Peptidoglycan binding protein specificity and bacterial cell division

细菌的细胞分裂需要大量蛋白质的协同作用，这些蛋白质形成一种称为 隔膜环未来的主要挑战包括识别所有这些蛋白质，并确定它们是什么。 在生物化学层面上是这样的我们的长期目标是识别和描述以前被忽视的 对细胞分裂很重要的细菌蛋白质。在这里，我们集中在一类新的细胞分裂蛋白， 包含“SPOR”域。SPOR结构域存在于来自1000多种细菌物种的3000多种蛋白质中， 包括许多严重的病原体。SPOR结构域结合间隔肽聚糖（PG），从而靶向许多 分裂蛋白质到分裂隔膜。SPOR结构域结合的PG的具体形式是“裸露的”PG。 聚糖，PG的氨基糖骨架的区域，没有肽侧链。这种不寻常的目标 这种机制使SPOR结构域蛋白与许多通过蛋白质- 蛋白质相互作用本研究将使用四种大肠杆菌SPOR结构域蛋白（DamX、DedD、FtsN和 （a）不同的SPOR结构域不同地结合裸露的聚糖，以及 (b)这些差异在生理上是重要的。在Aim 1生物层干涉法（BLI）和等温 滴定量热法（ITC）将用于确定SPOR结构域对裸露聚糖的亲和力， 对结合很重要的聚糖的特征。此外，NMR将用于确定第一个 与PG配体复合SPOR结构域的结构。在目标2中，四个E. coli SPOR结构域蛋白将 使用复杂的单分子显微镜方法在体内进行检查。这一目标包括： 确定不同的SPOR结构域蛋白是否定位于隔膜上的不同位点， 更详细的中隔环整体结构图。目标3将侧重于结构和功能 的DamX，使用结构域交换和缺失分析来识别该蛋白质中的重要结构域， 这些结构域与特定的功能，并确定SPOR结构域是否是功能上可互换的。 这些实验将把目标1和2中的生物化学和显微镜与SPOR的实际功能联系起来 蛋白质在细胞分裂中的作用。我们将特别努力去了解 DamX抑制细胞分裂，因为这是隔环蛋白的一种不寻常的活性，可能与 致病性E.感染膀胱的大肠杆菌。

Cell division in bacteria requires the concerted action of a large number of proteins that form a complex called the septal ring. Major challenges for the future include identifying all of these proteins and determining what they do on a biochemical level. Our long-term goal is to identify and characterize previously overlooked bacterial proteins that are important for cell division. Here we focus on a new class of cell division proteins that contain a “SPOR” domain. SPOR domains are found in over 3000 proteins from over 1000 bacterial species, including many serious pathogens. SPOR domains bind septal peptidoglycan (PG) and thereby target many division proteins to the division septum. The specific form of PG to which SPOR domains bind is a “denuded” glycan, a region of the amino sugar backbone of PG devoid of peptide side-chains. This unusual targeting mechanism sets SPOR domain proteins apart from the many septal ring proteins that localize via protein- protein interactions. This study will use four Escherichia coli SPOR domain proteins (DamX, DedD, FtsN and RlpA) to test two interrelated hypotheses: (a) different SPOR domains bind denuded glycans differently, and (b) these differences are physiologically important. In Aim 1 biolayer interferometry (BLI) and isothermal titration calorimetry (ITC) will be used to determine the affinity of SPOR domains for denuded glycans and the features of the glycan that are important for binding. In addition, NMR will be used to determine the first structure of a SPOR domain in complex with a PG ligand. In Aim 2, the four E. coli SPOR domain proteins will be examined in vivo using sophisticated single-molecule microscopy methods. Objectives of this aim include ascertaining whether different SPOR domain proteins localize to different sites at the septum and developing a more detailed picture of the overall architecture of the septal ring. Aim 3 will focus on the structure and function of DamX, using domain swapping and deletion analysis to identify important domains in that protein, ascribe those domains to specific functions and determine whether SPOR domains are functionally interchangeable. These experiments will connect the biochemistry and microscopy in Aims 1 & 2 to the actual function of SPOR domain proteins during cell division. Particular effort will be devoted to understanding the mechanism by which DamX inhibits cell division, because this is an unusual activity for a septal ring protein and may be relevant to pathogenesis by E. coli that infect the bladder.