A dual substrate-accessing mechanism of a major facilitator superfamily protein facilitates lysophospholipid flipping across the cell membrane.

Lysophospholipid transporter (LplT) is a member of the major facilitator superfamily present in many Gram-negative bacteria. LplT catalyzes flipping of lysophospholipids (LPLs) across the bacterial inner membrane, playing an important role in bacterial membrane homeostasis. We previously reported that LplT promotes both uptake of exogenous LPLs and intramembranous LPL flipping across the bilayer. To gain mechanistic insight into this dual LPL-flipping activity, here we implemented a combination of computational approaches and LPL transport analyses to study LPL binding of and translocation by LplT. Our results suggest that LplT translocates LPLs through an elongated cavity exhibiting an extremely asymmetric polarity. We found that two D(E)N motifs form a head group-binding site, in which the carboxylate group of Asp-30 is important for LPL head group recognition. Substitutions of residues in the head group-binding site disrupted both LPL uptake and flipping activities. However, alteration of hydrophobic residues on the interface between the N- and C-terminal domains impaired LPL flipping specifically, resulting in LPLs accumulation in the membrane, but LPL uptake remained active. These results suggest a dual substrate-accessing mechanism, in which LplT recruits LPLs to its substrate-binding site via two routes, either from its extracellular entry or through a membrane-embedded groove between transmembrane helices, and then moves them toward the inner membrane leaflet. This LPL-flipping mechanism is likely conserved in many bacterial species, and our findings illustrate how LplT adjusts the major facilitator superfamily translocation pathway to perform its versatile lipid homeostatic functions.