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Structure of nevanimibe-bound tetrameric human ACAT1

Abstract

Cholesterol is an essential component of mammalian cell membranes, constituting up to 50% of plasma membrane lipids. By contrast, it accounts for only 5% of lipids in the endoplasmic reticulum (ER)1. The ER enzyme sterol O-acyltransferase 1 (also named acyl-coenzyme A:cholesterol acyltransferase, ACAT1) transfers a long-chain fatty acid to cholesterol to form cholesteryl esters that coalesce into cytosolic lipid droplets. Under conditions of cholesterol overload, ACAT1 maintains the low cholesterol concentration of the ER and thereby has an essential role in cholesterol homeostasis2,3. ACAT1 has also been implicated in Alzheimer’s disease4, atherosclerosis5 and cancers6. Here we report a cryo-electron microscopy structure of human ACAT1 in complex with nevanimibe7, an inhibitor that is in clinical trials for the treatment of congenital adrenal hyperplasia. The ACAT1 holoenzyme is a tetramer that consists of two homodimers. Each monomer contains nine transmembrane helices (TMs), six of which (TM4–TM9) form a cavity that accommodates nevanimibe and an endogenous acyl-coenzyme A. This cavity also contains a histidine that has previously been identified as essential for catalytic activity8. Our structural data and biochemical analyses provide a physical model to explain the process of cholesterol esterification, as well as details of the interaction between nevanimibe and ACAT1, which may help to accelerate the development of ACAT1 inhibitors to treat related diseases.

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Fig. 1: Functional characterization of human ACAT1 and nevanimibe in vitro.
Fig. 2: Overall structure of human ACAT1 holoenzyme.
Fig. 3: Overall structure of monomeric ACAT1 and its ligands.
Fig. 4: The cholesterol entrance.

Data availability

The 3D cryo-EM density map has been deposited in the Electron Microscopy Data Bank under the accession number EMD-21390. Atomic coordinates for the atomic model have been deposited in the Protein Data Bank under the accession number 6VUM. Source Data for Figs. 1, 3, 4 and Extended Data Fig. 9 are provided with the paper. All other data are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank M. Brown and J. Goldstein for support throughout the project. 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 D. Stoddard for assistance with data collection; R. DeBose-Boyd, L. Esparza and L. Friedberg for technical support; E. Coutavas and E. Debler for discussion; and A. Lemoff at the UT Southwestern Proteomics Core for mass spectrometry identification. This work was supported by National Institutes of Health grants P01 HL020948, R01 GM134700 and R01 GM135343; the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center; O’Donnell Junior Faculty Funds; and Welch Foundation grant I-1957 to X.L. X.Q. is the recipient of a DDBrown Fellow of the Life Sciences Research Foundation. X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53-19) and a Rita C. and William P. Clements Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.

Author information

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Authors

Contributions

X.L. conceived the project and designed the research with T.L. T.L. and A.H. performed the biochemical study. T.L., Y.S. and X.Q. carried out cryo-EM work, built the model and refined the structure. T.L., Y.S., X.Q. and X.L. analysed the data, and all authors contributed to the preparation of the manuscript. X.L. wrote the manuscript.

Corresponding author

Correspondence to Xiaochun Li.

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The authors declare no competing interests.

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Peer review information Nature thanks David Drew, Savvas N. Savvides and Alan A. Tall for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Sequence alignment of human ACAT1 and ACAT2, hamster ACAT1 and ACAT2, chicken ACAT1 and zebrafish ACAT1.

The transmembrane helices and the residue numbers of human ACAT1 are indicated above the protein sequence. The specific residues necessary for ligand binding are labelled with an asterisk. Hs, Homo sapiens (human); cg, Cricetulus griseus (Chinese hamster); gg, Gallus gallus (chicken); dr, Danio rerio (zebrafish).

Extended Data Fig. 2 Expression and purification of human ACAT1 with nevanimibe.

Representative gel-filtration chromatogram of ACAT1 (Superose 6 Increase 10/30 column). The SDS–PAGE gel (with molecular weight markers) is shown on the right, and ACAT1 is indicated with an arrow. The experiment was reproduced three times on separate occasions with similar results.

Extended Data Fig. 3 Processing of data acquired from 200 kV cryo-TEM.

a, Representative gel-filtration chromatogram (Superose 6 Increase 10/30 column) of cross-linked ACAT1. The SDS–PAGE gel (with molecular weight markers) is shown on the right, and ACAT1 is indicated with an arrow. The experiment was reproduced twice on separate occasions with similar results. b, The data processing workflow in RELION-3. The 3D classes and refinement results from the cryo-EM data are shown.

Extended Data Fig. 4 Processing of data acquired from 300 kV cryo-TEM.

The data processing workflow in RELION-3. The cryo-EM 3D classes as well as the masks used for the refinement are shown. After RELION-3 refinement, the final cryo-EM map was sharpened using post-process with a B-factor value of −150 Å2.

Extended Data Fig. 5 FSC curve and estimation of the local resolution.

a, FSC curve as a function of resolution using RELION-3 output. b, Density maps of the structure of ACAT1, coloured by local resolution estimation using ResMap.

Extended Data Fig. 6 Cryo-EM map of the structural elements of ACAT1.

a, The FSC curves calculated between the refined structure model and the half map used for refinement (orange), the other half map (green) and the full map (blue). b, The major helices of ACAT1-A and ACAT1-B. The density map and model of the complex are shown as mesh and cartoons, respectively. Cryo-EM maps are contoured at the 5σ level.

Extended Data Fig. 7 Structural comparison of ACAT1 and DltB.

a, Structural comparison from parallel to the membrane view (left) and from the lumen view (right). b, Structural comparison of the catalytic sites of ACAT1 and DltB. The catalytic histidine residues are shown as sticks.

Extended Data Fig. 8 Purification of human ACAT1 variants for activity assays.

Representative gel-filtration chromatograms (Superose 6 Increase 10/30 column) of ACAT1 variants in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 1% CHAPS. The experiment was reproduced twice on separate occasions with similar results.

Extended Data Fig. 9 The putative allosteric sites of ACAT1.

a, b, Putative allosteric sites 1 (a) and 2 (b). Cholesterol (yellow) may form hydrophobic contacts that result in the stabilization of TM4 and TM6, respectively. c, Putative allosteric site 2 in the ACAT1 dimer. The cryo-EM maps of cholesterol are contoured at the 5σ level. d, Functional validation of the putative allosteric sites. Data are mean ± s.d. (n = 3 technically independent experiments). Each experiment was reproduced at least twice on separate occasions with similar results.

Source Data

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Long, T., Sun, Y., Hassan, A. et al. Structure of nevanimibe-bound tetrameric human ACAT1. Nature 581, 339–343 (2020). https://doi.org/10.1038/s41586-020-2295-8

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