Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA.

The 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 reenter 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 from <i>Bacillus subtilis</i>, with C₅₅-P, membrane phospholipids, and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in <i>Escherichia coli</i>, forms monomer-dimer equilibria, and binds to C₅₅-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favorably with C₅₅-P over shorter-chain analogs 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.