Abstract
AbstractThe peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C55-P), across the membrane to re-enter the pathway. This translocation of C55-P is facilitated by DedA and DUF368 domain-containing family membrane proteins via unknown mechanisms. Here we employ native mass spectrometry to investigate the interactions of UptA, a member of the DedA family of membrane protein fromBacillus subtilis, with C55-P, membrane phospholipids and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified inE. coli, forms monomer-dimer equilibria, and binds to C55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favourably with C55-P over shorter-chain analogues and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly inhibit UptA function by out-competing the substrate for the protein binding, in addition to their propensity to form complex with free C55-P. Overall, this study shows that UptA-mediated translocation of C55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.
Publisher
Cold Spring Harbor Laboratory