Fig. 4. Proposed model for Mfsd2a lipid transport cycle.

a The ligand-bound, outward-facing mouse Mfsd2a structure docked with ALA-LPC is bound in a lateral position Chamber₁ (see Supplementary discussion). The LPC headgroup is trapped by Z_A in an outward pointing orientation. b The proposed and unknown occluded ligand-bound conformation. Mfsd2a rocks from an outward to inward-facing conformation. c–e The various conformations during flipping of the LPC headgroup. The lipid tail is held in position by Chamber₁ while the LPC headgroup samples multiple configurations between Z_A and Z_B before it is moved to Z_B. This movement flips the LPC headgroup from an outward to inward-pointing orientation. This process results in a substrate with the lipid tail pointing out and the LPC headgroup pointing in, a ligand configuration that is aligned with the inner membrane leaflet. f The translocation of the lysolipid to Chamber₂ and Z_B. The lipid tail is translocated from Chamber₁ to Chamber₂. The LPC headgroup is shifted inwards. This process translocates the entire lipid-LPC substrate closer to the cytoplasmic exit. g The release of the substrate to the cytoplasmic side. LPC headgroup is moved to Z_C and the lipid tail is translocated to Chamber₃. Chamber₃ and Z_C are located at the cytoplasmic exit. h The substrate-free structure of drMfsd2a in the inward-facing conformation. This structure represents the conformation after lysolipid release from Mfsd2a. i–j The conformations during resetting of the transporter. In the absence of the substrate, Mfsd2a resets to the outward-open conformation (j) by first transitioning through a ligand-free occluded state (i).