Chemical Methods to Characterize Penicillin-Binding Protein Function and Interactions

细胞壁合成和重塑是细菌生长和分裂的中心，是许多细菌的靶标。 抗生素。尽管经过了几十年的研究，但我们对基本机制的理解仍然存在巨大差距 控制和协调细胞壁的生物合成，包括肽聚糖(PG)的组装。PG生物合成 利用多蛋白质复合体来协调微生物何时以及如何生长和分裂。批判性的阶级 在这个过程中的蛋白质是青霉素结合蛋白(Pbps)，它拉长和交联pg链。 是β-内酰胺类抗生素的靶标。蛋白质标记(例如，荧光融合)和超分辨率 成像策略极大地加强了对PG结构的研究，包括PBPS。然而，a 这些研究中缺少一条关键信息：每个PBP同系物何时何地具有催化活性 在组织的时候？我们率先开发了基于活动的探测器(ABP)，使我们能够跟踪 基于b-内酰胺和b-内酯支架的特定PBP同系物的催化活性 PBP转肽酶(TP)结构域。在这里，我们将利用现有的和新的ABP来评估PBP活动，通过 细胞分裂的过程，跟踪PBP的定位，并确定关键的调节蛋白伙伴是必不可少的 合适的细胞壁结构。这些目标将通过追求三个目标来实现。目标1.绘制 特定PBPs在细胞分裂过程中催化活性的定位、时间选择和调节。目前还不清楚 当每个PBP同系物积极促进PG生物合成时。我们将利用现有的选择性APB来 用超分辨成像研究细胞分裂过程中PBP的激活并评价多蛋白 调节PBP活动和运动的复合体。目的2.扩大PBP选择性ABPs文库 已知的和新型的亲电支架，结合蛋白质结晶学和分子建模。我们 将结合分子建模和共结晶研究来确定PBP同源物的关键特征 差异化。通过合理的探针设计和靶向文库的合成，我们将扩大 我们特定于PBP的血压。目的3.绘制PBP活性中心拓扑图，以便更深入地了解底物和 抑制物识别和发展等位基因特异的化学遗传学方法。一大笔钱 选择性ABPs开发中的挑战是PBP TP结构域的结构同源性。我们可以的 利用这一特性来开发一种等位基因特异的化学遗传学方法，也被称为“凹坑”， 其中，将保守活性中心残基突变以产生一个“空穴”，并对WT抑制剂或底物进行修饰 伴随着一种补充的化学“碰撞”。我们将调查保守的活性中心残基对 抑制物结合和PBPS中天然底物周转效率以确定适当的突变和 生成同源词“颠簸的”ABP(S)，用于同源词的特定研究。总体而言，在 这项拟议的工作将阐明在PG合成过程中如何利用每个PBP同系物以及 这些复合体的成分可能是未来药物开发的重要靶点。

Cell wall synthesis and remodeling are central to bacterial growth and division, and are targeted by numerous antibiotics. Despite decades of study, there are still huge gaps in our understanding of the basic mechanisms that control and coordinate cell wall biosynthesis, including the assembly of peptidoglycan (PG). PG biosynthesis utilizes multi-protein complexes to coordinate when and how a microbe grows and divides. A critical class of proteins in this process is the penicillin-binding proteins (PBPs), which elongate and crosslink the PG strands and are the targets of b-lactam antibiotics. Protein tagging (e.g., fluorescent fusions) and super-resolution imaging strategies have dramatically enhanced the study of PG construction, including the PBPs. However, a key piece of information is missing from these studies: when and where is each PBP homolog catalytically active during division? We have pioneered the development of activity-based probes (ABPs) that enable tracking of the catalytic activity of specific PBP homologs based on b-lactam and b-lactone scaffolds, which target the conserved PBP transpeptidase (TP) domain. Here, we will utilize existing and novel ABPs to evaluate PBP activity through the process of cell division, track PBP localization, and identify key regulatory protein partners that are essential to proper cell wall construction. These goals will be achieved by pursuit of three Aims. Aim 1. Map the localization, timing, and regulation of the catalytic activity of specific PBPs throughout cell division. It is not clear when each PBP homolog is actively contributing to PG biosynthesis. We will use existing selective APBs to investigate PBP activation during cell division with super-resolution imaging and evaluate the multi-protein complex(es) that regulate PBP activity and movement. Aim 2. Expand the library of PBP-selective ABPs utilizing known and novel electrophilic scaffolds, in combination with protein crystallography and molecular modeling. We will combine molecular modeling and co-crystallization studies to identify key features for PBP homolog differentiation. Through rational probe design and the synthesis of targeted libraries we will expand the scope of our PBP-specific ABPs. Aim 3. Map PBP active site topology for a deeper understanding of substrate and inhibitor recognition and the development of an allele-specific chemical genetics approach. A substantial challenge in the development of selective ABPs is the structural homology of the PBP TP domains. We can leverage this characteristic to develop an allele-specific chemical genetics approach, also known as “bump-hole,” in which a conserved active site residue is mutated to create a “hole” and a WT inhibitor or substrate is modified with a complementary chemical “bump.” We will investigate the contribution of conserved active site residues to inhibitor binding and native substrate turnover efficiency in the PBPs to identify an appropriate mutation and generate cognate “bumped” ABP(s) for homolog-specific studies. In total, the knowledge and tools generated in the proposed work will shed light on how each PBP homolog is utilized throughout PG synthesis, as well as point to components of these complexes that may be important targets for future drug development.