Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA

• 成果类型: Article
• 署名作者: Oluwole, Abraham O.; Kalmankar, Neha V.; Guida, Michela; Bennett, Jack L.; Poce, Giovanna; Bolla, Jani R.; Robinson, Carol V.
• 署名单位: University of Oxford; University of Oxford; Sapienza University Rome; University of Oxford
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10202
• DOI: 10.1073/pnas.2408315121
• 发表日期: 2024-10-03
• 关键词: cell-wall biosynthesis; pyrophosphate phosphatase; crystal-structure; membrane; mechanism; mray

摘要

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C-55-P), across the membrane to reenter the pathway. This translocation of C-55-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 Bacillus subtilis, with C-55-P, membrane phospholipids, and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in Escherichia coli, forms monomer-dimer equilibria, and binds to C-55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favorably with C-55-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 C-55-P. Overall, this study shows that UptA-mediated translocation of C-55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.