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Cholesterol efflux mechanism revealed by structural analysis of human ABCA1 conformational states

Abstract

ATP-binding cassette transporter A1 (ABCA1) utilizes energy derived from ATP hydrolysis to export cholesterol and phospholipids from macrophages. ABCA1 plays a central role in the biosynthesis of high-density lipoprotein (HDL), which mediates reverse cholesterol transport and prevents detrimental lipid deposition. Mutations in ABCA1 cause Tangier disease characterized by a remarkable reduction in the amount of HDL in blood. Here we present cryo-electron microscopy structures of human ABCA1 in ATP-bound and nucleotide-free states. Structural comparison reveals that ATP molecules pull the nucleotide-binding domains together, inducing movements of transmembrane helices 1, 2, 7 and 8 through a series of salt-bridge interactions. Subsequently, extracellular domains (ECDs) undergo a rotation and introduce conformational changes in the ECD–transmembrane interface. In addition, while we observe a sterol-like molecule in ECDs, no such density was observed in the structure of an HDL-deficiency mutant ABCA1Y482C, demonstrating the physiological importance of ECDs and a putative interaction mode between ABCA1 and its lipid acceptors. Thus, these structures, along with cholesterol efflux assays, advance the understanding ABCA1-mediated reverse cholesterol transport.

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Fig. 1: Overall structure of human ATP-bound ABCA1QQ and ABCA1WT in the nucleotide-free state.
Fig. 2: Structural comparison of the NBDs and TMDs between the nucleotide-free ABCA1WT in nanodiscs and the ATP-bound ABCA1QQ.
Fig. 3: Structural comparison of the ECDs and TMDs between the nucleotide-free ABCA1WT in nanodiscs and the ATP-bound ABCA1QQ.
Fig. 4: The putative function of ECDs in cholesterol engagement is revealed by structural comparison between ABCA1Y482C and nucleotide-free ABCA1WT in nanodiscs.

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Data availability

The 3D cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession nos. EMD-25800 (ATP-bound ABCA1QQ), EMD-25801 (ABCA1WT, nanodiscs), EMD-25802 (ABCA1Y482C) and EMD-25803 (ABCA1WT, detergent). Atomic coordinates for the atomic model have been deposited in the Protein Data Bank under accession nos. 7TBW (ATP-bound ABCA1QQ), 7TBY (ABCA1WT, nanodiscs), 7TBZ (ABCA1Y482C) and 7TC0 (ABCA1WT, detergent). Additional data supporting the findings in this study are provided as source data and supplementary information to this manuscript.

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Acknowledgements

Cryo-EM data were collected at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award RP170644). We thank our colleagues D. Stoddard and J. Diaz for assistance in data collection, E. Debler and P. Schmiege for editing during manuscript preparation and Y. Liu for advising on the cholesterol efflux assay. This work was supported by National Institutes of Health grants P01HL020948, R01HL072304 and R01GM135343 (to X.L.). X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53S-19) and a Rita C. and William P. Clements Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.

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X.L. conceived the project and designed the research with Y.S. Y.S. carried out the cryo-EM work and performed the biochemical analysis. Y.S. and X.L. analyzed the data and wrote the manuscript.

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Correspondence to Xiaochun Li.

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Nature Cardiovascular Research thanks Alan Tall and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Integrated supplementary information

Extended Data Fig. 1 Data processing and cryo-EM maps of ABCA1QQ.

a. Representative Superose 6 increase 10/300 gel-filtration chromatogram of ABCA1WT and ABCA1QQ. The peak fraction is shown on SDS–PAGE with molecular markers. b. The data processing workflow. c. The major structural elements of ABCA1QQ. EM density maps are shown in mesh at 5σ level. d. Fourier shell correlation (FSC) curve as a function of resolution (blue) from RELION-3 and the FSC curves calculated between the refined structure model and the full map (orange) from Phenix. e. Density maps of structures colored by local resolution estimation using RELION-3.

Extended Data Fig. 2 Data processing and cryo-EM maps of ABCA1WT in nanodiscs.

a. Representative Superose 6 increase 10/300 gel-filtration chromatogram of ABCA1WT reconstituted in nanodiscs. The peak fraction is shown on SDS–PAGE with molecular markers. b. ATPase activity of ABCA1WT in nanodiscs. Data are mean ± s.d. (n=2 independent experiments). c. The data processing workflow. d. Fourier shell correlation (FSC) curve as a function of resolution (blue) from RELION-3 and the FSC curves calculated between the refined structure model and the full map (orange) from Phenix. e. The major structural elements of ABCA1wt in nanodiscs. EM density maps are shown in mesh at 5σ level. f. Density maps of structures colored by local resolution estimation using RELION-3.

Extended Data Fig. 3 Data processing and cryo-EM maps of ABCA1WT in detergent.

The data processing workflow. b. The major structural elements of ABCA1wt in digitonin detergent. EM density maps are shown in mesh at 5σ level. c. Fourier shell correlation (FSC) curve as a function of resolution (blue) from RELION-3 and the FSC curves calculated between the refined structure model and the full map (orange) from Phenix. d. Density maps of structures colored by local resolution estimation using RELION-3. e. ABCA1WT in detergent (gray) shares a similar fold with that in nanodiscs (colored) with an R.M.S.D of 0.66 Å.

Extended Data Fig. 4 Discrepancies between the present model and the previously published ABCA1 structure (PDB: 5XJY).

a. The disulfide bonds in the ECDs. The disulfide bonds are shown in spheres. The different disulfide bonds in the current model are indicated underlined. b. The topological differences of the NBD-RD connections.

Extended Data Fig. 5 The movements of TM1, TM2, TM7 and TM8 between the ATP-bound ABCA1QQ and the nucleotide-free ABCA1WT.

The helices are highlighted in green (a) and yellow (b). The ATP molecules are shown in spheres.

Extended Data Fig. 6 The ECDs have a hydrophobic tunnel for cholesterol transfer.

a. A cholesterol bound to ECD1 of ABCA1WT. The cholesterol in the ECD1 is shown in yellow sticks. The ECDs accommodate several sterol-like molecules are indicated by red dashed box. b. The electrostatic surface representation of the ABCA1WT-ECDs shows an extracellular cavity. c. The electrostatic surface representation of the ABCA1QQ-ECDs shows an extracellular cavity. d. The electrostatic surface representation of the ABCA1Y284C-ECDs. The shrunk cavity is indicated by red arrow. The cryo-EM maps of the putative lipid molecules are shown at 5σ level and indicated by black arrows.

Extended Data Fig. 7 Data processing and cryo-EM maps of ABCA1Y482C.

a. Representative Superose 6 increase 10/300 gel-filtration chromatogram of ABCA1Y482C in digitonin. The peak fraction is shown on SDS-PAGE with molecular markers. b. The data processing workflow. c. Fourier shell correlation (FSC) curve as a function of resolution (blue) from RELION-3 and the FSC curves calculated between the refined structure model and the full map (orange) from Phenix. d. The major structural elements of ABCA1Y482C. EM density maps are shown in mesh at 5σ level. e. Density maps of structures colored by local resolution estimation using RELION-3.

Extended Data Fig. 8 Cholesterol efflux assays in HEK293T cells.

a. The efflux of [3H]-cholesterol from HEK293T cells that overexpress different ABCA1 proteins. Significance relative to ABCA1WT in unpaired t-test is indicated by asterisks. P values of the variants starting from ABCA1QQ are: <0.0001, 0.0017, 0.0001, 0.021, <0.0001, 0.160, 0.0424 by t-test. Data are mean ± s.d. (n=4 independent experiments). Expression of the ABCA1-Flag proteins was detected by western blotting. b. The surface localization of different ABCA1 variants visualized by immunofluorescence. (blue: DAPI; red: Flag) The experiments were performed twice with similar results.

Extended Data Fig. 9 The putative cholesterol-binding site in the cytosolic leaflet of ABC transporters.

a. The cholesterol-binding site in the cytosolic leaflet of G5G8. b. The cholesterol-binding site in the cytosolic leaflet of ABCG1. c. A cavity in the cytosolic leaflet of ABCA1 in digitonin. The putative cholesterol observed in the structure is shown in yellow sticks. The residue that is involved in cholesterol binding is shown in sticks.

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Sun, Y., Li, X. Cholesterol efflux mechanism revealed by structural analysis of human ABCA1 conformational states. Nat Cardiovasc Res 1, 238–245 (2022). https://doi.org/10.1038/s44161-022-00022-y

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