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 它们是在生化水平上起作用的。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，我们关注一类新的细胞分裂蛋白，它 包含一个“Spor”域名。在1000多个细菌物种的3000多个蛋白质中发现了Spor结构域， 包括许多严重的病原体。Spor结构域与隔膜肽聚糖(PG)结合，从而靶向许多 分裂蛋白到分裂隔膜。Spor结构域所结合的特定形式的PG是一种“剥离的” 葡聚糖，PG的氨基糖主干中没有多肽侧链的区域。这一不同寻常的目标 该机制将Spor结构域蛋白与许多通过蛋白质定位的隔环蛋白区分开来- 蛋白质的相互作用。本研究将使用四种大肠杆菌Spor结构域蛋白(DamX、DedD、FtsN和 RLPA)来检验两个相互关联的假设：(A)不同的Spor结构域以不同的方式结合去皮的多糖，以及 (B)这些差异在生理上是重要的。在AIM 1中，生物分子层干涉法(BLI)和等温 滴定量热法(ITC)将被用来确定Spor结构域与去皮多糖的亲和力 对于结合很重要的糖链的特征。此外，核磁共振将被用来确定第一个 具有PG配体的络合物中Spor结构域的结构。在目标2中，四个大肠杆菌Spor结构域蛋白将 使用尖端的单分子显微镜方法进行活体检查。这一目标的目标包括 确定不同的Spor结构域蛋白是否定位于隔膜的不同位置，并形成 更详细地描绘了隔环的整体结构。目标3将侧重于结构和功能 使用结构域交换和缺失分析来确定该蛋白中的重要结构域，归因于 将这些域映射到特定功能，并确定SPOR域在功能上是否可互换。 这些实验将把AIMS 1和2中的生化和显微镜与芽胞的实际功能联系起来 细胞分裂过程中的结构域蛋白。特别将致力于了解通过什么机制 DamX抑制细胞分裂，因为这对隔环蛋白来说是一种不寻常的活性，可能与 由感染膀胱的大肠杆菌引起的致病机制。

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.