Activation and substrate specificity of the human P4-ATPase ATP8B1
Abstract 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 exo...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-11-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-42828-9 |
| _version_ | 1797558027697520640 |
|---|---|
| author | Thibaud Dieudonné Felix Kümmerer Michelle Juknaviciute Laursen Charlott Stock Rasmus Kock Flygaard Syma Khalid Guillaume Lenoir Joseph A. Lyons Kresten Lindorff-Larsen Poul Nissen |
| author_facet | Thibaud Dieudonné Felix Kümmerer Michelle Juknaviciute Laursen Charlott Stock Rasmus Kock Flygaard Syma Khalid Guillaume Lenoir Joseph A. Lyons Kresten Lindorff-Larsen Poul Nissen |
| author_sort | Thibaud Dieudonné |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-10T17:25:34Z |
| format | Article |
| id | doaj.art-aa9dfcfc11924f8586ac72110b5b9aa1 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-10T17:25:34Z |
| publishDate | 2023-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-aa9dfcfc11924f8586ac72110b5b9aa12023-11-20T10:12:26ZengNature PortfolioNature Communications2041-17232023-11-0114111410.1038/s41467-023-42828-9Activation and substrate specificity of the human P4-ATPase ATP8B1Thibaud Dieudonné0Felix Kümmerer1Michelle Juknaviciute Laursen2Charlott Stock3Rasmus Kock Flygaard4Syma Khalid5Guillaume Lenoir6Joseph A. Lyons7Kresten Lindorff-Larsen8Poul Nissen9DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus UniversityStructural Biology and NMR Laboratory & Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of CopenhagenDANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus UniversityDANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus UniversityDANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus UniversityDepartment of Biochemistry, University of OxfordUniversité Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC)Department of Molecular Biology and Genetics, Aarhus UniversityStructural Biology and NMR Laboratory & Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of CopenhagenDANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus UniversityAbstract 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.https://doi.org/10.1038/s41467-023-42828-9 |
| spellingShingle | Thibaud Dieudonné Felix Kümmerer Michelle Juknaviciute Laursen Charlott Stock Rasmus Kock Flygaard Syma Khalid Guillaume Lenoir Joseph A. Lyons Kresten Lindorff-Larsen Poul Nissen Activation and substrate specificity of the human P4-ATPase ATP8B1 Nature Communications |
| title | Activation and substrate specificity of the human P4-ATPase ATP8B1 |
| title_full | Activation and substrate specificity of the human P4-ATPase ATP8B1 |
| title_fullStr | Activation and substrate specificity of the human P4-ATPase ATP8B1 |
| title_full_unstemmed | Activation and substrate specificity of the human P4-ATPase ATP8B1 |
| title_short | Activation and substrate specificity of the human P4-ATPase ATP8B1 |
| title_sort | activation and substrate specificity of the human p4 atpase atp8b1 |
| url | https://doi.org/10.1038/s41467-023-42828-9 |
| work_keys_str_mv | AT thibauddieudonne activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT felixkummerer activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT michellejuknaviciutelaursen activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT charlottstock activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT rasmuskockflygaard activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT symakhalid activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT guillaumelenoir activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT josephalyons activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT krestenlindorfflarsen activationandsubstratespecificityofthehumanp4atpaseatp8b1 AT poulnissen activationandsubstratespecificityofthehumanp4atpaseatp8b1 |