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 Bacillus subtilis, with C55-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 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.

Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA / Oluwole, Abraham O.; Kalmankar, Neha V.; Guida, Michela; Bennett, Jack L.; Poce, Giovanna; Bolla, Jani R.; Robinson, Carol V.. - In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - ISSN 1091-6490. - 121:41(2024), pp. 1-10. [10.1073/pnas.2408315121]

Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA

Michela Guida;Giovanna Poce;
2024

Abstract

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 Bacillus subtilis, with C55-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 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.
2024
peptidoglycan; undecaprenyl phosphate transporter a; upta; native mass spectrometry
01 Pubblicazione su rivista::01a Articolo in rivista
Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA / Oluwole, Abraham O.; Kalmankar, Neha V.; Guida, Michela; Bennett, Jack L.; Poce, Giovanna; Bolla, Jani R.; Robinson, Carol V.. - In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - ISSN 1091-6490. - 121:41(2024), pp. 1-10. [10.1073/pnas.2408315121]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1721966
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