MprF from Pseudomonas aeruginosa is a promiscuous lipid scramblase with broad substrate specificity.

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.