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The research group of Professor Zhou Congzhao and Professor Chen Yuxing of our hospital analyzed the three-dimensional structure of human phospholipid flippase ATP8B1-CDC50 and its complex with the physiological substrate phosphatidylserine by single particle cryo-electron microscopy, and clarified the ATP8B1 through a series of biochemical experiments .
Substrate specificity and molecular mechanisms of its activity regulation
.
The related research results were published online on March 30 , 2022 in the Proceedings of the National Academy of Sciences ( PNAS ) under the title " Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate binding" .
Asymmetry of lipid bilayers is a hallmark feature of eukaryotic cell membranes, maintaining 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 localized on the bile duct cells and canalicular membranes of the liver.
It uses the energy generated by ATP hydrolysis to turn phospholipids from the outer membrane to the inner membrane, which plays an important role in maintaining the homeostasis of cell membranes such as liver and intestine
.
Defects in ATP8B1 function are associated with severe human diseases, such as progressive familial intrahepatic cholestasis type I ( PFIC1 ), which in severe cases can develop into diseases such as hepatocholangiocarcinoma
.
Combining the in vitro ATP hydrolysis activity and the detection of lipid turnover activity based on fluorescent labeling, the researchers demonstrated for the first time that phosphatidylserine is the true physiological substrate of
ATP8B1 by biochemical means .
Structural comparison revealed that the ligand-free ATP8B1 was in a natural phosphorylated state, and its amino- and carboxyl-terminus were inserted between the three intracellular domains, respectively, in an autoinhibited state
.
This new conformation, discovered for the first time, renews our understanding of the P4 -type ATPase transport cycle
.
Upon physiological substrate binding, the autoinhibited state of
ATP8B1 will be released .
Further enzymatic experiments found that cholate abundant in physiological environment can greatly enhance the enzymatic activity of ATP8B1 , indicating that cholate plays an important role in the regulation of
ATP8B1 activity .
Structural analysis revealed that a unique positively charged flexible structure ( P-loop ) on the intracellular P domain of ATP8B1 is closely related to cholate response
.
These findings elucidate the molecular mechanism of membrane lipid asymmetry repair during ATP8B1 -mediated cholate cycling, revealing the "auto-inhibition - inhibition -release-" effect of ATP8B1 under the combined action of substrates and intrahepatic cholate .
It also provides a structural basis for therapeutic intervention and drug design for diseases such as cholestasis
.
Figure 1.
Three-dimensional structures of ligand-free and substrate-bound ATP8B1-CDC50A complexes
Professor Zhou Congzhao, Professor Chen Yuxing and Associate Professor Hou Wentao are the co-corresponding authors of the paper, and Ph.
D.
students Cheng Mengting and Chen Yu are the co-first authors
.
Cryo-EM data collection was completed at the Cryo-EM Center of the University of Science and Technology of China and the Institute of Biophysics, Chinese Academy of Sciences
.
The research was funded by the Ministry of Science and Technology, the Chinese Academy of Sciences and the National Natural Science Foundation of China
.
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