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

Matters Arising to this article was published on 22 July 2021

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

This article has been updated

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.

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

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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

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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.

Corresponding author

Correspondence to Ronen Marmorstein.

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The authors declare no competing interests.

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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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