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
Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
