Transport mechanism of class-3 P4 ATPase lipid flippases

The P4 ATPases are a large family of membrane-embedded enzymes that use ATP hydrolysis to transport large lipid substrates across lipid bilayers. P4 ATPases differ in their cellular membrane location and their substrates. The structures of the endosome- and Golgi-localized class-1 phosphatidylserine flippases--such as the yeast Drs2 and human ATP8A1--have recently been reported, revealing a substrate binding site on the lumenal side and several transport states. However, a substrate binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases in other classes are still unknown. Here we report a systematic structural and functional study on two plasma-membrane localized, class-3 P4 ATPases that have broader substrate specificity, the S. cerevisiae Dnf1-Lem3 and Dnf2-Lem3 complexes. We have captured substrate lipids on both the exoplasmic and cytosolic sides, and we found that these two enzymes have very similar structures, consistent with their high sequence identity and redundant function. Unexpectedly, Lem3 contributes to substrate binding near the cytosolic surface. We found that the conformational transitions through the substrate transport cycle of these two class-3 enzymes match those of the class-1 enzymes, suggesting a conserved lipid-flipping mechanism among all classes of the P4 ATPases. Our study also revealed a helix-turn-helix insertion in the cytosolic P domain that is unique to the class-3 enzymes and plays a crucial role in their function. Therefore, the P4 ATPases may have retained an overall transport mechanism while evolving distinct features for cellular membrane localization, regulatory mechanisms, and transporting different lipid substrates.