Structural basis for the lipid asymmetry of the Gram-negative bacterial outer membrane

革兰氏阴性菌的特征是具有由两层膜组成的细胞膜，一层是内（或细胞质）膜（IM），另一层是外膜（OM）。这两种膜非常不同：内膜的两个小叶都含有磷脂（PL），因此可以认为是对称的。相比之下，OM是一个不对称的双分子层，内部的小叶是PL，外部的、表面暴露的小叶几乎完全由脂多糖（LPS）组成。LPS的性质和结构与PL有很大的不同，LPS存在的主要后果是革兰氏阴性菌被一层极层包围，这层极层对中性和疏水分子形成了非常有效的屏障。鉴于大多数药物具有中度疏水性，OM独特的不对称结构是革兰氏阴性菌对抗生素和其他有害化合物具有高内在耐药性的主要因素。因此，从细菌的角度来看，OM的不对称是非常重要的。然而，由于原因尚不清楚，但可能是正常细胞生长的结果，PL可积聚在OM的外层小叶。这些PL在LPS的海洋中形成了“岛屿”，增加了有毒化合物的渗透性。因此，PL需要从外层叶中移除，以恢复OM的渗透性屏障。Mla（维持脂质不对称）系统在革兰氏阴性菌中广泛存在，可能是维持OM脂质不对称最重要的系统。它由六种蛋白质组成，这些蛋白质被认为形成了PL从OM到IM的“反向”运输系统。MlaA蛋白是Mla系统的OM组分，被认为可以选择性地从外层OM小叶中提取PL。这是如何发生的尚不清楚，主要是由于缺乏关于MlaA的结构信息。我们已经确定了MlaA蛋白的第一个结构（通过x射线晶体学），以了解MlaA的功能。这些初步数据非常有趣，因为它们表明MlaA具有独特的结构，但更重要的是，它们提供了关于功能的清晰和可测试的线索。在本提案中，我们将以这些结构为起点，确定MlaA如何从OM外小叶中提取PL，并随后将其转移到质周空间的结合蛋白（MlaC）。我们将使用跨学科的方法，包括功能分析、生物物理方法、计算方法和x射线晶体学。我们的项目将阐明革兰氏阴性菌的一个基本和重要的过程，并将为瞄准革兰氏阴性病原体的Mla系统作为降低毒力和增强各种抗生素的手段提供前景。

Gram-negative bacteria are characterised by having a cell envelope consisting of two membranes, an inner (or cytoplasmic) membrane (IM) and an outer membrane (OM). The two membranes are very different: the two leaflets of the inner membrane both contain phospholipids (PL) and can therefore be considered symmetric. By contrast, the OM is an asymmetric bilayer with an inner leaflet of PL and an outer, surface-exposed leaflet composed almost exclusively of lipopolysaccharide (LPS). The properties and structure of LPS are very different from those of PL, and the main consequence of the presence of LPS is that Gram-negative bacteria are surrounded by a polar layer that forms a very effective barrier for neutral and hydrophobic molecules. Given that the majority of drugs are moderately hydrophobic, the unique, asymmetric structure of the OM is a major factor why Gram-negative bacteria have a high intrinsic resistance towards antibiotics and other harmful compounds. Thus, from the bacterium's point of view, the asymmetry of the OM is very important. However, due to reasons that are not yet clear but likely are a consequence of normal cell growth, PL can accumulate in the outer leaflet of the OM. Those PL form "islands" amid a sea of LPS that increase the permeability of noxious compounds. Thus, PL need to be removed from the outer leaflet to restore the OM permeability barrier.The Mla (maintenance of lipid asymmetry) system is widespread in Gram-negative bacteria and is likely the most important system for maintaining OM lipid asymmetry. It consists of six proteins that are thought to form a "reverse" transport system for PL from the OM to the IM. The protein MlaA is the OM component of the Mla system and is thought to selectively extract PL from the outer OM leaflet. How this happens is unclear, mainly due to a lack of structural information on MlaA. We have determined the first structures of MlaA proteins (by X-ray crystallography) to understand how MlaA functions. These preliminary data are very interesting in that they show that MlaA has a unique structure, but more importantly they provide clear and testable clues regarding function. In this proposal we will use the structures as starting points to determine how MlaA extracts PL from the OM outer leaflet and subsequently transfers them to a binding protein (MlaC) in the periplasmic space. We will use an interdisciplinary approach with functional assays, biophysical methods, computational approaches and X-ray crystallography. Our project will elucidate a fundamental and important process in Gram-negative bacteria and will inform on the prospect of targeting the Mla system of Gram-negative pathogens as a means to decrease virulence and to potentiate various antibiotics.