Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2
| Autor: | Hanayo Nakanishi, Kenichi Hayashida, Tomohiro Nishizawa, Atsunori Oshima, Kazuhiro Abe |
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| Rok vydání: | 2021 |
| Předmět: |
MSP
membrane scaffolding protein SEC size-exclusion chromatography Lipid Bilayers DOPS dioleoylphosphatidylserine Biochemistry MβCD methyl-β-cyclodextrin active transport PtdCho phosphatidylcholine cryo-EM cryo-electron microscopy Molecular Biology membrane Phospholipids Adenosine Triphosphatases DOPC dioleoylphosphatidylcholine Cell Membrane Cryoelectron Microscopy apoptosis Membrane Transport Proteins PtdEtn phosphatidylethanolamine Cell Biology flippase PtdSer phosphatidylserine P-type ATPases P4-ATPases cryo-EM lipids (amino acids peptides and proteins) Nanodisc Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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