Activation and substrate specificity of the human P4-ATPase ATP8B1

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Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases.
OriginalsprogEngelsk
Artikelnummer7492
TidsskriftNature Communications
Vol/bind14
Udgave nummer1
Antal sider14
ISSN2041-1723
DOI
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
We thank Thomas Boesen, Andreas Bøggild, and Taner Drace for technical support during EM data collection at the EMBION Danish National cryo-EM facility of Aarhus University (5072-00025B, Danish Agency for Research and Higher Education) and Jesper Lykkegaard Karlsen for scientific computing support. We are grateful to Anna Marie Nielsen, Tanja Klymchuk, and Bente Andersen for their technical assistance. We also wish to thank, Tomás Heger, Line Marie Christiansen, Ronja Driller, Cédric Montigny, Christine Jaxel, and David Stokes for discussion and advice. T.D. also warmly thank Camille and Côme for their patience during the revision process. This work was supported by a Marie Skłodowska-Curie Actions Individual Fellowship — LivFlip (101024542) and by a Fondation Recherche Médicale (ARF202209015714) grant to T.D., by Engineering and Physical Sciences Research Council grants (EP/X035603 and EP/V030779) to S. K., by an ANR grant (ANR-14-CE09-0022) to G.L., by the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05) to G.L., by the Center National de la Recherche Scientifique (CNRS) to G.L., by a Lundbeckfonden Fellow grant (R335-2019-2053) to J.A.L, by the Lundbeck Foundation to the BRAINSTRUC structural biology initiative (155-2015-2666) to K.L.-L. and P.N., and by a Lundbeckfonden Professorship grant (R310-2018-3713) to P.N.

Funding Information:
We thank Thomas Boesen, Andreas Bøggild, and Taner Drace for technical support during EM data collection at the EMBION Danish National cryo-EM facility of Aarhus University (5072-00025B, Danish Agency for Research and Higher Education) and Jesper Lykkegaard Karlsen for scientific computing support. We are grateful to Anna Marie Nielsen, Tanja Klymchuk, and Bente Andersen for their technical assistance. We also wish to thank, Tomás Heger, Line Marie Christiansen, Ronja Driller, Cédric Montigny, Christine Jaxel, and David Stokes for discussion and advice. T.D. also warmly thank Camille and Côme for their patience during the revision process. This work was supported by a Marie Skłodowska-Curie Actions Individual Fellowship — LivFlip (101024542) and by a Fondation Recherche Médicale (ARF202209015714) grant to T.D., by Engineering and Physical Sciences Research Council grants (EP/X035603 and EP/V030779) to S. K., by an ANR grant (ANR-14-CE09-0022) to G.L., by the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05) to G.L., by the Center National de la Recherche Scientifique (CNRS) to G.L., by a Lundbeckfonden Fellow grant (R335-2019-2053) to J.A.L, by the Lundbeck Foundation to the BRAINSTRUC structural biology initiative (155-2015-2666) to K.L.-L. and P.N., and by a Lundbeckfonden Professorship grant (R310-2018-3713) to P.N.

Publisher Copyright:
© 2023, The Author(s).

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