Membrane Potential Is Required for MurJ Function
Frederick A Rubino1, Sujeet Kumar2, Natividad Ruiz2
1Department of Chemistry and Chemical Biology , Harvard University , Cambridge , Massachusetts 02138 , 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.
Insights
New antibiotics targeting MurJ (a flippase) are crucial for Gram-negative infections. Researchers developed a novel assay to quantify MurJ inhibition by measuring Lipid II accumulation, aiding antibiotic development.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- MurJ is a bacterial flippase essential for exporting Lipid II, a key component of the cell wall.
- Developing new antibiotics against Gram-negative bacteria is a critical unmet medical need.
- Existing methods to quantify MurJ activity are limited due to the lack of substrate chemical alteration during transport.
Purpose of the Study:
- To develop a quantitative assay for monitoring MurJ flippase activity.
- To identify conditions and compounds that inhibit MurJ function.
- To understand the conformational changes of MurJ in response to inhibition.
Main Methods:
- A biotin-tagging strategy was employed to quantify intracellular accumulation of Lipid II as a measure of MurJ inhibition.
- The assay was used to test the effect of membrane potential on MurJ activity.
- Cysteine accessibility probing was performed to analyze MurJ conformation.
Main Results:
- A novel assay successfully quantified MurJ inhibition by measuring intracellular Lipid II accumulation.
- MurJ activity was inhibited by compounds that dissipate the membrane potential.
- Inhibition under membrane potential dissipation led to MurJ adopting an inactive, outward-facing conformation.
Conclusions:
- Membrane potential is essential for MurJ function in *E. coli*.
- The developed assay allows for the quantification of MurJ inhibitors.
- The inactive, outward-facing conformation of MurJ provides a structural target for novel antibiotic design.
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