Structures of a P4-ATPase lipid flippase in lipid bilayers

He, Yilin; Xu, Jinkun; Wu, Xiaofei; Long, Li

doi:10.1007/s13238-020-00712-y

Cited by 37 publications

(34 citation statements)

References 17 publications

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“…During preparation of this manuscript, the cryo-EM structure of Dnf1–Cdc50 of a thermophilic fungus, Chaetomium thermophilum , was reported in the E1-ATP and E2P states at 3.4–3.5 Å resolution ( He et al, 2020 ). That Dnf1 structure contained two lipids, but no evidence was provided that they were substrate lipids.…”

Section: Resultsmentioning

confidence: 99%

Transport mechanism of P4 ATPase phosphatidylcholine flippases

Bai

You

Jain

et al. 2020

eLife

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The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases—such as the yeast Drs2 and human ATP8A1—have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the S. cerevisiae Dnf1–Lem3 and Dnf2–Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.

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Section: Resultsmentioning

confidence: 99%

Transport mechanism of P4 ATPase phosphatidylcholine flippases

Bai

You

Jain

et al. 2020

eLife

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“…This association is required for both proper localization and activity of the flippase complex [ 12 , 13 ] but seems not to affect its substrate specificity [ 14 ]. Recent cryo-electron microscopy structures of four P4-ATPases in complex with their respective Cdc50 subunits have revealed the structural basis of P4-ATPase-Cdc50 protein association [ 15 , 16 , 17 , 18 , 19 , 20 ]. Genes encoding P4-ATPases are highly conserved in evolution, and members of this P-type ATPase subfamily have been found in genomes of unicellular eukaryotes, plants, and animals.…”

Section: Introductionmentioning

confidence: 99%

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

Stanchev

Rizzo

Peschel

et al. 2021

JoF

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Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

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“…S1). As substrates were reported to greatly enhance the ATPase activity of P4-ATPases (14,16,24), we performed ATPase activity in the presence or absence of different phospholipids at 300 μM. DDM (1%) was added as the hydrotropic agent for the dissolution of lipids to produce clear and homogeneous stock solutions at room temperature.…”

Section: Resultsmentioning

confidence: 99%

“…It is puzzling that the Vmax of ATP8B1 is less than 1/6 of the previously reported P4-ATPases (14,16,24). Notably, the yeast P4-ATPase Drs2p in complex with Cdc50p with a non-detectable ATPase activity in the presence of substrate, could be activated by an activator molecule, lipid phosphatidylinositol 4-phosphate (PI4P) (25).…”

Section: Bile Acids Can Further Augment the Atpase Activitymentioning

confidence: 98%

Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate binding

Cheng

Chen

et al. 2021

Preprint

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The human P4-type ATPase ATP8B1 in complex with the auxiliary noncatalytic protein CDC50A or CDC50B mediates the transport of cell membrane lipids from the outer to the inner membrane leaflet, which is crucial to maintain the asymmetry of membrane lipid. Its dysfunction usually leads to imbalance of bile acid circulation, and eventually causing intrahepatic cholestasis diseases. Here we found that both ATP8B1-CDC50A and ATP8B1-CDC50B possess a higher ATPase activity in the presence of the most favored substrate phosphatidylserine (PS); and moreover, the PS-stimulated activity could be augmented upon the addition of bile acids. The cryo-electron microscopy structures of ATP8B1-CDC50A at 3.36 Å and ATP8B1-CDC50B at 3.39 Å enabled us to capture an unprecedented phosphorylated and autoinhibited state, with the N- and C-terminal tails separately inserting into the cytoplasmic inter-domain clefts of ATP8B1. The PS-bound ATP8B1-CDC50A structure at 3.98 Å indicated the autoinhibited state could be released upon PS binding. Structural analysis combined with mutagenesis revealed the residues that determine the substrate specificity, and a unique positively charged loop in the phosphorylated domain of ATP8B1 for the recruitment of bile acids. Altogether, we updated the Post-Albers transport cycle, with an extra autoinhibited state of ATP8B1, which could be activated upon substrate binding. These findings not only provide structural insights into the ATP8B1-mediated restoration of human membrane lipid asymmetry during bile acid circulation, but also advance our understanding on the molecular mechanism of P-type ATPases.

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Structures of a P4-ATPase lipid flippase in lipid bilayers

Abstract: contains supplementary material, which is available to authorized users.

Cited by 37 publications

References 17 publications

Transport mechanism of P4 ATPase phosphatidylcholine flippases

Transport mechanism of P4 ATPase phosphatidylcholine flippases

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate binding

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