Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

Abstract

P-type ATPases from the P4 subfamily (P4-ATPases) are primary active transporters that maintain lipid asymmetry in eukaryotic cell membranes by flipping lipids from the exoplasmic to the cytosolic leaflet. Mutations in several human P4-ATPase genes are associated with severe diseases. For instance, mutations in the ATP8B1 gene result in progressive familial intrahepatic cholestasis, a rare inherited disorder that usually progresses 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 the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. The lipid flippase complex is autoinhibited by the N- and C-termini of ATP8B1, which in concert form extensive interactions with the catalytic sites and flexible domain interfaces of ATP8B1. Consistently, ATP hydrolysis by the ATP8B1-CDC50A complex requires truncation of its C-terminus as well as the presence of phosphoinositides, with a marked preference for phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), 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-terminus further suggests molecular communication between N- and C-termini in the autoinhibition process and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A conserved (G/A)(Y/F)AFS motif in the C-termini of several P4-ATPase subfamilies suggests that this mechanism is employed widely across P4-ATPase lipid flippases, including both plasma membrane and endomembrane P4-ATPases.

Fast-forward on P-type ATPases: recent advances on structure and function

“…Rather, the actual inhibition seems to be mediated by the downstream part of the C-terminal tail, which interacts both with the N-and the A-domains thus preventing the A-domain rotation. For ATP8B1, the N-terminus was also observed and presumably reinforces the autoinhibition mechanism (85,86). The relief of these autoinhibition mechanisms in vivo is not fully understood yet.…”

“…The structural field of P4-ATPases has proliferated within the last four years with more than fifty structures determined for five P4A (Drs2, Dnf1, hATP8A1, hATP11C, hATP8B1) and one P4C (Neo1) (Fig. 7A) (75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86). These structures revealed critical steps of the P4-ATPase catalytic cycle, including the lipid entry path and occlusion (Fig.…”

“…For the yeast flippase Drs2p, specific partners have been shown to interact directly with the N-and C-termini, however there is no evidence that these interactions are sufficient for activation (91). For ATP8A2 and ATP8B1, kinase-mediated phosphorylation sites have been identified and are likely to be part of the activation mechanism (85,92). Another P4A regulatory mechanism was described for Drs2 and ATP8B1 with the activating role of phosphoinositides (85,(93)(94)(95).…”

“…For ATP8A2 and ATP8B1, kinase-mediated phosphorylation sites have been identified and are likely to be part of the activation mechanism (85,92). Another P4A regulatory mechanism was described for Drs2 and ATP8B1 with the activating role of phosphoinositides (85,(93)(94)(95). In Drs2, PI(4)P binding in a cavity formed by TM5, TM7, TM8 and TM10 induced a conformational change of a C-terminal amphipathic helix, linking PI(4)P and the autoinhibition mechanism (75,77).…”

“…ATP8B1 has been shown to have a high affinity for PI(3,4,5)P3. The PI(3,4,5)P3 binding site has not been identified but the structural analysis of ATP8B1 seems to indicate that it could bind similarly to PI(4)P in Drs2 (85). Although the activating role of phosphoinositide in Drs2 and ATP8B1 has been functionally characterized, the mechanistic basis of this activation remains to be investigated.…”