Structural basis of phosphatidylglycerol recognition and trafficking at the outer membrane

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》杂志编辑布鲁斯·阿尔伯特（Bruce Albert）最近对我们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，我们的理解也存在巨大的差距。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？”[Science，2012，337：6102]。我们打算通过定义大肠杆菌蛋白质YraP的功能和机制来填补这一生物学知识的空白。根据Interpro数据库，至少有6746种与YraP相似的蛋白质已经从包括许多病原体在内的广泛细菌物种中测序，因此与许多微生物和传染病有更广泛的相关性。此外，作为一种独特的细菌基因，表达的压力，YraP提供了潜在的机会，设计的抗菌剂，特别是一旦合适的筛选试验和机制的见解是可用的。YraP相关蛋白的多样性，其通常由一对BON结构域组成，包括溶血素、机械敏感通道、膜孔形成蛋白Secretin和几种真核蛋白。这表明一个共同的功能，涉及脂质识别或膜操纵，首先提出的C叶芝和A贝特曼在2003年，虽然他们是如何在结构和机制的水平仍然不清楚。我们的主要重点是解决原子分辨率结构和分子间的相互作用YraP，提供了关于BON结构域如何与包围所有革兰氏阴性菌的保护性外膜的磷脂组分结合的第一个实验见解。外膜的主要功能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）的流入和流出。插入外膜的蛋白质包括90种脂质修饰的蛋白质，如YraP，以及作为免疫反应靶点的孔、通道和抗原。这些脂蛋白嵌入由免疫原性脂多糖、磷脂和糖脂组成的双层中。我们将研究YraP如何结合和组织脂质，并有助于将它们运输到细菌的外膜。为了发展对YraP功能的第一个机制性理解，我们将使用包括核磁共振光谱（NMR）在内的生物物理方法。可视化YraP的蛋白质结构和配体相互作用的原子将提供有价值的见解，了解它们接触的氨基酸残基和脂质的功能作用。PG识别和外膜特异性的这些原理可以帮助发现分子抑制剂和新型抗菌剂，YraP在控制细胞渗透性和确保细菌应激期间的活力方面发挥关键作用。涉及一个联合结构-由来自微生物和感染研究所和亨利惠康生物分子核磁共振光谱学大楼（HWB-NMR）的两个研究小组对一种新靶标进行的功能分析，分别我们将结合联合收割机伊恩亨德森的细菌学小组的分子和细胞的见解，该小组刚刚发现了YraP在脂质运输中的作用，沿着迈克尔Overduin小组的结构生物学专业知识，该小组专门研究阐明蛋白质：脂质识别和运输机制。

The overall aim of this project is to determine how bacteria recognize and organize lipids in their outer membraneThe challenge we are tackling here is best described by Bruce Albert, the Editor of Science, who recently 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?" [Science, 2012, 337: 6102]. We intend to fill this gap of biological knowledge by defining the function and mechanism of an E.coli coli protein called YraP. According to the Interpro database there are at least 6746 proteins with similarity to YraP which have been sequenced from a wide range of bacterial species including many pathogens, and hence there is broader relevance to many micro-organisms and infectious diseases. Moreover, as a unique bacterial gene that is expressed in response to stress, YraP offers potential opportunities for the design of antimicrobial agents, especially once suitable screening assays and mechanistic insights are available. The diversity of YraP-related proteins, which typically are composed of a pair of BON domains, includes haemolysins, mechanosensitive channels, the membrane-pore forming protein Secretin, and several eukaryotic proteins. This suggests a common function involving lipid recognition or membrane manipulation, as first proposed by C Yeats and A Bateman in 2003, although how they are related at a structural and mechanistic level remains unclear.Our primary focus is on solving the atomic resolution structures and molecular interactions of YraP, providing the first experimental insights into how the BON domain engages the phospholipid components of the protective outer membrane that surrounds all Gram negative bacteria. The outer membrane's main function is to form a semi-permeable layer that controls the influx and efflux of nutrients and other materials including drug molecules. The proteins inserted into outer membranes include 90 lipid modified proteins like YraP as well as pores, channels and antigens that act as targets of immune responses. These lipoproteins are embedded into a bilayer composed of immunogenic lipopolysaccharides, phospholipids and glycolipids. We will investigate how YraP binds and organizes lipids and contributes to trafficking them to the outer membrane of bacteria. In order to develop the first mechanistic understanding of YraP function, we will use biophysical methods including nuclear magnetic resonance spectroscopy (NMR). Visualising the atoms of the protein structures and ligand interactions of YraP will provide valuable insights into the functional roles of the amino acid residues and lipids they contact. These principles of PG recognition and outer membrane specificity could aid in the discovery of molecular inhibitors and new classes of antimicrobial agents, with YraP playing a key role in controlling cell permeability and ensuring viability during bacterial stress.This project represents collaborative science, involving a joint structure-function analysis of a novel target by two research groups from the Institute of Microbiology and Infection and the Henry Wellcome Building for Biomolecular NMR Spectroscopy (HWB-NMR), respectively. We will combine the molecular and cellular insights of Ian Henderson's bacteriology group, which has just discovered the role of YraP in lipid trafficking, along with the structural biological expertise within Michael Overduin's group, which specialises in elucidating protein:lipid recognition and trafficking mechanisms.