A unique gating mechanism revealed by the cryo-EM structure of monomeric ATP9A flippase.

Among mammalian P4-ATPase flippases, only ATP9A and ATP9B do not require the auxiliary subunit CDC50 protein. Whilst its yeast homolog, Neo1, is essential for cell survival, little is known about mammalian ATP9A. We present cryo-EM structures of human monomeric ATP9A at a resolution reaching 2.2 Å, in the outward-facing E2P state. Two distinguishable conformations were obtained from a single sample, one with its outward gate open and the other in its closed form. Unlike canonical gating observed for most P-type ATPases, which is driven by the movement of transmembrane (TM) helices 1 and 2 linked to the A domain, outward gating in ATP9A is achieved by the movement of TM6-10 helices, likely initiated by the unwinding of TM6. As a result, the volume of the phospholipid binding cavity in the open state surpasses that of other flippases, which could allow binding of phospholipids with larger hydrophilic headgroups than that of phosphatidylserine. ATP9A shows an ATPase activity that is significantly increased by the addition of phospholipids that retain the overall negative charge, including phosphatidylserine, phosphatidylinositol, and its phosphorylated species, compared with other electroneutral phospholipids. The observation of spontaneous binding of phosphorylated species of phosphatidylinositol in molecular simulation reinforces this fact. Our data provide mechanistic rationales for ATP9A gating, achieved by the rearrangement of the second half of the TM helices. Since TM4-TM10 is anchored by the CDC50 protein subunit in other flippases, the here-observed outward gating mechanism is unique to P4B-type flippases, which function as a monomer.