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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

基本信息

批准号：
10254419
负责人：
Erin Elizabeth Carlson
金额：
$ 30.68万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
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/10254419
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 Image Individual Investigation Knowledge Lactones Libraries Light 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 Resolution Rod Streptococcus pneumoniae Structure Work base beta-Lactams chemical genetics combat crosslink design drug development genetic approach genetic regulatory protein inhibitor/antagonist knowledge of results molecular modeling mutant novel protein activation protein function scaffold tool

项目摘要

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.

细胞壁的合成和重塑是细菌生长和分裂的核心，是许多细菌的靶标