Spaciotemporal Regulation of Specific Penicillin Binding Protein (PBP) Function Determined by New Activity-Based Approaches

细胞壁的合成和重塑是细菌生长和分裂的重要过程。的 这一基础科学提案的长期目标是填补有关生物学的基础知识的主要空白。 合成肽聚糖（PG）的单个青霉素结合蛋白（PBP）的功能和调节 在细菌细胞壁中。一个主要的未满足的挑战，在映射PBP激活和定位，以及 PBPs与其相关的调节蛋白之间的关系，一直无法评估 存在于每种细菌物种中的单独PBP同系物的功能状态 成长与分裂我们建议开发新的工具和方法，以促进全面的 通过追求以下两个目标来理解PBP的功能和调节。目标1：发展选择性 在肺炎链球菌模型中，利用两种探针， 已知的和新的亲电支架，结合蛋白质晶体学和分子生物学， 建模针对生物体中每个PBP同源物的探针设计需要鉴定支架 选择性抑制每种酶。我们已经绘制了β-内酰胺文库的PBP选择性，提供了一个 为开发基于活性的探针奠定了坚实的基础;然而，许多PBP被 现有的β-内酰胺。为了解决这一额外的挑战，我们将采用分子建模和结构分析。 生物学研究，以及我们新发现的PBP选择性β-内酯支架，用于开发一套 的PBPs的探针。肺炎，这是一个强大的细胞生物学模型，以建立和验证 使用这种新型探针。目标2.绘制转肽酶的定位、定时和调控图 通过使用基于活性的探针和互补方法来检测特定PBPs的活性。我们将使用 目标1中基于特定活动的探针，以回答有关功能，定位， 以及S.肺炎模型;这些问题无法用现有的 战略布局在这个目标中，我们将使用尖端的高分辨率显微技术来确定空间 和时间分布的特定的，功能的PBP在不同阶段的S。pneumoniae细胞周期在 此外，我们还将确定细胞内活性PBPs的数量在假定的突变体中是如何改变的。 PBP调节器。最后，我们将通过使用基于活动和 互补的方法。这项赠款将回答有关PBP功能的基本问题， 时空调控的“超级细菌”pneumoniae模型，并提供新的工具和方法， 剖析PG合成在其他真细菌，从而导致更深入地了解这一复杂的 过程一般。此外，这项提议产生的新知识将揭示新的目标， PG合成中的步骤，可能被用作未来药物开发的弱点，以对抗 越来越多的抗药性细菌病原体的出现。

Cell wall synthesis and remodeling are essential processes central to bacterial growth and division. The long-term goal of this basic-science proposal is to fill in major gaps in fundamental knowledge about the functions and regulation of the individual penicillin-binding proteins (PBPs) that synthesize peptidoglycan (PG) in bacterial cell walls. A major unmet challenge in mapping PBP activation and localization, as well as the relationships between the PBPs and their associated regulatory proteins, has been the inability to assess the functional state of the separate PBP homologs that are present in each bacterial species over the course of growth and division. We propose to generate new tools and approaches that will contribute to a comprehensive understanding of PBP function and regulation by pursuit of the following two Aims. Aim 1: Develop selective activity-based probes for each PBP homolog in a Streptococcus pneumoniae model utilizing both known and novel electrophilic scaffolds, in combination with protein crystallography and molecular modeling. Design of probes to target each PBP homolog in an organism requires the identification of scaffolds that selectively inhibit each enzyme. We have mapped the PBP selectivity of a library of β-lactams, providing a solid foundation for the development of activity-based probes; however, many PBPs are poorly inhibited by existing β-lactams. To address this additional challenge, we will employ molecular modeling and structural biology studies, as well as our newly identified PBP-selective β-lactone scaffold for the development of a suite of probes for the PBPs of S. pneumoniae, which is a powerful cell biology model to establish and validate the use of this new type of probe. Aim 2. Map the localization, timing, and regulation of the transpeptidase activity of specific PBPs by using activity-based probes and complementary approaches. We will use the specific activity-based probes from Aim 1 to answer fundamental questions about the function, localization, and interactions of the PBPs in a S. pneumoniae model; these questions cannot be answered with existing strategies. In this Aim, we will use cutting-edge high-resolution microscopic techniques to determine the spatial and temporal distributions of specific, functional PBPs at different stages of the S. pneumoniae cell cycle. In addition, we will determine how cellular amounts of active PBPs are altered in mutants defective in putative PBP regulators. Finally, we will determine the interactors of PBPs by using both activity-based and complementary approaches. This grant will answer fundamental questions about PBP function and spaciotemporal regulation in a “superbug” S. pneumoniae model and provide new tools and approaches for dissecting PG synthesis in other eubacteria, and thereby lead to a deeper understanding of this complex process in general. In addition, the new knowledge produced by this proposal will reveal novel targets and steps in PG synthesis that may be exploited as vulnerabilities for future drug development to combat the increasing emergence of antibiotic-resistant bacterial pathogens.