Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last but critical step being the flipping of carrier lipid, undecaprenyl phosphate (C ₅₅ -P), across the membrane to renter the pathway. This translocation of C ₅₅ -P is facilitated by DedA and DUF386 domain-containing family membrane proteins via unknown mechanisms. Here we employ native mass spectrometry to investigate the interactions of UptA, a putative flippase from Bacillus subtilis , with C ₅₅ -P, membrane phospholipids and cell wall targeting antibiotics. Our results show that UptA forms monomer-dimer equilibria and binds to ligands in a pH-dependent fashion. Specifically, we show that UptA interacts more favourably with C ₅₅ -P over shorter-chain analogues and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly intercept UptA function by outcompeting the substrate for the protein binding site. Overall, this study shows that UptA-mediated translocation of C ₅₅ -P is potentially regulated by anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.