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.
This grant will generate new chemical tools and approaches needed to build a comprehensive understanding of bacterial cell wall synthesis, which is central to growth and survival. This work will fill in major gaps in fundamental knowledge about bacterial cell wall synthesis, critical for the future development of novel antibiotics and strategies to combat antibiotic-resistant bacterial diseases.