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

摘要

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.