Flippases transport phospholipids between leaflets of the plasma membrane bilayer to establish and maintain appropriate membrane asymmetry. How flippases are able to move such large substrates is not well understood. The flippase ATP8A2 is a P-type ATPase that has not been structurally characterized but is thought to resemble both in structure and in catalytic cycle the structurally characterized P-type ATPase cation pumps, containing a transmembrane domain with ten helices (M1–M10). As the M4 segment of the cation-transporting ATPases and, specifically, a glutamate within the segment have been implicated in substrate binding, Vestergaard et al. focused on this segment within ATP8A2 to gain new insights into the flippase mechanism. They began by mutating the position equivalent to the glutamate, I364, and residues adjacent to it. Among six I364 mutations, the authors saw a direct correlation between the apparent affinity for the substrate phosphatidylserine and ATPase activity levels. Another M4 mutant, N359A, significantly affected ATPase activity and apparent substrate affinity. Homology models refined by molecular dynamics revealed a groove bordered by M1, M2, M4 and M6 that is substantially larger than that in the cation-transporting ATPases. The groove contained a substantial amount of water filling in two distinct pockets, which were differentially placed in the two modeled conformations. Further mutagenesis of a hydrophobic cluster of residues in this region including I115 and F88 suggest that the groove forms a path for lipid head group transport from the exoplasm to a cytoplasmic site in the bilayer, with I364 playing a part in the release of the phospholipid during its transfer.
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Bucci, M. A phospholipid path. Nat Chem Biol 10, 409 (2014). https://doi.org/10.1038/nchembio.1537