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Structural basis for catalysis and substrate specificity of human ACAT1


As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer’s disease and cancer2,3,4,5,6,7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family8.

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Fig. 1: Cryo-EM structure of human ACAT1 as a dimer of dimers.
Fig. 2: The C tunnel in the ACAT1 protomer may accommodate an oleoyl-CoA molecule.
Fig. 3: Structure-based working model for ACAT1.

Data availability

The atomic coordinates of the tetrameric and dimeric ACAT1 have been deposited in the PDB under accession codes 6P2P and 6P2J, respectively. The corresponding electron microscopy maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-20239 and EMD-20238, respectively. For uncropped SDS–PAGE gels, see Supplementary Fig. 1. Source Data for Figs. 13 and Extended Data Figs. 2, 7 are provided with the paper. The raw electron micrographs for structural analysis are available from the corresponding authors upon reasonable request.


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We thank P. Shao for technical support during electron microscopy image acquisition; S. Kyin for technical support during the mass spectrometry analysis of cholesteryl oleate; and S. Dang for assistance with directional FSC analysis. We acknowledge the use of Princeton’s Imaging and Analysis Center, which is partially supported by the Princeton Center for Complex Materials, and the National Science Foundation (NSF)-MRSEC programme (DMR-1420541). This work was supported in part by the Ara Parseghian Medical Research Foundation (N.Y). H.Q. is supported by the New Jersey Council for Cancer Research. N.Y. is supported by the Shirley M. Tilghman endowed professorship from Princeton University.

Author information




N.Y., R.Y. and H.Q. conceived and N.Y. supervised the project. H.Q. and X.Z. designed the experiments. H.Q., X.Z. and R.Y. performed cloning and protein purification. H.Q. prepared cryo-EM samples, collected data and determined the structures. X.Z. and H.Q. performed the fluorescence-based activity assays. X.Z. validated the fluorescence-based assay by detecting cholesterol ester by mass spectrometry. X.S. and C.C.L.W. analysed lipid extractants from the enzymes by LC–MS. S.G. and X.Y. performed molecular docking of cholesterol. X.D. and H.Y. contributed to data analysis. N.Y., H.Q. and X.Z. wrote the manuscript.

Corresponding authors

Correspondence to Hongwu Qian or Nieng Yan.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks David Drew, Savvas N. Savvides and the other, anonymous, reviewer(s) 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 and mouse ACAT1, ACAT2 and DGAT1.

Secondary structural elements of human ACAT1 are indicated above the sequences according to the present cryo-EM structure. Invariant and highly conserved residues are shaded yellow and grey, respectively. The conserved residue His460 at the active site is shaded red and coloured white. The conserved FYXDWWN motif and predicted cholesterol binding motif are indicated with blue and orange boxes, respectively. Sequences from human (h) and mouse (m) were aligned using the online MultiAlin server (

Extended Data Fig. 2 Enzymatic activity of recombinant human ACAT1.

a, Schematic of our fluorescence-based activity assay for ACAT1. Top, the chemical structure of CoASH; bottom, a schematic illustration of the fluorescence-based activity assay. b, Interference of detergents on the enzymatic activity of ACAT1. The proteins used for the assay were purified by SEC in the presence of 1% CHAPS or 0.02% GDN. c, Allosteric activation of ACAT1 by cholesterol. The sigmoidal plot of catalytic activity with increasing concentrations of cholesterol is consistent with the proposed allosteric activation of ACAT1 by cholesterol32. Data in b, c are mean ± s.d. of three independent experiments.

Source Data.

Extended Data Fig. 3 Cryo-EM analysis of the structure of human ACAT1.

a, A representative micrograph (left) and 2D class averages (right) of cryo-samples of ACAT1 in GDN micelles. The box size for 2D averages is 310 Å. b, FSC curves for the 3D electron microscopy reconstructions of tetrameric and dimeric ACAT1. c, Local resolution map of tetrameric (top) and dimeric (bottom) ACAT1 calculated using RELION 3.0. The resolution bars on the right are labelled in Å. d, Directional FSC (dFSC) for the dimeric reconstruction. Each purple curve indicates a different direction. In total, 500 dFSC curves were generated, which were averaged and shown by the green curve (average dFSC)51. e, FSC curves of the refined model versus the summed map that it was refined against (black); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (green) for the dimeric reconstruction.

Extended Data Fig. 4 Flowchart for structural determination.

a, Flowchart of data processing; see Methods for details. b, Electron microscopy maps of representative structural elements. The densities, contoured at 10–13σ, were prepared in PyMOL. c, Structure of an ACAT1 protomer. The structure is rainbow-coloured on the left (blue for the amino terminus and red for and the carboxyl terminus) and domain-coloured on the right. d, Topological structure of ACAT1. The structural elements are colour-coded to match the domain colours in c.

Extended Data Fig. 5 NTD is responsible for tetramerization.

a, Electron microscopy map of the tetrameric ACAT1, displayed at low threshold (0.004) in Chimera, reveals extra cytosolic densities that may belong to the NTD. b, Tetrameric ACAT1 shown in the lumenal (left) and cytosolic (right) views. The insets show residues on the tetrameric interface. c, Validation of the oligomeric states of dimeric and monomeric mutants using SEC. SEC profiles and corresponding SDS–PAGE gels for wild-type ACAT1 and two variants, ACAT1(ΔNTD) and ACAT1(ΔNTD-3A), in GDN micelles are shown. The experiment was independently repeated twice with similar results. d, A representative micrograph (left) and representative 2D averages (right) of ACAT1(ΔNTD). The box size for the 2D averages is 220 Å, whereas that for wild-type ACAT1 is 310 Å. e, The two protomers in each dimer are nearly identical. Superimposition of the two protomers in one dimer is shown. f, Lumenal view of the dimeric ACAT. An open cavity is formed by TM1, TM5, TM6 and TM9 from two protomers around the C2 axis, which is indicated by the black oval in the centre. g, The lumenal cavity in the centre of each dimer is highly hydrophobic. The electrostatic surface potential, calculated in PyMOL, is shown in a cut-open side view.

Extended Data Fig. 6 Structural comparison of ACAT1 and DltB.

a, ACAT1 and DltB share an identical structural core. TM2–TM9 of ACAT1 can be superimposed onto TM3–TM10 of DltB (PDB ID: 6BUI) with an r.m.s.d. of 5.0 Å over 272 Cα atoms. Superimposition of ACAT1 onto DltB is shown in two perpendicular side views. The major conformational shifts of TM8 and TM9in ACAT1 from the corresponding segments in DltB are indicated with orange arrows. TM1 of ACAT1 and the corresponding segments TM1 and TM2 (dark grey) in DltB adopt different structures. b, Loop1 and Loop2 constitute the major cytosolic segments in both ACAT1 and DltB. The cytosolic views of the two proteins, with corresponding structural segments coloured the same, are shown here. c, There is no C tunnel in DltB as there is in ACAT1. The electrostatic surface potentials of ACAT1 and DltB are shown in the same cut-open side views. The conserved His residue is shown as magenta sticks in both structures.

Extended Data Fig. 7 LC–MS identification of the ligand to which the linear density in the structure might belong.

a, Electron microscopy densities for oleoyl-CoA in the ACAT1-A protomer from the dimer reconstruction. The densities for oleoyl-CoA (shown as blue mesh) and surrounding residues (shown as grey mesh) are contoured at 6σ. Two perpendicular views are shown. b, Electron microscopy densities for oleoyl-CoA in the tetrameric reconstruction. All the densities were contoured at 5σ. Two perpendicular views of ACAT1-A (left) and ACAT1-B (right) are shown. The densities in the other two protomers are not shown because of the C2 symmetry. c, LC profiles of commercial oleoyl-CoA (top), and lipids extracted from wild-type enzymes (middle) and the QQ mutant (bottom). d, MS/MS spectrum of commercial oleoyl CoA (top) and extracted oleoyl-CoA from wild-type enzymes (middle) and QQ mutant (bottom). Fractions 1–10 represent the same fragments as those in e. e, Potential MS/MS fragmentation pattern of oleoyl-CoA. f, The QQ mutant shows nearly complete loss of enzymatic activity. Data are mean ± s.d. of three independent experiments.

Source Data.

Extended Data Fig. 8 SEC profiles of the ACAT1 mutants in activity assays.

a, SEC profiles of enzymes with mutations related to oleoyl-CoA coordination. b, SEC profiles of enzymes with mutations related to the T tunnel. The experiments were independently repeated twice with similar results.

Extended Data Fig. 9 The T tunnel may serve as the cholesterol entry site.

a, Cholesterol may access the active site through the T tunnel. Left, a side view of one protomer looking through the T tunnel. The black box indicates the position of the T tunnel. Right, a stretched density is found in the T tunnel. The contour of the density is not reminiscent of cholesterol or GDN. It may result from a mixture of molecules. Nevertheless, the presence of such density suggests that a hydrophobic molecule can enter this tunnel. The density, shown as green mesh, is contoured at 6σ. The density for the potentially bound oleoyl-CoA (blue mesh) is also shown at 6σ as a reference. b, Residues constituting the T tunnel. The density is shown to indicate the tunnel. c, A conserved histidine residue is found in the active site in the crystal structures of carnitine acetyltransferase (PDB ID: 2H3P), cholesterol sulfotransferase (PDB ID: 1Q20), and UDP-N-acetylglucosamine acyltransferase (LpxA) (PDB ID: 2JF3). This residue is highlighted as magenta sticks in all three panels. The bound substrates—carnitine, pregnenolone and UDP-GlcNAc—are all coloured light pink. The crucial histidine residue may activate the nucleophilic substrate through deprotonation.

Extended Data Table 1 Data collection, 3D reconstruction and model statistics

Supplementary information

Supplementary Figure

This file contains raw gels for Fig. 1a and Extended Data Fig. 5c.

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Qian, H., Zhao, X., Yan, R. et al. Structural basis for catalysis and substrate specificity of human ACAT1. Nature 581, 333–338 (2020).

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