Activation and substrate specificity of the human P4-ATPase ATP8B1

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for maintaining cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. In this study, we present a total of nine cryo-electron microscopy structures at a resolution ranging from 2.4 to 3.1 Å, along with functional and computational studies of the human flippase ATP8B1-CDC50A complex, describing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as its regulation by phosphoinositides. Our findings show that the P4-ATPase transport site is filled with water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identified and characterized the PI(3,4,5)P3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of ATP8B1 broad specificity for lipids and identifies a new transport substrate for P4-ATPases, phosphatidylinositol (PI). We report the critical role of the sn-2 ester bound of glycerophospholipids in substrate recognition by ATP8B1. These findings provide fundamental insights into ATP8B1 regulation, the catalytic cycle, and substrate recognition in P4-ATPases. ### Competing Interest Statement The authors have declared no competing interest.