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

Abstract

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.

Dissecting the conformational complexity and flipping mechanism of a prokaryotic heme transporter

“…These results suggest that CydDC operates via a trap-and-flip mechanism similar to that described for the MFS type proton-dependent lipid transporter LtaA and other primary and secondary active lipid transporters (41)(42)(43)(44)(45). In MsbA and PfMATE, lipid flipping is primarily driven by electrostatic interactions between lipid head groups and side chains of the respective transporters (42,45).…”

CHARMM-GUI Membrane Builder: Past, Current, and Future Developments and Applications

“…MD simulations can capture substrate movement in transporters. Lambert et al used MD simulations to study LtaA, a proton-dependent major facilitator superfamily lipid transporter that is essential for lipoteichoic acid synthesis in the pathogen Staphylococcus aureus . In their study, both inward-open and outward-open conformations of the transporter were illustrated by examining the solvent accessibility of the cavities (Figure C).…”

“…From the applications in the CHARMM-GUI community, we note that the membrane simulations are increasingly serving as a computational microscopy with statistical reliability . Processes occurring in or out of the membrane surface, including vesicle fusion, DNA transfection, virus infection, substrate translocation, nanopore sequencing, drug delivery, protein detergent extraction, and pollutant toxicity, as well as mutation and drug-induced protein conformational changes, can all be visualized and analyzed at the atomistic level. ,,,,,,,,,, …”

“…Lambert et al used MD simulations to study LtaA, a proton-dependent major facilitator superfamily lipid transporter that is essential for lipoteichoic acid synthesis in the pathogen Staphylococcus aureus. 170 In their study, both inward-open and outward-open conformations of the transporter were illustrated by examining the solvent accessibility of the cavities (Figure 4C). Notably, the simulations revealed that the lipid substrate (gentiobiosyl-diacylglycerol) permeates into the lower region of the lateral gate through the lateral opening of the translocation channel.…”

Structural basis for triacylglyceride extraction from mycobacterial inner membrane by MFS transporter Rv1410

“…The ~2300 Å 3 outward-facing central cavity of MHAS2168 is well-equipped to accommodate lipids, in that the cavity walls of both domains are very hydrophobic, even compared to other MFS lipid transporters LtaA 30,31 and MFSD2A [32][33][34] (Extended Data Fig. 3).…”