Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p

Abstract

The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.

Structures of a P4-ATPase lipid flippase in lipid bilayers

“…In both E1-ATP and E2P structures, ctCdc50 has extensive interactions with ctDnf1. On the cytoplasmic side, the N-terminal peptide (residues 23-46) of ctCdc50 runs along one face of ctDnf1, interacting with the cytosolic loops of ctDnf1, including the segment connecting TM4 and the P domain, the loop between TMs 6 and 7 (L6/7), L8/9, and a C-terminal amphipathic helix that was suggested to undergo conformational changes upon PI4P activation in scDrs2 24,25 (Fig. S6).…”

“…Recently, the electron cryo-microscopy (cryo-EM) structures of S. cerevisiae Drs2-Cdc50 (scDrs2-Cdc50) 24,25 and human ATP8A1-CDC50a (hATP8A1-CDC50a) 26 solubilized in detergents were reported. The structures of scDrs2-Cdc50 were determined in the E2P state and the hATP8A1-CDC50a structures in several E1 and E2 intermediate states.…”

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

“…We performed single-particle cryo-EM on purified samples in apo form (E1 state) and on samples incubated with AMPPCP (E1-ATP state), ALF4 − -ADP (E1P-ADP and E2P-transition states), or BeF3 − (E2P state). The substrate transport states were assigned based on the established nomenclature in the P-type ATPase field (Bai et al, 2019a;Hiraizumi et al, 2019;Timcenko et al, 2019). We obtained cryo-EM 3D maps of the S. cerevisiae Dnf1-Lem3 in the E1, E1P-ADP, and E2P states and Dnf2-Lem3 in the E1, E1-ATP, E1P-ADP, E2P-transition, and E2P states, at resolutions of 2.8 Å to 4.0 Å (Supplemental Table 1).…”

“…Interestingly, this ordered Dnf1 C-tail segment contains a short -helix, the equivalent of which was reported to function as a switch that converts the Drs2 flippase from its auto-inhibitory state to the active state in a unique PI4P-binding-dependent manner (Bai et al, 2019a). However, the Dnf1 helix is stabilized by the C-tail of Lem3 in a conformation corresponding to the active state of Drs2, suggesting that Dnf1 and Dnf2 do not require binding to a phosphoinositide at this site for activation.…”

“…The first structural and mechanistic insights into the P4 ATPases came from recent cryo-EM structures of the S. cerevisiae class-1 flippase Drs2-Cdc50 in its auto-inhibited and active E2P states (Bai et al, 2019a;Timcenko et al, 2019), and of its human homolog, ATP8A1-CDC50a, in six transport states (E1, E1-ATP, E1P-ADP, E1P, E2P, and E2Pi-PL) (Hiraizumi et al, 2019).…”