Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2

Hanayo Nakanishi,Kenichi Hayashida,Tomohiro Nishizawa,Atsunori Oshima,Kazuhiro Abe

doi:10.1016/j.jbc.2021.101498

Abstract

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-β-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.

Highlights

In the plasma membrane of eukaryotic cells, phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) are localized in the inner leaflet, whereas phosphatidylcholine (PtdCho) and sphingomyelin (SM) are abundant in the outer leaflet [1, 2]
Nanodisc assembly is likely driven by the detergent removal, so we tried to determine the key parameters for reconstitution by making “empty-Nanodiscs” in a simplified system (Fig. 1A), using only membrane scaffolding proteins (MSP), phospholipid, and detergent, notably excluding target protein ATP11C
MSP1D1 has been utilized for Nanodisc reconstitution of other P-type ATPases such as SERCA and Na+,K+-ATPase, and it is expected to be suitable for ATP11C too because of its similar size

Summary

Introduction

In the plasma membrane of eukaryotic cells, phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) are localized in the inner leaflet (cytosolic side), whereas phosphatidylcholine (PtdCho) and sphingomyelin (SM) are abundant in the outer leaflet (exoplasmic side) [1, 2]. This asymmetric distribution of phospholipids is a prerequisite for vesicular trafficking [3], cell signaling, and other cellular processes [4]. Three classes of membrane transporters mediate phospholipid translocation across the lipid bilayer; scramblases, flippases, and floppases [8,9,10,11]. Cryo-EM structure of ATP11C-Nanodisc transition of four cornerstone states, E1–E1P–E2P–E2, according to the Post–Albers-type reaction scheme [13, 21]

Results

Discussion

Conclusion