Molecular mechanism of phospholipid transport at the bacterial outer membrane interface

Abstract The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer with outer leaflet lipopolysaccharides (LPS) exposed to extracellular milieu and inner leaflet phospholipids (PLs) facing the periplasm. This unique lipid asymmetry is the key to its innate drug resistance, rendering the OM impermeable to external insults, including antibiotics and bile salts. To maintain this OM barrier, the OmpC-Mla system removes mislocalized PLs from the OM outer leaflet, and transports them back to the inner membrane (IM); in the first step, the OM OmpC-MlaA complex transfers PLs to the periplasmic chaperone MlaC. This process likely occurs via a hydrophilic channel in MlaA, yet mechanistic details have remained elusive. Here, we biochemically and structurally characterize the architecture of the MlaA-MlaC transient complex. We map the interaction surfaces between MlaA and MlaC in Escherichia coli , revealing that MlaC binds MlaA at the periplasmic face in a manner that possibly juxtaposes the MlaA channel and the MlaC lipid binding cavity. In addition, we show that electrostatic interactions between the putative C-terminal tail helix of MlaA and a surface patch on MlaC are important for recruitment of the latter to the OM. We further provide biochemical evidence for conformational changes in the MlaA channel that correlate with interactions with MlaC and OM porins, as well as functional states of MlaA. Finally, we solve a 2.9-Å cryo-EM structure of OmpC-MlaA in nanodiscs in a disulfide-trapped complex with MlaC, reinforcing the mechanism of MlaC recruitment, and highlighting membrane thinning as a plausible strategy for directing lipids into the MlaA channel. Our work offers critical insights into how the OmpC-MlaA complex catalyzes retrograde transport of PLs to the IM to maintain OM lipid asymmetry.