Structure And Regulatory Mechanism Of Human Phospholipid Flippase ATP8B1-CDC50A/B

Lipid asymmetry across the membrane bilayer is a hall-mark feature of eukaryotic cell membranes.Phosphatidylcholine,sphingomyelin and glycolipids are enriched at the outer leaflet of plasma membrane,while phosphatidylethanolamine,phosphatidylserine and phosphatidylinositol are mainly restricted to the inner leaflet.The asymmetric distribution of lipids is essential to maintain the cellular biological function,such as cell shape determination,membrane stability,cell signaling,and bile and cholesterol homeostasis,et al.To maintain membrane asymmetry,eukaryotic cells express a series of cooperatively functioning lipid transporters,such as scramblases,floppases and flippases.The P4-ATPases are lipid flippases that generate and maintain the membrane asymmetry by transporting phospholipids from the outer to the inner leaflet.ATP8B1 is a member of P4-ATPases.ATP8B1 form a complex with an auxiliary non-catalytic protein CDC50A or CDC50B for its proper localization and the integral function.ATP8B1-CDC50A complex co-localizes in the cholangiocyte and canalicular membrane of the hepatocyte with the primary bile salt export pump ABCB11.ATP8B1 has an important role in maintaining the membrane asymmetry homeostasis to ensure the function of ABCB 11.Defects in ATP8B1 are associated with severe human diseases,such as the intrahepatic cholestasis diseases progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1.Recently,a series of structures of human P4-ATPases(ATP8A1 and ATP 11C)complexed with CDC50A have been reported.However,the complex structure of P4-ATPase with CDC50B remains unknown.In this project,ATP8B1-CDC50A and ATP8B1-CDC50B complexes were coexpressed in HEK293F cells.The ATPase activity of purified complexes is stimulated by phosphatidylserine(PS)and phosphatidylcholine;however,ATP8B1 possesses a higher specificity towards PS.Using cryo-electron microscopy(cryo-EM)technology,the apo ATP8B1-CDC50A,apo ATP8B1-CDC50B and PS-bound ATP8B1-CDC50A complex structures were determined to 3.36 A,3.39 A and 3.98 A,respectively.ATP8B1 consists of a transmembrane domain and three cytoplasmic domains:N doamin,P domain and A domain.The N domain is responsible for binding to ATP,which donates the phosphate group for autophosphorylation of the P domain,generating a phosphorylated intermediate during the transport cycle.The A domain can dephosphorylate the phosphorylated P domain,and finally triggering substrate translocation.CDC50A/B is composed of two transmembrane helix and a highly glycosylated extracelluar domain.Comparison with the structure of known P4-ATPase showed that apo ATP8B1 adopts the natural phosphorylated E2P state and autoinhibited with its own N-and C-terminal tails inserting into the three cytoplasmic domains.Moreover,the PS-stimulated activity could be augmented upon the addition of bile acid.Structural analysis showed that ATP8B1 harbors an unique postively charged P-loop,truncation of which led to a significant reduction in taurocholic acid stimulation,suggesting the important role of this P-loop in sensing bile acids.All together,we proposed a putative regulatory mechanism of ATP8B1 in the presence of bile acids.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 P4-ATPases.