Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders
- Université Paris-Saclay, CEA, CNRS, France
- Ruhr University Bochum, Germany
- Aarhus University, Denmark
Abstract
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, e.g. 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.
Data availability
Refined coordinates for the atomic model of the autoinhibited state of ATP8B1 have been deposited in PDB under the accession code 7PY4.The cryo-EM map of autoinhibited ATP8B1 has been deposited in EMDB under the accession code EMD-13711.
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Atomic model of ATP8B1-CDC50A in the E2P autoinhibited stateProtein Data Bank ID 7PY4.
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Cryo-EM map of ATP8B1-CDC50A in the E2P autoinhibited stateElectron Microscopy Data Bank ID EMD-13711.
Article and author information
Author details
Funding
EMBO (Short-term fellowship,7881)
- Thibaud Dieudonné
French Infrastructure for Integrated Structural Biology (FRISBI,ANR-10-INSB-05)
- Christine Jaxel
- Cédric Montigny
- Guillaume Lenoir
French ministry for higher education (PhD fellowship)
- Thibaud Dieudonné
European Commission (Marie Sklodowska-Curie individual fellowship)
- Thibaud Dieudonné
Agence Nationale de la Recherche (Young investigator grant,ANR-14-CE09-0022)
- Guillaume Lenoir
Lundbeckfonden (Professorship grant)
- Poul Nissen
Deutsche Forschungsgemeinschaft (GU 1133/11-1)
- Thomas Günther Pomorski
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Dieudonné et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders
eLife 11:e75272.
https://doi.org/10.7554/eLife.75272
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Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.
