they share a common function based on lipid recognition. ******Our focus is on understanding the cellular function, molecular interactions and 3D structures of YraP. We will reveal how this lipoprotein engages the phospholipids found in the membrane that surrounds all Gram negative bacteria. The outer membrane's role is to form a semi-permeable layer that controls the entry and exit of nutrients and other materials including drug molecules. The proteins inserted into this membrane include pores, channels and antigens that act as targets of immune responses. We propose that they all depend on YraP to deliver a specific lipid to the outer membrane. We will investigate how YraP recognizes and organizes this lipid, and how it traffics lipid from inside the cell. We will use methods including magnetic resonance spectroscopy to visualize the atoms of the structures and binding interfaces to expose the specific roles of its amino acid residues and lipids they contact. The resulting principles of ligand recognition and membrane biogenesis will inform the discovery of inhibitors, vaccines and antimicrobial agents. This project is collaborative, involving detailed structure-function analysis of a novel target by researchers at Canada's national NMR facility and the UK-based Institute of Microbiology and Infection. We will capitalize on our joint discovery of the role of YraP in lipid trafficking, along with a new NMR system geared to elucidate how this superfamily of proteins recognizes lipids, shapes membranes and protect cells.