Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

Elife. 2022 Apr 13:11:e75272. doi: 10.7554/eLife.75272.


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. 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 ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and 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 C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

Keywords: Cryo-EM; P4-ATPases; S. cerevisiae; autoinhibition; biochemistry; chemical biology; lipid flippase; molecular biophysics; phosphoinositides; progressive familial intrahepatic cholestasis; structural biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases* / metabolism
  • Cell Membrane / metabolism
  • Cholestasis, Intrahepatic* / genetics
  • Cholestasis, Intrahepatic* / metabolism
  • Humans
  • Mutation
  • Phosphatidylinositols* / metabolism
  • Phospholipid Transfer Proteins / metabolism


  • Phosphatidylinositols
  • Phospholipid Transfer Proteins
  • Adenosine Triphosphatases
  • ATP8B1 protein, human

Grants and funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.