Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

LtaA catalyzes glycolipid translocation by a 'trap-and-flip' mechanism, pointing to a shared mechanistic model among MFS lipid transporters. Asymmetric lateral openings allow access of the entire lipid substrate to the amphipathic central cavity. Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters take part in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acid synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a 'trap-and-flip' mechanism that might be shared among MFS lipid transporters.

Automated summary

LtaA is a proton-dependent MFS lipid transporter that catalyzes the translocation of glycolipids through a 'trap-and-flip' mechanism. LtaA has asymmetric lateral openings and its entire amphipathic central cavity can bind to lipids, while its hydrophilic pocket determines substrate specificity.