A novel regulatory mechanism of human phospholipid turnover enzyme ATP8B1 was discovered by the University of Science and Technology of China

Source: China University of Science and Technology News

The three-dimensional structure of human phospholipid invertase ATP8B1-CDC50 and its complex with physiological substrate phosphatidylserine was analyzed by single-particle cryo-electron microscopy technique by Professor Zhou Conzhao and Professor Chen Yuxing from the School of Life Sciences and Medicine, University of Science and Technology of China. The molecular mechanism of substrate specificity and activity regulation of ATP8B1 was elucidated by a series of biochemical experiments. the relevant research results were summarized as "Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate. The title "binding" was published online March 30, 2022 in the Proceedings of the National Academy of Sciences (PNAS).

The asymmetry of the lipid bilayer is a hallmark feature of eukaryotic cell membranes and supports a range of cellular functions, such as membrane stability, cell shape, cell signaling, and bile and cholesterol homeostasis. Human ATP8B1 is a P4 type ATPase, which is mainly located in bile duct cells and tubule membranes of the liver. It uses the energy generated by ATP hydrolysis to turn phospholipids from the outer cell membrane to the inner membrane, which plays an important role in maintaining cell membrane homeostasis such as liver and intestine. Deficiency of ATP8B1 is associated with serious human diseases, such as progressive familial intrahepatic cholestasis type I (PFIC1), which in severe cases will develop into hepatobiliary duct cancer.

Combining in vitro ATP hydrolytic activity with fluorescently labeled lipid turnover activity, we demonstrated for the first time that phosphatidylserine is the true physiological substrate of ATP8B1 by biochemical means. Structure comparison showed that ATP8B1 without ligands was in a natural phosphorylated state, and its amino terminal and carboxyl terminal were inserted between the three intracellular domains, respectively, and were in a self-inhibition state. This new conformation, discovered for the first time, updates our understanding of the transport cycle of P4 ATPase. When the physiological substrate binds, the self-inhibition state of ATP8B1 will be removed. Further enzyme activity experiments showed that the presence of cholic acid in the physiological environment can greatly enhance the enzyme activity of ATP8B1, indicating that cholic acid plays an important role in the regulation of ATP8B1 activity. Structural analysis showed that ATP8B1 has a unique positive flexible structure (P-loop) on the P-domain, which is closely related to the cholate response. These results elucidate the molecular mechanism of membrane lipid asymmetry repair mediated by ATP8B1 in the process of cholic acid circulation, reveal the fine regulatory mechanism of "self-inhibition - disinhibition - enhancement of enzyme activity" under the combined action of ATP8B1 substrate and intrahepatic cholate, and also provide a structural basis for therapeutic intervention and drug design for diseases such as cholestasis.

Figure 1. Three-dimensional structure of ATP8B1-CDC50A complex without ligands and substrates