Characterization of Lipidated New Delhi Metallo-β-lactamase Using Synthetic Nanodiscs

Carbapenem-resistant bacteria present a serious challenge to current treatment methods for bacterial infections. Particularly concerning are metallo-carbapenemases, a subset of plasmid-borne β-lactamases, active against nearly all β-lactam antibiotics and insusceptible to available β-lactamase inhibitors. The most widespread metallo-carbapenemase is New Delhi metallo-β-lactamase (NDM). NDM is unique among all known metallo-β-lactamases due to a post-translational lipidation that anchors it to the outer membrane. Most β-lactamases, including all other metallo-carbapenemases, exist as soluble proteins in the periplasm of Gram-negative bacteria. The functional impact of membrane anchoring remains unclear because most biochemical studies are performed on truncated soluble proteoforms that may have structural and functional differences from the full-length form. To study the functional impact of lipidation, the overexpression of lipidated NDM was optimized in Escherichia coli and the lipoprotein was solubilized into synthetic nanodiscs, consisting of lipid bilayer segments encircled by a stabilizing polymer. Highlighting the evolution of metallo-β-lactamases for improved fitness under zinc-limiting conditions, membrane anchoring improves zinc affinity while thermostability is found to be differentially altered in different clinical variants of NDM. These results improve the understanding of the function of a unique membrane-anchored β-lactamase as well as establish a platform for the use of nanodisc-based approaches for the study of bacterial lipoproteins.