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Scientific result | Molecular mechanisms | Structural biology
An I2BC team, in collaboration with the Universities of Aarhus, Bochum and Copenhagen, unveils the mechanism of autoinhibition of the human flippase ATP8B1, as well as its regulation by lipids and phosphorylation. The study, which combines biochemical and structural approaches by cryo-electron microscopy, provides a better understanding of the impact of flippase mutations responsible for intrahepatic cholestasis on its enzymatic activity.
Biological membranes are dynamic objects whose protein and lipid composition varies over time to ensure many cellular processes. In particular, there is a difference in lipid composition between the two layers of the membrane (lipid asymmetry) which is finely regulated by proteins, including flippases. These proteins catalyze the transport of lipids from the outer (exoplasmic) layer to the inner (cytosolic) layer of the membranes.
In humans, mutations in the ATP8B1 flippase are responsible for several forms of intrahepatic cholestasis (familial progressive intrahepatic cholestasis, intrahepatic cholestasis of pregnancy and benign recurrent intrahepatic cholestasis).
Significant efforts are made to understand the lipid transport mechanisms used by ATP8B1 family proteins (P4 ATPase family). In 2019, a European collaboration involving a team from the Protein and Membrane Systems Laboratory (I2BC) unveiled three high-resolution structures (obtained by cryo-electron microscopy) of the yeast homolog (Drs2p) interacting with its Cdc50p subunit. This work revealed the mechanism by which the C-terminus of Drs2p inhibits the activity of the complex and the first steps in the removal of this auto-inhibition by PI4P (see "Lipid transmembrane transport: very first structures are revealed!").
In collaboration with the Universities of Aarhus, Bochum and Copenhagen, the I2BC researchers have this time studied the mechanism of ATP8B1 autoinhibition, as well as its regulation by lipids and phosphorylation.
Using cryo-electron microscopy, they established the structure of the ATP8B1-CDC50A complex in an autoinhibited state. They also measured the enzymatic activity of the wild-type protein and of mutants truncated at its N- and/or C-termini. The researchers show that both ends, and particularly the C-terminus, play an essential role in the auto-inhibition of the enzyme activity. The efficiency of the inhibition is dependent on the phosphorylation of residue S1223 located in the C-terminal segment. In addition, phosphoinositides, primarily phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), are activators of the enzyme. The use of a synthetic peptide mimicking the C-terminal segment of the protein restores the inhibition of truncated forms of ATP8B1 but differently depending on whether the truncated form contains the N-terminal segment or not. These data suggest a molecular communication between the N- and C-terminal segments in autoinhibition. This result also demonstrates that it is possible to design compounds capable of regulating the activity of the complex. The resolution of the structure of ATP8B1/CDC50A in an autoinhibited state allows to localize the mutations of ATP8B1 responsible for the different forms of intrahepatic cholestasis. The mutations are uniformly distributed over the sequence but most are located in key areas that directly impact the catalytic properties of the enzyme. The presence of the (G/A)(Y/F)AFS motif in the auto-inhibitory C-terminal segment, already identified in other ATPases of the same family and directly involved in the auto-regulation of the enzymatic activity, suggests that this mechanism is widely employed among the flippases of the P4 ATPase family.
Thibaud Dieudonné, Sara Abad Herrera, Michelle Juknaviciute Laursen, Maylis Lejeune, Charlott Stock, Kahina Slimani, Christine Jaxel, Joseph A Lyons, Cédric Montigny, Thomas Günther Pomorski, Poul Nissen, Guillaume Lenoir. Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders. | eLife 2022;11:e75272
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