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

Abstract

AbstractThe 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.