Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis for catalysis and substrate specificity of human ACAT1

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Chang, T. Y., Chang, C. C. & Cheng, D. Acyl-coenzyme A:cholesterol acyltransferase. Annu. Rev. Biochem. 66, 613–638 (1997).

    CAS  PubMed  Google Scholar 

  2. 2.

    Ohshiro, T. et al. Pyripyropene A, an acyl-coenzyme A:cholesterol acyltransferase 2-selective inhibitor, attenuates hypercholesterolemia and atherosclerosis in murine models of hyperlipidemia. Arterioscler. Thromb. Vasc. Biol. 31, 1108–1115 (2011).

    CAS  PubMed  Google Scholar 

  3. 3.

    Hartmann, T., Kuchenbecker, J. & Grimm, M. O. Alzheimer’s disease: the lipid connection. J. Neurochem. 103 (Suppl 1), 159–170 (2007).

    CAS  PubMed  Google Scholar 

  4. 4.

    Jiang, Y. et al. Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma. Nature 567, 257–261 (2019).

    ADS  CAS  PubMed  Google Scholar 

  5. 5.

    Li, J. et al. Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer. Oncogene 35, 6378–6388 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Yue, S. et al. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. Cell Metab. 19, 393–406 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Yang, W. et al. Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism. Nature 531, 651–655 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Hofmann, K. A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling. Trends Biochem. Sci. 25, 111–112 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Rudel, L. L. & Shelness, G. S. Cholesterol esters and atherosclerosis-a game of ACAT and mouse. Nat. Med. 6, 1313–1314 (2000).

    CAS  PubMed  Google Scholar 

  10. 10.

    Bhatt-Wessel, B., Jordan, T. W., Miller, J. H. & Peng, L. Role of DGAT enzymes in triacylglycerol metabolism. Arch. Biochem. Biophys. 655, 1–11 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Zhao, Y. et al. Identification and characterization of a major liver lysophosphatidylcholine acyltransferase. J. Biol. Chem. 283, 8258–8265 (2008).

    CAS  PubMed  Google Scholar 

  12. 12.

    Kojima, M., Hamamoto, A. & Sato, T. Ghrelin O-acyltransferase (GOAT), a specific enzyme that modifies ghrelin with a medium-chain fatty acid. J. Biochem. 160, 189–194 (2016).

    CAS  PubMed  Google Scholar 

  13. 13.

    Buglino, J. A. & Resh, M. D. Palmitoylation of Hedgehog proteins. Vitam. Horm. 88, 229–252 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chang, C. C. Y., Sun, J. & Chang, T.-Y. Membrane-bound O-acyltransferases (MBOATs). Front. Biol. 6, 177 (2011).

    ADS  CAS  Google Scholar 

  15. 15.

    Chang, C. C., Huh, H. Y., Cadigan, K. M. & Chang, T. Y. Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J. Biol. Chem. 268, 20747–20755 (1993).

    CAS  PubMed  Google Scholar 

  16. 16.

    Cases, S. et al. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization. J. Biol. Chem. 273, 26755–26764 (1998).

    CAS  PubMed  Google Scholar 

  17. 17.

    Chang, C. C. et al. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J. Biol. Chem. 275, 28083–28092 (2000).

    CAS  PubMed  Google Scholar 

  18. 18.

    Guo, Z. Y., Lin, S., Heinen, J. A., Chang, C. C. & Chang, T. Y. The active site His-460 of human acyl-coenzyme A:cholesterol acyltransferase 1 resides in a hitherto undisclosed transmembrane domain. J. Biol. Chem. 280, 37814–37826 (2005).

    CAS  PubMed  Google Scholar 

  19. 19.

    Yu, C. et al. Role of the N-terminal hydrophilic domain of acyl-coenzyme A:cholesterol acyltransferase 1 on the enzyme’s quaternary structure and catalytic efficiency. Biochemistry 41, 3762–3769 (2002).

    CAS  PubMed  Google Scholar 

  20. 20.

    Das, A., Davis, M. A. & Rudel, L. L. Identification of putative active site residues of ACAT enzymes. J. Lipid Res. 49, 1770–1781 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Liu, J., Chang, C. C., Westover, E. J., Covey, D. F. & Chang, T. Y. Investigating the allosterism of acyl-CoA:cholesterol acyltransferase (ACAT) by using various sterols: in vitro and intact cell studies. Biochem. J. 391, 389–397 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Rogers, M. A. et al. Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase. J. Biol. Chem. 287, 17483–17492 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Zhang, Y. et al. Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1. J. Biol. Chem. 278, 11642–11647 (2003).

    CAS  PubMed  Google Scholar 

  24. 24.

    Ghosh, S., Zhao, B., Bie, J. & Song, J. Macrophage cholesteryl ester mobilization and atherosclerosis. Vascul. Pharmacol. 52, 1–10 (2010).

    CAS  PubMed  Google Scholar 

  25. 25.

    Tardif, J. C. et al. Effects of the acyl coenzyme A:cholesterol acyltransferase inhibitor avasimibe on human atherosclerotic lesions. Circulation 110, 3372–3377 (2004).

    CAS  PubMed  Google Scholar 

  26. 26.

    Chang, C. et al. Human acyl-CoA:cholesterol acyltransferase (ACAT) and its potential as a target for pharmaceutical intervention against atherosclerosis. Acta Biochim. Biophys. Sin. (Shanghai) 38, 151–156 (2006).

    CAS  Google Scholar 

  27. 27.

    Nissen, S. E. et al. Effect of ACAT inhibition on the progression of coronary atherosclerosis. N. Engl. J. Med. 354, 1253–1263 (2006).

    CAS  PubMed  Google Scholar 

  28. 28.

    Meuwese, M. C. et al. ACAT inhibition and progression of carotid atherosclerosis in patients with familial hypercholesterolemia: the CAPTIVATE randomized trial. J. Am. Med. Assoc. 301, 1131–1139 (2009).

    CAS  Google Scholar 

  29. 29.

    Shibuya, Y., Chang, C. C. & Chang, T. Y. ACAT1/SOAT1 as a therapeutic target for Alzheimer’s disease. Future Med. Chem. 7, 2451–2467 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Ma, D. et al. Crystal structure of a membrane-bound O-acyltransferase. Nature 562, 286–290 (2018).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Chang, C. C. et al. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner. J. Biol. Chem. 273, 35132–35141 (1998).

    CAS  PubMed  Google Scholar 

  32. 32.

    Chang, C. C. et al. Purification of recombinant acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) from H293 cells and binding studies between the enzyme and substrates using difference intrinsic fluorescence spectroscopy. Biochemistry 49, 9957–9963 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Cao, J. et al. Targeting acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases. J. Biol. Chem. 286, 41838–41851 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Yu, C. et al. Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro. J. Biol. Chem. 274, 36139–36145 (1999).

    CAS  PubMed  Google Scholar 

  35. 35.

    Guo, Z., Cromley, D., Billheimer, J. T. & Sturley, S. L. Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences. J. Lipid Res. 42, 1282–1291 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Hsiao, Y. S., Jogl, G. & Tong, L. Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates. J. Biol. Chem. 281, 28480–28487 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Lee, K. A. et al. Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence of pregnenolone and 3′-phosphoadenosine 5′-phosphate. Rationale for specificity differences between prototypical SULT2A1 and the SULT2BG1 isoforms. J. Biol. Chem. 278, 44593–44599 (2003).

    CAS  PubMed  Google Scholar 

  38. 38.

    Ulaganathan, V., Buetow, L. & Hunter, W. N. Nucleotide substrate recognition by UDP-N-acetylglucosamine acyltransferase (LpxA) in the first step of lipid A biosynthesis. J. Mol. Biol. 369, 305–312 (2007).

    CAS  PubMed  Google Scholar 

  39. 39.

    Miyazaki, M., Kim, Y. C., Gray-Keller, M. P., Attie, A. D. & Ntambi, J. M. The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1. J. Biol. Chem. 275, 30132–30138 (2000).

    CAS  PubMed  Google Scholar 

  40. 40.

    Guo, Z. Y., Chang, C. C. & Chang, T. Y. Functionality of the seventh and eighth transmembrane domains of acyl-coenzyme A:cholesterol acyltransferase 1. Biochemistry 46, 10063–10071 (2007).

    CAS  PubMed  Google Scholar 

  41. 41.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Google Scholar 

  42. 42.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Scheres, S. H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Scheres, S. H. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016).

    CAS  PubMed  Google Scholar 

  49. 49.

    Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    CAS  Google Scholar 

  50. 50.

    Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Dang, S. et al. Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Nature 552, 426–429 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Google Scholar 

  53. 53.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  Google Scholar 

  54. 54.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  Google Scholar 

  55. 55.

    Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    DeLano, W. L. The PyMOL Molecular Graphics System, http://www.pymol.org(2002).

  57. 57.

    Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44 (W1), W344–W350 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Ethics declarations

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 (http://multalin.toulouse.inra.fr).

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.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qian, H., Zhao, X., Yan, R. et al. Structural basis for catalysis and substrate specificity of human ACAT1. Nature 581, 333–338 (2020). https://doi.org/10.1038/s41586-020-2290-0

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing