# Chemical Methods to Characterize Penicillin-Binding Protein Function and Interactions

> **NIH NIH R01** · UNIVERSITY OF MINNESOTA · 2020 · $305,252

## Abstract

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 cogna...

## Key facts

- **NIH application ID:** 10135597
- **Project number:** 1R01GM140486-01
- **Recipient organization:** UNIVERSITY OF MINNESOTA
- **Principal Investigator:** Erin Elizabeth Carlson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $305,252
- **Award type:** 1
- **Project period:** 2020-09-18 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10135597

## Citation

> US National Institutes of Health, RePORTER application 10135597, Chemical Methods to Characterize Penicillin-Binding Protein Function and Interactions (1R01GM140486-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10135597. Licensed CC0.

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