The maintenance of (phospho)lipid (PL) asymmetry (Mla) transporter was named based on its role in maintaining outer membrane (OM) PL asymmetry in Escherichia coli (Ec). The Mla transporter is an ATP-binding cassette transport system proposed to traffic PLs. It consists of six core components, including the OM lipoprotein, MlaA; the periplasmic PL chaperone, MlaC; the ATP-binding cassette, MlaE, MlaF; and two auxiliary proteins, MlaD, MlaB. Deletion of any mla gene in Acinetobacter baumannii (Ab) or E. coli largely reduced the minimum inhibitory concentration of all antibiotics tested, showing that the Mla transporter is essential for the maintenance of OM integrity. Therefore, the Mla transporter is necessary for bacterial survival and recognized as an attractive drug target. MlaC and the periplasmic facing portion of MlaD play a key role in PL transport.
MlaC transports PLs between the inner membrane and OM, and MlaD receives or transfers PLs from or to MlaC. Although Mla components from E. coli and A. baumannii are structurally similar and both have substrates as PLs, AbMlaD has an additional region of 47-amino acids (aa), which is not found in any other Mla orthologues. In this study, PL-free MlaC was incubated with PL-bound MlaD (or vice versa), the Mla proteins were separated via size exclusion chromatography, and PLs were then extracted from the purified Mla proteins where thin layer chromatography was used to identify the bound PLs. We show that AbMlaD and EcMlaD can transfer PLs to AbMlaC and EcMlaC, respectively (but not vice versa), and that EcMlaD can transfer PLs to AbMlaC (but not vice versa). In contrast, PL transfer between AbMlaD and EcMlaC was observed in both directions. When the structures of AbMlaD and EcMlaD are superimposed, the 47-aa additional region of AbMlaD is found in a similar position to the periplasmic facing β6-β7 loop of EcMlaD that contains two hydrophobic residues required for the EcMlaC-EcMlaD interaction. Together these data suggest that the 47-aa additional region of AbMlaD is spatially positioned to interact with AbMlaC and EcMlaC via a larger hydrophobic surface and, therefore, hypothesize that this region may enhance the MlaC-MlaD interaction.