Gram-negative bacteria have become a serious threat to public health because their resistance to ?- lactam antibiotics, the most widely used and successful class of antibiotics worldwide. These pathogens resist multiple ??lactams chiefly through the acquisition of ??lactamase proteins, which hydrolytically destroy the drug. Moreover, under drug pressure, the ??lactamases are evolving broader ?-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 ?-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 ?-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.
Peng, Jeffrey, W. Narrative This proposal describes studies to understand inter-relationships between sequence, flexibility, and function in gram-negative pathogenic bacteria. The goal is to understand the physical basis for mutations in flexible regions leading to increased levels of antibiotic resistance.