Targeting and assembly of E. coli cell division proteins

 描述（由申请人提供）：细菌细胞分裂的研究很重要，原因有二。首先，这是一个需要理解的基本细胞过程。其次，随着目前新型抗生素的缺乏，细菌胞质分裂的普遍和必要过程 作为抗微生物药物的靶点越来越重要。在细菌如大肠杆菌中，胞质分裂由两种必需且高度保守的细胞骨架蛋白：微管蛋白样FtsZ和肌动蛋白样FtsA协调。这些蛋白质在细胞分裂位点的内膜上共同组装成一个环形聚合结构，称为Z环。一旦组装好，这个环就会招募一个由其他蛋白质组成的大复合体到膜上，可能分布在单个的亚复合体中。这种蛋白质机器，通常被称为分裂体，诱导合成隔膜肽聚糖，同时在生长的隔膜的前缘收缩，最终将细胞一分为二。这台机器需要强大，但对各种输入反应灵敏， 因此过度建造。这项建议的重点是FtsA及其与FtsZ和后来的分裂体蛋白的相互作用，因为最近的研究结果表明，FtsA调节FtsZ的组装，除了其在束缚FtsZ聚合物的膜和招聘后来的分裂体组件的作用。FtsZ可以在膜上施加收缩力，但这种力需要调节和协调，与其后面的分裂隔膜的生长。我们假设，FtsA是高度动态的，是FtsZ组装和回收的主要调节器，它稳定了机器，并最大限度地提高了其灵活性。然而，我们不知道是什么调节FtsA的活性。我们的工作假设是，FtsA的能力之间的平衡，与自己的广告FtsZ的互动是至关重要的，它的活动。我们在此提出阐明FtsA的ATP结合、ATP酶活性、寡聚化状态以及与动力学相互作用和调节动力学的能力之间的联系。 的Z环。FtsZ、FtsA和第三种蛋白质ZipA的复合物构成了所谓的“原环”。为了阐明原环蛋白之间的关系，我们建议研究FtsA中的突变，更有效地结合FtsZ，FtsA和ZipA之间的竞争FtsZ结合位点的影响，以及ZipA和FtsA在调节成束的FtsZ原丝的作用。我们最近发现了一种新的ftsZ突变体，该突变体形成了过量的纤维束并绕过了对ZipA的需求，并通过了解一种小肽抑制FtsZ纤维束的机制，对FtsZ纤维束的研究将有所帮助。最后，我们现在知道，FtsA，这是一个“早期”分裂体蛋白，直接招募一个“晚期”分裂体蛋白，FtsN，也许是为了不断增长的分裂隔膜和收缩Z环之间的反馈。我们建议确定这种相互作用的分子细节，这种潜在的早期招募FtsN是如何重要的divisome活动，以及如何可以规避这种相互作用。

 DESCRIPTION (provided by applicant): The study of bacterial cell division is important for two reasons. First, it is a basic cellular process that needs to be understood. Second, with the current scarcity of novel antibiotics, the universal and essential process of bacterial cytokinesis is increasingly relevant as a target of antimicrobial drugs. In bacteria such as Escherichia coli, cytokinesis is orchestrated by two essential and highly conserved cytoskeletal proteins: tubulin-like FtsZ and actin-like FtsA. These proteins coassemble into a circumferential polymeric structure, called the Z ring, on the inner membrane at the site of cell division. Once assembled, the ring then recruits a large complex of other proteins to the membrane, probably distributed in individual subcomplexes. This protein machine, often called the divisome, induces synthesis of septal peptidoglycan while constricting at the leading edge of the growing septum, eventually splitting the cell into two. The machine needs to be robust, yet responsive to a variety of inputs, and is therefore overbuilt. This proposal focuses on FtsA and its interactions with FtsZ and with later divisome proteins, because recent results indicate that FtsA regulates assembly of FtsZ, in addition to its role in tethering FtsZ polymers to the membrane and recruiting later divisome components. FtsZ can impart a constriction force on the membrane, but this force needs to be regulated and coordinated with growth of the division septum behind it. We hypothesize that FtsA is highly dynamic and a major regulator of FtsZ assembly and recycling, which stabilizes the machine and maximizes its flexibility. However, we do not understand what regulates FtsA's activity. Our working hypothesis is that a balance between FtsA's ability to interact with itself ad FtsZ is crucial for its activity. We propose here to elucidate the connection between FtsA's ATP binding, ATPase activity, oligomerization state, and ability to interact with and regulate dynamics of the Z ring. The complex of FtsZ, FtsA and a third protein, ZipA, constitutes the so-called "proto-ring". To elucidate the relationship among proto-ring proteins, we propose to investigate the effect of mutations in FtsA that bind FtsZ more efficiently, competition between FtsA and ZipA for FtsZ binding sites, and the role of ZipA and FtsA in regulating the bundling of FtsZ protofilaments. Studies of FtsZ filament bundling will be aided by a new ftsZ mutant we recently discovered that forms excess bundles and bypasses the requirement for ZipA, and by understanding the mechanism by which a small peptide inhibits FtsZ filament bundling. Finally, we now know that FtsA, which is an "early" divisome protein, directly recruits a "late" divisome protein, FtsN, perhaps for the purpose of feedback between the growing division septum and the constricting Z ring. We propose to determine the molecular details of this interaction, how important this potentially early recruitment of FtsN is for divisome activity, and how this interaction can be circumvented.