Phospholipid flipping involves a central cavity in P4 ATPases

P4 ATPase flippases translocate phospholipids across biomembranes, thus contributing to the establishment of transmembrane lipid asymmetry, a feature important for multiple cellular processes. The mechanism by which such phospholipid flipping occurs remains elusive as P4 ATPases transport a giant substrate very different from that of other P-type ATPases such as Na⁺/K⁺- and Ca²⁺-ATPases. Based on available crystal structures of cation-transporting P-type ATPases, we generated a structural model of the broad-specificity flippase ALA10. In this model, a cavity delimited by transmembrane segments TM3, TM4, and TM5 is present in the transmembrane domain at a similar position as the cation-binding region in related P-type ATPases. Docking of a phosphatidylcholine headgroup in silico showed that the cavity can accommodate a phospholipid headgroup, likely leaving the fatty acid tails in contact with the hydrophobic portion of the lipid bilayer. Mutagenesis data support this interpretation and suggests that two residues in TM4 (Y374 and F375) are important for coordination of the phospholipid headgroup. Our results point to a general mechanism of lipid translocation by P4 ATPases, which closely resembles that of cation-transporting pumps, through coordination of the hydrophilic portion of the substrate in a central membrane cavity.