Chemical Methods to Characterize Penicillin-Binding Protein Function and Interactions

细胞壁的合成和重塑是细菌生长和分裂的核心，并且是许多细菌生长和分裂的靶点。 抗生素尽管经过了几十年的研究，我们对基本机制的理解仍然存在巨大差距 控制和协调细胞壁生物合成，包括肽聚糖（PG）的组装。PG生物合成 利用多蛋白复合物来协调微生物何时以及如何生长和分裂。一个关键的类 在这个过程中的蛋白质是青霉素结合蛋白（PBP），它延长和交联PG链 并且是β-内酰胺抗生素的靶点。蛋白质标记（例如，荧光融合）和超分辨率 成像策略显著地增强了对PG构建（包括PBP）的研究。但 这些研究中缺少一个关键信息：每个PBP同系物在何时何地具有催化活性 在组织里吗我们率先开发了基于活动的探针（ABP），可以跟踪 基于b-内酰胺和b-内酯骨架的特定PBP同系物的催化活性，其靶向保守的 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活性， 细胞分裂过程，跟踪PBP定位，并确定关键的调控蛋白伴侣是必不可少的 到细胞壁的正确构建这些目标将通过追求三个目标来实现。目标1.映射 在整个细胞分裂过程中，特定PBP的催化活性的定位、定时和调节。目前尚不清楚 当每个PBP同源物积极地促进PG生物合成时。我们会利用现有的选择性通告， 用超分辨率成像研究细胞分裂过程中PBP的激活，并评估多蛋白 调节PBP活性和运动的复合物。目标二。扩大PBP选择性ABPs库， 已知的和新的亲电支架，结合蛋白质晶体学和分子建模。我们 将结合联合收割机分子建模和共结晶研究，以确定PBP同系物的关键特征 分化通过合理的探针设计和靶向文库的合成，我们将扩大 我们的PBP特异性ABPs目标3。绘制PBP活性位点拓扑图，以更深入地了解底物和 抑制剂识别和等位基因特异性化学遗传学方法的发展。大幅 选择性ABP开发中的挑战是PBP TP结构域的结构同源性。我们可以 利用这一特性来开发等位基因特异性化学遗传学方法，也称为“bump-hole”， 其中保守的活性位点残基被突变以产生“洞”，并且WT抑制剂或底物被修饰 有一个互补的化学“碰撞”我们将研究保守的活性位点残基对 PBPs中的抑制剂结合和天然底物周转效率，以鉴定适当的突变， 生成同源“碰撞”ABP，用于同源物特异性研究。总体而言， 所提出的工作将阐明每个PBP同系物在整个PG合成过程中是如何被利用的， 这些复合物的成分可能是未来药物开发的重要目标。

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.