Chemical Methods to Characterize Penicillin-Binding Protein Function and Interactions

表征青霉素结合蛋白功能和相互作用的化学方法

• 批准号: 10645143
• 负责人: Erin Elizabeth Carlson
• 金额: $ 30.63万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10645143
• 关键词: Active Sites; Alleles; Anabolism; Antibiotics; Bacillus subtilis; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Binding; Biochemical; Cell Division Process; Cell Wall; Cell division; Characteristics; Chemicals; Chemistry; Complex; Crystallization; Crystallography; Data; Development; Engineering; Evaluation; Foundations; Future; Genetic; Goals; Grant; Growth; Homologous Gene; Homologous Protein; Individual; Investigation; Knowledge; Lactams; Lactones; Libraries; Maps; Methods; Microbe; Microbiology; Microscopic; Molecular Machines; Monobactams; Movement; Multiprotein Complexes; Mutate; Mutation; Organism; Penicillin-Binding Proteins; Peptidoglycan; Peptidyltransferase; Planet Earth; Polymers; Process; Protein Isoforms; Proteins; Regulation; Research; Rod; Shapes; Streptococcus pneumoniae; Structure; Work; beta-Lactams; chemical genetics; combat; crosslink; design; drug development; genetic approach; genetic regulatory protein; inhibitor; knowledge of results; molecular modeling; mutant; novel; protein activation; protein function; scaffold; superresolution imaging; tool

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.

细胞壁合成和重塑是细菌生长和分裂的核心，是许多细菌的靶点。 抗生素。尽管经过几十年的研究，我们对其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 利用多蛋白质复合物来协调微生物生长和分裂的时间和方式。批判性的一类 此过程中的蛋白质是青霉素结合蛋白 (PBP)，它会延长并交联 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 成像策略极大地增强了 PG 结构（包括 PBP）的研究。然而，一个 这些研究中缺少关键信息：每个 PBP 同系物何时何地具有催化活性 分裂期间？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-内酯支架的特定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 活动： 细胞分裂过程，跟踪 PBP 定位，并识别重要的关键调节蛋白伙伴 适当的细胞壁构建。这些目标将通过追求三个目标来实现。目标 1. 绘制地图 整个细胞分裂过程中特定 PBP 催化活性的定位、时间安排和调节。目前还不清楚 当每个 PBP 同源物积极促进 PG 生物合成时。我们将利用现有的选择性 APB 通过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 活动和运动的复合物。目标 2. 利用扩展 PBP 选择性 ABP 库 已知的和新颖的亲电子支架，与蛋白质晶体学和分子建模相结合。我们 将结合分子建模和共结晶研究来确定 PBP 同系物的关键特征 差异化。通过合理的探针设计和目标文库的合成，我们将扩大研究范围 我们的 PBP 特定 ABP。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开发。一个实质性的 开发选择性 ABP 的挑战是 PBP TP 结构域的结构同源性。我们可以 利用这一特性开发等位基因特异性化学遗传学方法，也称为“凹凸孔” 其中保守的活性位点残基发生突变以产生“孔”，并且 WT 抑制剂或底物被修饰 具有互补的化学“凸块”。我们将研究保守活性位点残基对 PBP 中的抑制剂结合和天然底物周转效率，以确定适当的突变和 生成同源“碰撞”ABP 用于同系物特异性研究。总的来说，产生的知识和工具 拟议的工作将阐明如何在整个 PG 合成过程中利用每个 PBP 同源物，以及点 这些复合物的成分可能是未来药物开发的重要目标。