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J Am Chem Soc. 2018 Apr 4;140(13):4481-4484. doi: 10.1021/jacs.8b00942. Epub 2018 Mar 26.

Membrane Potential Is Required for MurJ Function.

Author information

1
Department of Chemistry and Chemical Biology , Harvard University , Cambridge , Massachusetts 02138 , United States.
2
Department of Microbiology , The Ohio State University , Columbus , Ohio 43210 , United States.
3
Department of Microbiology and Molecular Genetics , Harvard Medical School , Boston , Massachusetts 02115 , United States.

Abstract

MurJ, the flippase that exports the bacterial cell wall monomer Lipid II to the periplasm, is a target for new antibiotics, which are desperately needed to treat Gram-negative infections. Quantitative methods to monitor MurJ activity are required to characterize inhibitors but are challenging to develop because the lipid-linked substrate is not chemically altered in a flippase reaction. Here we show that MurJ inhibition can be quantified by measuring the accumulation of intracellular Lipid II using a biotin-tagging strategy. We have exploited this assay to show that MurJ is inhibited in the presence of a compound that dissipates the membrane potential. By probing cysteine accessibility we have found that under this condition MurJ relaxes into an inactive, outward-facing conformation reminiscent of that targeted by the peptide antibiotic LysM. We conclude that membrane potential is required for MurJ function in E. coli, and we anticipate that the ability to accumulate this inactive conformation will lead to structures useful for inhibitor design.

PMID:
29558128
PMCID:
PMC5892188
[Available on 2019-04-04]
DOI:
10.1021/jacs.8b00942
[Indexed for MEDLINE]

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