Structural basis of the outer membrane protein assembly system by NMR spectroscopy

在这项研究项目中，将通过包括磁共振光谱在内的生物物理方法分析细菌细胞膜中发现的蛋白质的三维结构和功能。我们将表征与YaeT跨膜蛋白相互作用的四种大肠杆菌蛋白的分子机制。这种细菌蛋白质复合物跨越外膜并与来自周质空间的其他蛋白质相互作用，将它们分泌出细胞并进入外膜和周围环境。在这里，我们专注于四个辅助脂蛋白，NlpB，YfgL，YfiO和SmpA，这与YaeT形成一个基本的复合物，并发现在基本上所有的革兰氏阴性菌。它们一起募集并折叠所有形成桶状折叠的外膜蛋白。革兰氏阴性细菌的特征是围绕称为肽聚糖的聚合物网络的保护性外膜。外膜的主要功能是在肽聚糖周围形成半渗透层。例如，它控制营养物质和其他物质（包括药物分子）的流入和流出。包括孔和通道的蛋白质插入外膜中以调节其渗透性。插入外膜的一些蛋白质是重要的抗原，其充当保护性免疫应答的靶标，例如自转运蛋白，其是所有革兰氏阴性细菌内最广泛使用的蛋白质分泌系统。对许多细菌基因组的分析表明，我们正在研究的保守蛋白组装复合物普遍存在于革兰氏阴性细菌的外膜中，并且对其生存至关重要。这种基本系统也存在于一些革兰氏阳性细菌和线粒体和叶绿体中，反映了这些细胞器的细菌起源，它也是生存所必需的。因此，虽然我们专注于大肠杆菌系统，但我们的工作结果将对各种细胞类型和细胞器中的蛋白质组装和折叠产生广泛的影响。我们正在研究的脂蛋白负责以其折叠和功能形式产生外膜蛋白，因此对细菌细胞的活力和正常生理学非常重要。了解这四种脂蛋白如何识别和组装蛋白质对于靶向致病菌和在细菌感染期间操纵免疫反应非常重要。可视化它们的结构并表征它们的配体相互作用和结合口袋提供了有价值的机制见解，可以帮助发现分子抑制剂和新类别的抗菌剂。不同类型革兰氏阴性菌的外膜除了蛋白质外还含有多种脂质。外叶由不同菌种的脂多糖、糖脂和磷脂组成。外膜中的脂质对于蛋白质组装到膜中是必不可少的，并且在免疫应答中也具有重要作用。因此，我们还将研究脂质和膜与脂蛋白的相互作用，以更好地了解脂蛋白如何在膜表面取向和组装。

In this research project, the three dimensional structures and functions of proteins found in the membrane that surrounds bacterial cells will be analyzed by biophysical methods including magnetic resonance spectroscopy. We will characterize the molecular mechanisms of four Escherichia coli proteins which interact with the YaeT transmembrane protein. This bacterial protein complex spans the outer membrane and interacts with other proteins from the periplasmic space, secreting them out of the cell and into the outer membrane and surrounding environment. Here we focus on four accessory lipoproteins, NlpB, YfgL, YfiO and SmpA, which form an essential complex with YaeT and are found in essentially all Gram negative bacteria. Together they recruit and fold all outer membrane proteins which form barrel folds. Gram negative bacteria are characterized by a protective outer membrane that surrounds a polymeric network known as peptidoglycan. The outer membrane's main function is to form a semi-permeable layer around the peptidoglycan. For example, it controls the influx and efflux of nutrients and other materials including drug molecules. Proteins including pores and channels are inserted into the outer membrane in order to regulate its permeability. Some of the proteins inserted into the outer membrane are important antigens that act as targets of protective immune responses e.g. the autotransporter proteins, the most widely used protein secretion system within all Gram-negative bacteria. The analysis of many bacterial genomes has revealed that the conserved protein assembly complex we are studying is universally found in outer membranes of Gram negative bacteria, and is essential for their survival. This basic system is also found in some Gram positive bacteria and mitochondria and chloroplasts, reflecting the bacterial origins of such organelles, where it is also essential for survival. Thus, although we are focussing on the E coli system, the results of our work will have broad implications for protein assembly and folding in a diverse range of cell types and organelles. The lipoproteins we are studying are responsible for the production of outer membrane proteins in their folded and functional forms, and hence are important for the viability and normal physiology of the bacterial cell. Understanding how the four lipoproteins recognize and assemble proteins is important for targeting pathogenic bacteria and for manipulating the immune response during a bacterial infection. Visuallizing their structures and characterizing their ligand interactions and binding pockets provides valuable mechanistic insights that could aid in the discovery of molecular inhibitors and new classes of antimicrobial agents. The outer membranes of different types of Gram-negative bacteria contain a variety of lipids in addition to proteins. The outer leaflet is composed of lipopolysaccharides, glycolipids and phospholipids which differ between bacterial species. The lipids in the outer membrane are essential for the assembly of proteins into the membrane, and also have important roles in immune response. Consequently, we will also investigate the interactions of lipids and membranes with lipoproteins in order to better understand how the lipoproteins are oriented and assemble at the membrane surface.