TY - JOUR

T1 - Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

AU - Dieudonné, Thibaud

AU - Herrera, Sara Abad

AU - Laursen, Michelle Juknaviciute

AU - Lejeune, Maylis

AU - Stock, Charlott

AU - Slimani, Kahina

AU - Jaxel, Christine

AU - Lyons, Joseph A.

AU - Montigny, Cédric

AU - Pomorski, Thomas Günther

AU - Nissen, Poul

AU - Lenoir, Guillaume

N1 - Publisher Copyright: © 2022, Dieudonné et al.

PY - 2022/4

Y1 - 2022/4

N2 - 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.

AB - 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.

KW - autoinhibition

KW - biochemistry

KW - chemical biology

KW - Cryo-EM

KW - lipid flippase

KW - molecular biophysics

KW - P4-ATPases

KW - phosphoinositides

KW - progressive familial intrahepatic cholestasis

KW - S. cerevisiae

KW - structural biology

UR - http://www.scopus.com/inward/record.url?scp=85129778691&partnerID=8YFLogxK

U2 - 10.7554/eLife.75272

DO - 10.7554/eLife.75272

M3 - Journal article

C2 - 35416773

AN - SCOPUS:85129778691

VL - 11

JO - eLife

JF - eLife

SN - 2050-084X

M1 - e75272

ER -