Structure of the Bacterial Lipid ABC Transporter MlaFEDB Reveals Substrate Bound

Abstract

The outer membrane (OM) of Gram-negative bacteria acts as an important barrier that protects against external stressors, rendering double-membraned bacteria intrinsically more antibiotic resistant. The OM is composed of asymmetrically distributed lipopolysaccharide (LPS) and phospholipid (PL) molecules, which are required for building and maintaining the OM. Much has been learned about how LPS is moved between the inner and outer membranes, while less is known regarding PL transport. The Maintenance of Lipid Asymmetry (Mla) system has previously been implicated in OM maintenance by lipid transport across the cell envelope. This system includes an inner membrane (IM) ATP-Binding Cassette (ABC) transporter complex, MlaFEDB, which is thought to drive PL transport. The mechanism of PL transport by this system is poorly characterized, in part due to limited structural information of this transport complex. We determined the cryo-EM structure of MlaFEDB to 3.05 Å resolution, revealing the transmembrane domain of MlaFEDB, MlaE, to have a fold conserved with the LPS and MacAB transport systems. Additionally, we identified two PLs bound in an outward-open facing pocket at the periplasmic region of the MlaE dimer interface. These PLs are observed in two distinct orientations, which may represent intermediate states in the translocation of PL through the complex. Site-specific UV crosslinking confirmed the presence of lipids in this binding pocket in vivo. This structure's conserved fold and bound PLs provide structural insights into the MlaFEDB transport mechanism.