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MprF from Pseudomonas aeruginosa is a promiscuous lipid scramblase with broad substrate specificity
Matthew T K Hankins1, Matyas Parrag2, Alisa A Garaeva3
1Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Abstract:
The multiple peptide resistance factor (MprF) is a bifunctional membrane protein found in many bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus. MprF modifies inner leaflet lipid headgroups through aminoacylation and translocates modified lipid to the outer leaflet. This activity provides increased resistance to antimicrobial agents. MprF presents a promising target in multiresistant pathogens, but structural information is limited and both substrate specificity and energization of MprF-mediated lipid transport are poorly understood. Here, we present the cryo-EM structure of MprF from P. aeruginosa (PaMprF) bound to a synthetic nanobody. PaMprF adopts an "open" conformation with a wide, lipid-exposed groove on the periplasmic side that induces a local membrane deformation in molecular dynamics simulations. Using an in vitro liposome transport assay, we demonstrate that PaMprF translocates a wide range of different lipids without an external energy source. This suggests that PaMprF is the first dedicated lipid scramblase to be characterized in bacteria.
Insights
The multiple peptide resistance factor (MprF) protein
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- The multiple peptide resistance factor (MprF) is a bifunctional membrane protein crucial for antimicrobial resistance in bacteria like *Pseudomonas aeruginosa* and *Staphylococcus aureus*.
- MprF's activity involves modifying and translocating lipids, but its structure, substrate specificity, and transport mechanism remain poorly understood.
- Limited structural data hinders the development of MprF as a therapeutic target against multiresistant pathogens.
Purpose of the Study:
- To elucidate the structural basis of MprF function using cryo-electron microscopy (cryo-EM).
- To investigate the substrate specificity and energy requirements of MprF-mediated lipid transport.
- To characterize MprF as a potential lipid scramblase.
Main Methods:
- Determined the cryo-EM structure of *Pseudomonas aeruginosa* MprF (PaMprF) in complex with a synthetic nanobody.
- Utilized molecular dynamics simulations to analyze membrane interactions and conformational changes.
- Employed an in vitro liposome transport assay to assess lipid translocation capabilities.
Main Results:
- Revealed the cryo-EM structure of PaMprF in an "open" conformation, featuring a wide, lipid-exposed groove.
- Observed that this groove induces local membrane deformation, suggesting a mechanism for lipid interaction.
- Demonstrated that PaMprF efficiently translocates a diverse range of lipids without requiring an external energy source.
Conclusions:
- The structural and functional data suggest PaMprF functions as a dedicated lipid scramblase.
- MprF's ability to translocate lipids without external energy provides new insights into bacterial membrane dynamics.
- Understanding MprF's mechanism offers potential avenues for developing novel antimicrobial strategies targeting resistant bacteria.
