BPS2026 – Novel lipid recognition and activation mechanism of a P4-ATPase elucidated via cryo-EM

P4-ATPases are unidirectional lipid transporters in eukaryotes that transport lipids from exoplasmic to the cytosolic leaflet of membranes, maintaining lipid asymmetry. Therefore, they are also known as flippases. Defects in P4-ATPases lead to several forms of cholestasis and cerebral ataxia in humans. Recent structural characterization of several human and yeast flippases have identified key enzymatic states in the lipid-transporting E2 half-cycle; however, modes of initial substrate recognition and binding to the lipid entry site have not been clearly identified so far. Using Drosophila ATP8B as a candidate flippase, we examined the lipid-flipping behavior. The dATP8B can be activated by substrates PE and PS in the absence of modulators, unlike its human homolog which requires PIP lipids and removal of its auto-inhibitory domain for full activation. We therefore investigated the constitutive activation of dATP8B via cryo-EM. In our study, we identify novel E2P states where a large rearrangement of trans-membrane helices allows exposure of the binding site and initiate lipid binding followed by activation. We are also able to identify all of the previously characterized E2P intermediates, and therefore are able to provide a systematic rationale of lipid-binding initiation in the P4-ATPase activation pathway. Our proposed pathway re-contextualizes activation in terms of lipid-sensitive or insensitive states, rather than the presence or absence of the auto-inhibitory domain.