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Molecular basis for acetyl-CoA production by ATP-citrate lyase

Nature Structural & Molecular Biology volume 27, pages 33–41 (2020)Cite this article

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Matters Arising to this article was published on 22 July 2021

An Author Correction to this article was published on 02 April 2020

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Abstract

ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report cryo-EM structures of human ACLY, alone or bound to substrates or products. ACLY forms a homotetramer with a rigid citrate synthase homology (CSH) module, flanked by four flexible acetyl-CoA synthetase homology (ASH) domains; CoA is bound at the CSH–ASH interface in mutually exclusive productive or unproductive conformations. The structure of a catalytic mutant of ACLY in the presence of ATP, citrate and CoA substrates reveals a phospho-citryl-CoA intermediate in the ASH domain. ACLY with acetyl-CoA and oxaloacetate products shows the products bound in the ASH domain, with an additional oxaloacetate in the CSH domain, which could function in ACLY autoinhibition. These structures, which are supported by biochemical and biophysical data, challenge previous proposals of the ACLY catalytic mechanism and suggest additional therapeutic possibilities for ACLY-associated metabolic disorders.

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Fig. 1: Structure of ACLY in complex with CoA and citrate substrates.
Fig. 2: Structure of the ACLY tetramer with an asymmetric ASH subunit in complex with two CoA molecules.
Fig. 3: Structure of ACLY in its unliganded form.
Fig. 4: Structure of ACLY in the presence of acetyl-CoA and OAA products and citrate and OAA binding properties.
Fig. 5: Activity of the putative E599 catalytic residue.
Fig. 6: Structure of the ACLY-E599Q catalytic mutant in the presence of ATP, citrate and CoA co-substrates.
Fig. 7: Proposed model of metabolite regulation of ACLY activity.

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Data availability

Structures and EM maps of ACLY-apo (PDB-6POF, EMDB-20414), ACLY–citrate-CoA-D2 (PDB-6UUZ, EMDB-20903), ACLY–citrate-CoA-C1 assym open (PDB-6UIA, EMDB-20784), ACLY–citrate-CoA-C1 assym closed (PDB-6POE, EMDB-20413), ACLY–OAA–acetyl-CoA-C1 (PDB-6UV5, EMDB-20904), ACLY–OAA–acetyl-CoA-D2 (PDB-6UI9, EMDB-20783) and ACLY-E599Q–ATP-citrate-CoA (PDB-6UUW, EMDB-20902) have been deposited to the PDB and EMDB databases. Source data for Figs. 1f, 4d,e and 5a,b and Extended Data Fig. 1c are available with the online version of the paper.

Change history

  • 14 February 2020

    The callout to Extended Data Fig. 7c was linked to Fig. 4; the link has now been removed.

  • 02 April 2020

    A Correction to this paper has been published: https://doi.org/10.1038/s41594-020-0421-9

References

  1. Lee, J. V., Shah, S. A. & Wellen, K. E. Obesity, cancer and acetyl-CoA metabolism. Drug Discov. Today Dis. Mech. 10, e55–e61 (2013).

    Article  Google Scholar 

  2. Chypre, M., Zaidi, N. & Smans, K. ATP-citrate lyase: a mini-review. Biochem. Biophys. Res. Commun. 422, 1–4 (2012).

    Article  CAS  Google Scholar 

  3. Granchi, C. ATP citrate lyase (ACLY) inhibitors: an anti-cancer strategy at the crossroads of glucose and lipid metabolism. Eur. J. Med. Chem. 157, 1276–1291 (2018).

    Article  CAS  Google Scholar 

  4. Bilen, O. & Ballantyne, C. M. Bempedoic acid (ETC-1002): an investigational inhibitor of ATP citrate lyase. Curr. Atheroscler. Rep. 18, 61 (2016).

    Article  Google Scholar 

  5. Burke, A. C. & Huff, M. W. ATP-citrate lyase: genetics, molecular biology and therapeutic target for dyslipidemia. Curr. Opin. Lipidol. 28, 193–200 (2017).

    Article  CAS  Google Scholar 

  6. Pinkosky, S. L. et al. Liver-specific ATP-citrate lyase inhibition by bempedoic acid decreases LDL-C and attenuates atherosclerosis. Nat. Commun. 7, 13457 (2016).

    Article  CAS  Google Scholar 

  7. Ray, K. K. et al. Safety and efficacy of bempedoic acid to reduce LDL cholesterol. N. Engl. J. Med. 380, 1022–1032 (2019).

    Article  CAS  Google Scholar 

  8. Sanchez, L. B., Galperin, M. Y. & Muller, M. Acetyl-CoA synthetase from the amitochondriate eukaryote Giardia lamblia belongs to the newly recognized superfamily of acyl-CoA synthetases (nucleoside diphosphate-forming). J. Biol. Chem. 275, 5794–5803 (2000).

    Article  CAS  Google Scholar 

  9. Bond, D. R. et al. Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae. Appl. Environ. Microbiol. 71, 3858–3865 (2005).

    Article  CAS  Google Scholar 

  10. Nguyen, N. T. et al. Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes. Biochemistry 40, 13177–13187 (2001).

    Article  CAS  Google Scholar 

  11. Singh, M., Richards, E. G., Mukherjee, A. & Srere, P. A. Structure of ATP citrate lyase from rat liver. Physicochemical studies and proteolytic modification. J. Biol. Chem. 251, 5242–5250 (1976).

    Article  CAS  Google Scholar 

  12. Walsh, C. T. Jr. & Spector, L. B. Citryl phosphate and the mode of action of the citrate cleavage enzyme. J. Biol. Chem. 244, 4366–4374 (1969).

    Article  CAS  Google Scholar 

  13. Sun, T., Hayakawa, K., Bateman, K. S. & Fraser, M. E. Identification of the citrate-binding site of human ATP-citrate lyase using X-ray crystallography. J. Biol. Chem. 285, 27418–27428 (2010).

    Article  CAS  Google Scholar 

  14. Inoue, H., Fujio, S., Keihachi, F., Kozaburo, A. & Takeda, Y. Studies on ATP citrate lyase of rat liver: I. Purification and some properties. J. Biol. Chem. 60, 543–553 (1966).

    CAS  Google Scholar 

  15. Inoue, H., Suzuki, F., Tanioka, H. & Takeda, Y. Role of ATP in the ATP citrate lyase reaction. Biochem. Biophys. Res. Commun. 26, 602–608 (1967).

    Article  CAS  Google Scholar 

  16. Inoue, H., Suzuki, F., Tanioka, H. & Takeda, Y. Studies on ATP citrate lyase of rat liver. 3. The reaction mechanism. J. Biochem. 63, 89–100 (1968).

    Article  CAS  Google Scholar 

  17. Inoue, H., Tsunemi, T., Suzuki, F. & Takeda, Y. Studies on ATP citrate lyase of rat liver. IV. The role of CoA. J. Biochem. 65, 889–900 (1969).

    Article  CAS  Google Scholar 

  18. Srere, P. A. The citrate cleavage enzyme. I. Distribution and purification. J. Biol. Chem. 234, 2544–2547 (1959).

    Article  CAS  Google Scholar 

  19. Srere, P. A. The citrate cleavage enzyme: II. Stoichiometry substrate specificity and its use for coenzyme A assay. J. Biol. Chem. 236, 50–53 (1961).

    Article  CAS  Google Scholar 

  20. Srere, P. A. & Lipmann, F. An enzymatic reaction between citrate, adenosine triphosphate and coenzyme A1. J. Am. Chem. Soc. 75, 4874–4874 (1953).

    Article  CAS  Google Scholar 

  21. Bazilevsky, G. A. et al. ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis. J. Biol. Chem. 294, 7259–7268 (2019).

    Article  CAS  Google Scholar 

  22. Verschueren, K. H. G. et al. Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle. Nature 568, 571–575 (2019).

    Article  CAS  Google Scholar 

  23. Wei, J. et al. An allosteric mechanism for potent inhibition of human ATP-citrate lyase. Nature 568, 566–570 (2019).

    Article  CAS  Google Scholar 

  24. Hu, J., Komakula, A. & Fraser, M. E. Binding of hydroxycitrate to human ATP-citrate lyase. Acta Crystallogr. D Struct. Biol. 73, 660–671 (2017).

    Article  CAS  Google Scholar 

  25. Wiegand, G., Remington, S., Deisenhofer, J. & Huber, R. Crystal structure analysis and molecular model of a complex of citrate synthase with oxaloacetate and S-acetonyl-coenzyme A. J. Mol. Biol. 174, 205–219 (1984).

    Article  CAS  Google Scholar 

  26. Pentyala, S. N. & Benjamin, W. B. Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. Biochemistry 34, 10961–10969 (1995).

    Article  CAS  Google Scholar 

  27. Rokita, S. E., Srere, P. A. & Walsh, C. T. 3-Fluoro-3-deoxycitrate: a probe for mechanistic study of citrate-utilizing enzymes. Biochemistry 21, 3765–3774 (1982).

    Article  CAS  Google Scholar 

  28. Fan, F. et al. On the catalytic mechanism of human ATP citrate lyase. Biochemistry 51, 5198–5211 (2012).

    Article  CAS  Google Scholar 

  29. Tanner, K. G. et al. Catalytic mechanism and function of invariant glutamic acid 173 from the histone acetyltransferase GCN5 transcriptional coactivator. J. Biol. Chem. 274, 18157–18160 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    Article  CAS  Google Scholar 

  34. Kim, D. E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004).

    Article  CAS  Google Scholar 

  35. Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Delano, W. L. The PyMol Molecular Graphics System (DeLano Scientific, 2002).

  40. Linn, T. C. & Srere, P. A. Identification of ATP citrate lyase as a phosphoprotein. J. Biol. Chem. 254, 1691–1698 (1979).

    Article  CAS  Google Scholar 

  41. Michnik, A., Drzazga, Z., Kluczewska, A. & Michalik, K. Differential scanning microcalorimetry study of the thermal denaturation of haemoglobin. Biophys. Chem. 118, 93–101 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Negative staining and cryo-EM grids screening were carried out in EMRL at the Perelman School of Medicine, University of Pennsylvania. We thank B. Zuo and S. Molugu for their help with negative staining and cryo-EM grids screening. Cryo-EM data collection was carried out at the University of Massachusetts Cryo-EM Core Facility. Molecular graphics and structural analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. We thank K. Song, K. Lee, C. Xu and other staff members at the University of Massachusetts Cryo-EM Core Facility for their support with cryo-EM data collection; K. Wellen, A. Olia, S. Deng and M. Ricketts for helpful discussions; T. Eeuwen and K. Murakami for advice with EM data collection; and S. Zeng for organizing cryo-EM data collection trips. This work was supported by NIH grants R35 GM118090 and P01 AG031862 to R.M. and NIH grant no. F31CA189559 to G.B.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Xuepeng Wei, Austin Vogt & Ronen Marmorstein

  2. Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Xuepeng Wei, Kollin Schultz, Gleb A. Bazilevsky, Austin Vogt & Ronen Marmorstein

  3. Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Kollin Schultz

  4. Graduate Group in Cell and Molecular Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Gleb A. Bazilevsky

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  1. Xuepeng Wei
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Contributions

Conceptualization was provided by X.W., K.S., G.A.B., A.V. and R.M.; methodology by X.W., K.S., G.A.B., A.V. and R.M.; investigation by X.W., K.S., G.A.B. and A.V.; and formal analysis by X.W., K.S., G.A.B. and A.V. The original draft was written by X.W. Visualization was provided by X.W. and G.A.B. The manuscript was reviewed and edited by X.W., K.S., G.A.B., A.V. and R.M. Funding acquisition was carried out by R.M. Resources were provided by R.M. Supervision was performed by R.M.

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Correspondence to Ronen Marmorstein.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Production of recombinant human ACLY.

a, SDS-PAGE (12% acrylamide) verification of intact, homogeneous human ACLY protein produced in Escherichia coli. b, Size-exclusion chromatography of ACLY on a superdex 200 increase column. c, Differential Scanning Fluorimetry (DSF) of ACLY in the absence and presence of metabolite ligands (Mean + /- Standard Deviation, n = 3 biologically independent samples). P-values were calculated by two-way ANOVA with Dunnett’s multiple comparisons test.

Source data

Extended Data Fig. 2 Electron micrographs of ACLY.

a, Representative negative stain images of human ACLY. b, 2D class average from 3,000 negative stain ACLY. c, Representative cryo-EM images of human ACLY in complex with citrate-CoA. d, Representative 2D class averages of cryo-EM ACLY particles.

Extended Data Fig. 3

Image processing workflow for single particle reconstruction of ACLY–citrate-CoA.

Extended Data Fig. 4 Analysis of single particle cryo-EM reconstructions.

a, Fourier Shell Correlation (FSC) curves for 3D reconstructions of reported structures, marked with resolutions corresponding to FSC = 0.143. b, Cryo-EM density of representative helical segments (residues 1055-1077) from ACLY–citrate-CoA (left) and ACLY–OAA–acetyl-CoA structures. c, Local resolution estimation of cryo-EM maps of ACLY–citrate-CoA-D2 (left), ACLY–citrate-CoA-C1 asymm closed (middle) and ACLY–OAA–acetyl-CoA (right) by Resmap.

Extended Data Fig. 5 Structural comparison of the CSH module of ACLY with citrate synthase.

a, Side-by-side views of Sulfolobus solfataricus citrate synthase (left) with the ACLY CSH module. b, Superposition of CSH/OAA2 with pig heart citrate synthase bound to OAA.

Extended Data Fig. 6 Model of alternate acetyl-CoA orientations against the citrate synthase homology (CSH) domain.

a, Both the extended and ‘flipped’ orientations of acetyl-CoA with the observed cryo-EM density (blue chicken wire) are shown. b, The flipped orientation of acetyl-CoA is modeled into ACLY-apo showing no significant steric clashes.

Extended Data Fig. 7 Cryo-EM density of ACLY ligands.

a, CoA in ACLY–CoA–citrate. b, Acetyl-CoA in ACLY–OAA–acetyl-CoA. c, Phosphor-citryl-CoA in ACLY-E599Q–ATP-citrate-CoA. d, Superposition of Acetyl-CoA and Phosphor-citryl-CoA showing movement of F537 as a function of bound intermediate/product.

Supplementary information

Reporting Summary

Supplementary Video 1

Side view of ACLY morphed through four conformational states reported here (ACLY-apo, ACLY–citrate-CoA, ACLY-E599Q–ATP-citrate-CoA, ACLY–OAA-acetyl-CoA and back to ACLY-apo). CPK-white carbon coloring is used for bound ligands. The binding of ATP and citrate prior to CoA binding is modeled based on the ACLY ASH domain crystal structures bound to citrate (3MWD) and ADP (5TES).

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Wei, X., Schultz, K., Bazilevsky, G.A. et al. Molecular basis for acetyl-CoA production by ATP-citrate lyase. Nat Struct Mol Biol 27, 33–41 (2020). https://doi.org/10.1038/s41594-019-0351-6

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