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Thibaud Dieudonné, Sara Ursula Abad Herrera, Michelle Juknaviciute Laursen, Maylis Lejeune, Charlott Stock, Kahina Slimani, Christine Jaxel, Joseph A. Lyons, Cédric Montigny, Thomas Günther-Pomorski, Poul Nissen, Guillaume Lenoir

• P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. \(\it ATP8B1\) forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase \(\it {ATP8B1-CDC50A}\) at 3.1 Å resolution. We find that \(\it ATP8B1\) is autoinhibited by its \(\it N-\) and \(\it C\)-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the \(\it C\)-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5) P\(_3\)), and removal of both \(\it N\)- and \(\it C\)-termini results in full activation. Restored inhibition of \(\it ATP8B1\) truncation constructs with a synthetic peptide mimicking the \(\it C\)-terminal segment further suggests molecular communication between \(\it N\)- and \(\it C\)-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the \(\it C\)-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.