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其外膜中的脂质。《科学》杂志的编辑布鲁斯·艾伯茨（Bruce Alberts）对我们在这里面临的挑战进行了最好的描述，他说：“我痛苦地意识到，即使是最简单的细胞，我们的理解仍然存在巨大的差距。例如，考虑一下常见的细菌E。大肠杆菌，在分子生物学的早期作为主要的模式生物。50多年后的今天，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，这是非常发人深省的。通过对这些蛋白质的关注，是否会发现一些新的生物分子功能类别，这些生物分子是所有细胞都共有的？“** 我们将通过阐明E.大肠杆菌中的蛋白质YraP。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，因此对感染性疾病有意义。此外，作为一种独特的基因产物，表达的压力，YraP可以提供机会，设计的抗菌剂，一旦其结构，功能和筛选试验。YraP的进化亲属包括溶血素、机械敏感通道、膜孔形成蛋白Secretin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如何与所有革兰氏阴性菌周围的膜中发现的磷脂结合。外膜的作用是形成一个半渗透层，控制营养物质和其他物质（包括药物分子）的进出。插入该膜中的蛋白质包括孔、通道和抗原，它们充当免疫反应的靶点。我们认为它们都依赖于YraP将特定的脂质递送到外膜。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光谱法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的原则将通知抑制剂，疫苗和抗菌剂的发现。该项目是合作的，涉及加拿大国家NMR设施和英国微生物学和感染研究所的研究人员对一种新靶标的详细结构-功能分析。我们将利用我们对YraP在脂质运输中的作用的联合发现，沿着一个新的NMR系统，以阐明这个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.