Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders
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Evaluation Summary:
This manuscript reports the first high-resolution structure of the P4 flippase ATP8B1, which is associated with intrahepatic cholestatic disorder in humans. Using biochemical studies guided by the structure, the authors demonstrate ATP8B1's autoinhibition mechanism, its regulation by lipids and phosphorylation, and a plausible mechanism of disease-associated mutation. These results are an important contribution to the expanding literature in membrane protein dynamics and function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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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, for example 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)P 3 ), 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.
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Evaluation Summary:
This manuscript reports the first high-resolution structure of the P4 flippase ATP8B1, which is associated with intrahepatic cholestatic disorder in humans. Using biochemical studies guided by the structure, the authors demonstrate ATP8B1's autoinhibition mechanism, its regulation by lipids and phosphorylation, and a plausible mechanism of disease-associated mutation. These results are an important contribution to the expanding literature in membrane protein dynamics and function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Joint Public Review:
The human lipid flippase ATP8B1 transports and enriches phospholipids to the cytosolic leaflet of plasma membrane.
Diedonnne et al purified and determined the cryo-electron microscopic structure of human lipid flippase ATP8B1 in complex with CDC50A (and obligatory targeting subunit) at 3.1 Angstrom resolution. The cryoEM structure presents the architecture of ATP8B1 and the arrangement of its three cytosolic domains (A, N and P) with respect to each other.
The authors found that the conserved C-terminal motif locks ATP8B1 in an autoinhibited resting state, and that full activation of the enzyme requires release of both C- and N-termini as well as phosphoinositides binding. Impressively, they restored the inhibition of a truncated thus active protein by adding synthetic peptides and found that Ser-1223 …
Joint Public Review:
The human lipid flippase ATP8B1 transports and enriches phospholipids to the cytosolic leaflet of plasma membrane.
Diedonnne et al purified and determined the cryo-electron microscopic structure of human lipid flippase ATP8B1 in complex with CDC50A (and obligatory targeting subunit) at 3.1 Angstrom resolution. The cryoEM structure presents the architecture of ATP8B1 and the arrangement of its three cytosolic domains (A, N and P) with respect to each other.
The authors found that the conserved C-terminal motif locks ATP8B1 in an autoinhibited resting state, and that full activation of the enzyme requires release of both C- and N-termini as well as phosphoinositides binding. Impressively, they restored the inhibition of a truncated thus active protein by adding synthetic peptides and found that Ser-1223 phosphorylation reduced inhibition by C-terminal peptide, revealing a unique role of the C-terminus of ATP8B1. These findings of the human enzyme are largely consistent with the well-studied yeast homolog Drs2p flippase and are deemed significant and likely appealing to a broad audience, given the fact that ATP8B1 is associated with the liver disease intrahepatic cholestasis. The study is well designed; however, there are several suggestions for including control experiments, improving the presentation, and discussing the results and interpretations.
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