Abstract
Among mammalian P4-ATPase flippases, only ATP9A and ATP9B do not require the auxiliary subunit CDC50 protein. Whilst its yeast homolog, Neo1, is essential for cell survival, little is known about mammalian ATP9A. We present cryo-EM structures of human monomeric ATP9A at a resolution reaching 2.2 Å, in the outward-facing E2P state. Two distinguishable conformations were obtained from a single sample, one with its outward gate open and the other in its closed form. Unlike canonical gating observed for most P-type ATPases, which is driven by the movement of transmembrane (TM) helices 1 and 2 linked to the A domain, outward gating in ATP9A is achieved by the movement of TM6–10 helices, likely initiated by the unwinding of TM6. As a result, the volume of the phospholipid binding cavity in the open state surpasses that of other flippases, which could allow binding of phospholipids with larger hydrophilic headgroups than that of phosphatidylserine. ATP9A shows an ATPase activity that is significantly increased by the addition of phospholipids that retain the overall negative charge, including phosphatidylserine, phosphatidylinositol, and its phosphorylated species, compared with other electroneutral phospholipids. The observation of spontaneous binding of phosphorylated species of phosphatidylinositol in molecular simulation reinforces this fact. Our data provide mechanistic rationales for ATP9A gating, achieved by the rearrangement of the second half of the TM helices. Since TM4–TM10 is anchored by the CDC50 protein subunit in other flippases, the here-observed outward gating mechanism is unique to P4B-type flippases, which function as a monomer.
| Original language | English |
|---|---|
| Article number | 110631 |
| Journal | Journal of Biological Chemistry |
| Volume | 301 |
| Issue number | 10 |
| DOIs | |
| Publication status | Published - Oct 2025 |
| MoE publication type | A1 Journal article-refereed |
Funding
This work was partially funded by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under grant number JP24ama121004 to C.G., C.C.G., and H.S. P.M. gratefully acknowledges the use of the bioinformatics infrastructure facility supported by Biocenter Finland ; CSC-IT Center for Science (Project no.: 2000461) for the computational facility; Jukka Lehtonen (Structural Bioinformatics Laboratory) for the IT support; and NordicPOP Mobility funding to visit Southern University of Denmark during 2023 and 2025. Cryo-EM data are acquired using EM01CT of SPring-8 with approval of the Japan Synchrotron Radiation Research Institute (proposal numbers: 2023B2518 and 2024B2519 ). H.K. is supported by the Lundbeckfonden Ascending Investigator grant number #R344-2020-1023 . K.A. thanks Drs Hanayo Nakanishi, Katsumori Segawa, Yugo Miyata, and Xu Jing for the contribution at the initial stage of this project. Computations were performed on the Finnish supercomputer LUMI, with access obtained via the Danish e-Infrastructure Cooperations. This research was funded by a grant-in-aid for scientific research (grant no.: 24K01975) and Japan Science and Technology Agency CREST grant number (grant no.: JPMJCR22E4). K. A. conceptualization; K. A. methodology; K. A. C. C. G. and H. K. validation; K. A. P. M. Y. Q. C. C. G. K. T. and H. K. formal analysis; K. A. investigation; K. A. resources; K. A. P. M. Y. Q. C. C. G. C. G. and H. S. data curation; K. A. and H. K. writing–original draft; K. A. C. C. G. C. G. H. S. K. T. and H. K. writing–review & editing; K. A. C. C. G. and H. K. visualization; K. A. supervision; K. A. project administration; K. A. funding acquisition. This work was partially funded by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under grant number JP24ama121004 to C.G. C.C.G. and H.S. P.M. gratefully acknowledges the use of the bioinformatics infrastructure facility supported by Biocenter Finland; CSC-IT Center for Science (Project no.: 2000461) for the computational facility; Jukka Lehtonen (Structural Bioinformatics Laboratory) for the IT support; and NordicPOP Mobility funding to visit Southern University of Denmark during 2023 and 2025. Cryo-EM data are acquired using EM01CT of SPring-8 with approval of the Japan Synchrotron Radiation Research Institute (proposal numbers: 2023B2518 and 2024B2519). H.K. is supported by the Lundbeckfonden Ascending Investigator grant number #R344-2020-1023. K.A. thanks Drs Hanayo Nakanishi, Katsumori Segawa, Yugo Miyata, and Xu Jing for the contribution at the initial stage of this project. Computations were performed on the Finnish supercomputer LUMI, with access obtained via the Danish e-Infrastructure Cooperations. This research was funded by a grant-in-aid for scientific research (grant no.: 24K01975 ) and Japan Science and Technology Agency CREST grant number (grant no.: JPMJCR22E4 ).
Keywords
- ATPase
- cryo-EM
- lipid transport
- membrane protein
- molecular dynamics simulations
- phospholipid
- transporter