Structural basis of MsbA-mediated lipopolysaccharide transport

TLDR: This study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

Abstract: Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 A-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases. Cryo-electron microscopy snapshots of the E. coli flippase MsbA at discrete functional states reveal a ‘trap and flip’ mechanism for lipopolysaccharide flipping and the conformational transitions of MsbA during its substrate transport cycle. The translocation and flipping of lipids across membrane bilayers is important for maintaining lipid asymmetry, but it also plays a part in membrane trafficking and signalling. This process is catalysed by flippases, bacterial ATP-binding cassette transporters that flip lipopolysaccharide (LPS). Because of the importance of LPS transport to bacterial survival, these transporters are also a target for the development of antibiotics. Here, Maofu Liao and colleagues present the structural basis of LPS recognition by the Escherichia coli flippase MsbA and reveal the trajectory of LPS. Using cryo-electron microscopy the authors provide snapshots of several of the states involved in lipid translocation across the bacterial membrane. In one of these states, they detected electron density within the transmembrane domain of MsbA corresponding to LPS. On the basis of these insights, the researchers propose a 'trap and flip' mechanism in which LPS translocates far into the transporter without flipping.