# Defining flexibility and activity relationships for gram-negative antibiotic resistance proteins

> **NIH NIH R01** · UNIVERSITY OF NOTRE DAME · 2020 · $270,375

## Abstract

ABSTRACT
 Gram-negative bacteria have become a serious threat to public health because their resistance to β-
lactam antibiotics, the most widely used and successful class of antibiotics worldwide. These pathogens
resist multiple β−lactams chiefly through the acquisition of β−lactamase proteins, which hydrolytically
destroy the drug. Moreover, under drug pressure, the β−lactamases are evolving broader β-lactamase
activity. Understanding the mechanisms for these “gain-of-activity” mutations is crucial for anticipating and
curbing their effects.
 An intriguing clue has come from clinical isolates of Acinetobacter baumannii, a Gram-negative pathogen
and a global clinical scourge. A baumannii deploys a Class D β-lactamase, OXA-24, to inactivate penicillins
and carbapenems. Recently, clinical isolates of A. baumannii with expanded resistance were traced to
substitution mutations within flexible segments of OXA-24 associated with substrate recognition. These
results raise our overall hypothesis that conformational dynamics can influence the substrate spectrum of
Class-D β-lactamases, specifically, in the flexible recognition loops at the protein surface.
 We therefore propose investigating this hypothesis through flexibility-activity studies of OXA-24 and
substitution mutants already established to cause “gain-of-activity” phenotypes in the clinic. Our
investigations use liquid state NMR to characterize the conformational ensembles of the free enzyme and
substrate, and acyl-enzyme complex, for WT-OXA-24 and resistant variants.
Aim 1. Compare the conformational sampling of apo OXA-24/40 with that of its clinical variants.
Aim 2. Define the site-specific changes in ligand conformational flexibility caused by complex
formation.
Aim 3. Compare the conformational sampling of the OXA-24/40/ligand complexes with those of its
clinical variants.
 A predictive understanding of how flexible protein regions respond to resistance-expanding mutations
remains an open challenge. Our proposed research answers this challenge via investigations into the role of
protein flexibility in expanding gram-negative antibiotic resistance. Our results may suggest new strategies
for improved inhibitors, and new insights into how proteins evolve new functions.

## Key facts

- **NIH application ID:** 9898388
- **Project number:** 5R01GM123338-03
- **Recipient organization:** UNIVERSITY OF NOTRE DAME
- **Principal Investigator:** JEFFREY W PENG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $270,375
- **Award type:** 5
- **Project period:** 2018-05-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9898388

## Citation

> US National Institutes of Health, RePORTER application 9898388, Defining flexibility and activity relationships for gram-negative antibiotic resistance proteins (5R01GM123338-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9898388. Licensed CC0.

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