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Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle

Nature volume 568, pages 571–575 (2019)Cite this article

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Abstract

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8,9,10,11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.

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Fig. 1: Structure and mechanism of human ATP citrate lyase.
Fig. 2: Evolutionary origin of ACLY and its distinct metabolic functions.
Fig. 3: ACLY enzymes undergo conformational switching during catalysis.
Fig. 4: Citrate synthase evolved from an ancestral CCL module.

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Data availability

Protein expression constructs generated in this study are available via the BCCM/GeneCorner Plasmid Collection (http://bccm.belspo.be) through the following accession codes: LMBP 11277 (pTrcHis2-hACLY), LMBP 11131 (pET-DUET-hACLY-A/B), LMBP 11132 (pET11a-Mco-ACLY-A/B), LMBP 11133 (pET11a-Hth-CCL), LMBP 11134 (pET-Duet-Hth-CCSα/β), LMBP 11125 (pET11a-Cli-ACLY-A/B), LMBP 11128 (pET15b-hCCL) and LMBP 11129 (pET15b-Cli-CCL). X-ray crystallographic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession codes 6HXH (hACLY-A/B in space group P1), 6QFB (hACLY-A/B in space group C2), 6HXI (M. concilii ACLY-A/B), 6HXJ (C. limicola ACLY-A/B), 6HXK (CCL module of hACLY, space group P212121), 6HXL (CCL module of hACLY, space group P21), 6HXM (CCL module of hACLY, space group C2221), 6HXN (CCL module of C. limicola ACLY, space group P3121), 6HXO (CCL module of C. limicola ACLY, space group P21), 6QCL (CCL module of C. limicola ACLY in complex with acetyl-CoA and l-malate), 6HXP (H. thermophilus CCL) and 6HXQ (H. thermophilus CCS). SAXS data and models have been deposited in the Small Angle Scattering Biological Data Bank with accession codes SASDE36, SASDE46 and SASDE56 for hACLY-A/B; SASDFA3, SASDFB3 and SASDFC3 for hACLY; and SASDE66, SASDE76 and SASDE86 for C. limocola ACLY-A/B. Source Data for the SEC–MALLS analysis of hACLY-A/B (Extended Data Fig. 1d) and for the enzymatic assays for hACLY and hACLY-A/B (Extended Data Fig. 1e) are available online. Data are available from the corresponding author(s) upon reasonable request.

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Acknowledgements

K.V. and Y.B. are post-doctoral research fellows of the Research Foundation – Flanders (FWO) (fellowships 12A5517N and 12S0519N). J.F. is supported by an EMBO long-term post-doctoral fellowship (ALTF441-2017). This work was supported by grants from the FWO to K.V. (1524918N), a Concerted Research Action (GOA) grant from Ghent University to S.N.S. (BOF17-GOA-028), a Hercules Foundation infrastructure grant to S.N.S. (AUGE-11-029), a programme grant from the VIB to S.N.S., and the Horizon 2020 grants: Chap4Resp (to I.G., grant no. 647784), iNext (grant no. 653706) and CALIPSOplus (grant no. 730872). We acknowledge access to experimental facilities and technical support at the following synchrotron radiation facilities: PETRA III (beamlines P12, P13 and P14), SOLEIL (Proxima-2, Swing), ESRF (ID23-1, ID23-2 and ID30-B), SLS (PXI and PXIII). This work used the platforms of the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The electron microscope facility is supported by the Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), Fonds FEDER, the Centre National de la Recherche Scientifique (CNRS), the CEA, the University of Grenoble, EMBL, and the GIS-Infrastrutures en Biologie Sante et Agronomie (IBISA).

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Nature thanks Frank M. Raushel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations

  1. Unit for Structural Biology, VIB Center for Inflammation Research, Ghent, Belgium

    Koen H. G. Verschueren, Ann Dansercoer, Yehudi Bloch, Savvas N. Savvides & Kenneth Verstraete

  2. Unit for Structural Biology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Koen H. G. Verschueren, Ann Dansercoer, Yehudi Bloch, Savvas N. Savvides & Kenneth Verstraete

  3. European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DESY, Hamburg, Germany

    Clement Blanchet & Dmitri Svergun

  4. University of Grenoble Alpes, CNRS, CEA, CNRS, IBS, Grenoble, France

    Jan Felix & Irina Gutsche

  5. Integrated Molecular Plant Physiology Research, Department of Biology, University of Antwerp, Antwerp, Belgium

    Dirk De Vos

  6. Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Jozef Van Beeumen

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Contributions

K.H.G.V. conducted crystallographic experiments and refined crystal structures. C.B. analysed SAXS data with contributions from D.S. J.F. and I.G. performed negative-stain electron microscopy analysis of hACLY. A.D. performed molecular cloning and produced recombinant proteins. D.D.V. and J.V.B. initiated studies on hACLY and provided research tools, with contributions from Y.B. S.N.S. contributed to data analysis. K.V. directed and designed the study, conducted crystallographic, SAXS, size-exclusion chromatography coupled to multiangle laser light scattering (SEC–MALLS) and kinetic experiments, refined and analysed crystal structures, analysed SAXS data, and created the figures. K.V. and S.N.S. wrote the manuscript with contributions from all other authors.

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Correspondence to Kenneth Verstraete.

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Extended data figures and tables

Extended Data Fig. 1 Structure of the human ACLY.

a, Reaction scheme for ACLY. In the first step, ACLY undergoes autophosphorylation at His760. Citryl-phosphate (citryl-P) and citryl-CoA form non-covalent enzyme-intermediate complexes. b, Left, representative class averages for hACLY as obtained by negative-stain electron microscopy. The size of the box is 40 × 40 nm. Right, flowchart of the 3D reconstruction in C2 symmetry. Negative-stain electron microscopy analysis was performed on a single sample of purified hACLY (n = 1). SDG, stochastic gradient descent. c, Coomassie-stained SDS–PAGE gel for recombinantly produced ACLY enzymes. Lane 1, hACLY-A/B; lane 2, hACLY; lane 3, M. concilii ACLY-A/B; lane 4, C. limicola ACLY-A/B; lane 5, hACLY(His760Ala). In this study, each protein was purified several times, and the electrophoretic profile of each sample in the gel shown is representative for different protein batches. For gel source data, see Supplementary Fig. 1. d, Size-exclusion chromatography (SEC) elution profile of hACLY-A/B plotted as the light scattering intensity at 90° in function of the elution volume. The reported molecular mass by multiangle laser light scattering (MALLS) represents the average molecular mass ± s.d. across the elution peak. The theoretical mass for hACLY-A/B is 462 kDa. Number of samples for hACLY-A/B analysed: n = 1. e, Reaction rates for hACLY-A/B, hACLY and hACLY(His760Ala) plotted as a function of ATP concentration. For hACLY-A/B and hACLY, data replicates (n = 4, in which n represents a different protein batch) were fitted by a Michaelis–Menten equation and the obtained Michaelis constant (Km) and turnover number (kcat) values (mean + s.e.m) are shown. The kinetic parameters for hACLY-A/B and hACLY are significantly different via two-tailed unpaired t-tests: P = 0.0002 (comparing kcat); and P = 0.0156 (comparing Km). For the hACLY(His760Ala) mutant, the number of replicate batches: n = 1. f, Representative crystal structure for hACLY-A/B extracted from the P1 crystal form and coloured by chain. Bound substrates are shown as coloured spheres. g, View on the helical bundle core of the CCL module with the protruding two-helix stalk regions indicated. CoA-binding domains are omitted for clarity. h, Overlay of the four hACLY-A/B crystal structures extracted from the P1 and C2 crystal forms. The overlay is based on the superposition of the CCL modules. Structures are coloured according to the scheme in Fig. 1a. A zoom-in view shows the structural plasticity around the two-helix stalk region. i, View on the helical bundle core of CCL from H. thermophilus coloured by chain. The N and C termini of a single chain are indicated.

Source data

Extended Data Fig. 2 CoA-binding modes in the CCS-module of ACLY and related CCS.

a, View on the CoA-binding mode in hACLY-A/B crystal structures. b, Detail of the CoA-binding mode at the interface between the CCL and CCS modules. The so-called power helices in the CCS module are indicated. Dashed lines represent polar interactions. c, View on the CoA-binding mode in the crystal structure for ACLY-A/B from M. concilii. d, View on the CoA-binding modes in the crystal structure for ACLY-A/B from C. limicola. In this structure, the phosphopantheine tails of the CoA-molecules were partly disordered. e, View on the CoA-binding mode in CCSα/β from H. thermophilus. f, Cartoon representation of CCSα/β from H. thermophilus and succinyl-CoA synthetase α/β (SCSα/β) from E. coli, both in complex with CoA. α-subunits are coloured in blue and β-subunits in grey. The C-terminal tail extending from CCSα to CCSβ is in orange.

Extended Data Fig. 3 Structural plasticity in the CCL modules of human and C. limicola ACLY.

a, Crystal structure of the CCL module of hACLY in space group P212121, in complex with citrate. b, Crystal structure of the CCL module of hACLY in space group C2221, in complex with citrate and CoA. c, Crystal structure of the CCL module of hACLY in space group P21, in complex with citrate and CoA. This crystal form contained two tetramers in the crystallographic asymmetric unit (asu). In a–c, CoA-binding domains are coloured according to the structural state of the CCL active site: open (white), intermediate (blue) and closed (magenta), and substrates are shown as coloured spheres. d, Binding mode of CoA as seen in the hACLY-A/B crystal structure (left) compared to CoA binding in a closed CCL module protomer (right). Substrates are shown as coloured sticks and dashed lines indicate polar interactions. e, A CCL module protomer in the open state as seen in the hACLY-A/B structure (white CoA-binding domain) overlaid with a protomer in the closed state (magenta CoA-binding domain). The latter was extracted from a crystal structure for the isolated CCL module of hACLY (c). Arrows indicate structural transitions. f, Reaction itinerary in human ACLY. g, Crystal structure for the CCL module of C. limicola ACLY in space group P21, in complex with citrate. This crystal form contained two tetramers in the asu. In the second tetramer (right), one of the CoA-binding domains was not modelled owing to disorder. h, Crystal structure for the CCL module of C. limicola ACLY in space group P3121, in complex with CoA. i, CCL module of C. limicola ACLY as observed in the C. limicola ACLY crystal structure. j, Overlay of C. limicola CCL module protomers coloured according to the structural state of their active site: open (white), intermediate (blue) and closed (magenta).

Extended Data Fig. 4 ACLY structures across different domains of life.

Cartoon representations of the crystal structures of human ACLY-A/B (top), M. concilii ACLY-A/B (middle) and C. limicola ACLY-A/B (bottom). The CCS modules are shown in surface mode. Distinct structural regions are coloured according to the colouring scheme in Fig. 1a.

Extended Data Fig. 5 Sequence alignment of ACLY, CCS and CCL.

a, Homology relationships between the different enzymes for which crystal structures were determined in this manuscript. b, Sequence alignment for ACLY, CCL and CCS according to the scheme in a. Top secondary structure elements correspond to hACLY, bottom secondary structure elements correspond to CCSα/β and CCL from H. thermophilus. Strictly conserved residues are white against a black background. CCSα, CCSβ and CCL homology regions and CoA-binding domain are indicated by a coloured bar on top of the alignment. The CCSα β-hairpin (orange) and CCL stalk (green) regions in ACLY enzymes are highlighted. Conserved residues at the ACLY two-helix pivot are indicated with a purple arrow. Regulatory phosphorylation sites in the linker region (brown) that connects the ancestral ACLY-A and ACLY-B parts in hACLY are indicated by a letter P in yellow circles.

Extended Data Fig. 6 Conformational switching of ACLY during catalysis.

a, View of the interaction between the CCS and CCL modules in a representative hACLY-A/B crystal structure (space group P1), with the CCSα β-hairpin (orange) and CoA-binding domain (pink) in cartoon mode. Bound CoA is shown as coloured spheres. b, Overlay of a human CCL protomer in the closed state (pink CoA-binding domain) with the crystal structure of hACLY-A/B as in a (white CoA-binding domain). The resulting clash between the CoA-binding domain (with bound CoA-molecule) and the β-hairpin is indicated by a red box. c, View of the interaction between the CCS and CCL modules in the crystal structure of C. limicola ACLY-A/B, with the CCSα β-hairpin (orange) and CoA-binding domain (pink) in cartoon mode. d, Zoomed-in view of the stalk region in the crystal structures of hACLY-A/B and C. limicola ACLY-A/B based on the superposition of the helical core of the CCL modules. e, f, Interactions at the stalk region and β-hairpin as observed in the crystal structures of hACLY-A/B and C. limicola ACLY-A/B. g, Two-state rigid-body SAXS model for apo-hACLY-A/B (MultiFoXS, χ2 = 2.8) overlaid with the hACLY-A/B crystal structures in space groups P1 and C2 (grey). h, Single-state rigid-body SAXS model for hACLY-A/B (MultiFoXS, χ2 = 2.8) in the presence of both citrate and CoA overlaid with the hACLY-A/B crystal structures in space groups P1 and C2 (grey). i, Comparison between in-solution SAXS scattering profiles measured from linker-deleted hACLY-A/B and full-length hACLY. (i) Profiles recorded from hACLY-A/B (green) and hACLY (grey) in HBS buffer; (ii) profiles recorded from hACLY-A/B (purple) and hACLY (black) in HBS buffer supplemented with citrate and CoA; (iii) profiles recorded from hACLY in HBS buffer (grey) and HBS buffer supplemented with both citrate and CoA (black); (iv) profiles recorded from hACLY-A/B in HBS buffer (green) and HBS buffer supplemented with both citrate and CoA (purple); and (v) fit of the theoretical scattering profile (red) calculated from an AllosMod-FoXS model for hACLY (as shown in j) to the experimental scattering profile recorded in the presence of citrate and CoA (black). j, AllosMod-FoXS SAXS model for hACLY in HBS buffer supplemented with citrate and CoA, overlaid with the hACLY-A/B crystal structures in space groups P1 and C2 (grey). In g, h and j, the bottom numeric table presents an all-residue (Cα) r.m.s.d. matrix for the hACLY-A/B crystal structures and presented SAXS models, and for each crystal structure and model the calculated fit (χ2 value) against the recorded SAXS data are shown as calculated by FoXS, Crysol and Crysol 3.0. P1-hACLY-A/B_1 and P1-hACLY-A/B_2 denote structures for hACLY-A/B extracted from the P1 crystal form; C2-hACLY-A/B_1 and C2-hACLY-A/B_2 denote structures for hACLY-A/B extracted from the C2 crystal form.

Extended Data Fig. 7 Citrate synthase evolved from an ancestral CCL module.

a, Side-by-side comparison and overlay of the helical bundle cores of H. thermophilus CCL and P. furiosus CS (PDB accession 1AJ8). b, Two adjacent CCL protomers (CCL and CCL’) extracted from the H. thermophilus CCL tetramer. c, A CS protomer extracted from P. furiosus CS. d, CCL without its CoA-binding domain (residues 2–100 and 204–231) aligned with the N-terminal half of CS (residues 6–143). e, CCL’ (residues 30–256) aligned with the C-terminal half of CS (residues 154–376). f, CCL and CCL’ aligned with the CS protomer. g, Sequence alignment between CCL and CCL’ and CS sequences. Top secondary structure elements correspond to H. thermophilus CCL and CCL’, bottom secondary structures correspond to P. furiosus CS. The active site residues of CS are indicated by a purple arrow. h, i, Details of the overlay between CCL and CCL’ and the CS protomer. j, Side view of the CCL module and CS highlighting the pseudo-two-fold symmetry in the CS protomer. CoA is shown by sticks.

Extended Data Fig. 8 Homology between ACLY and citrate synthase.

a, Sequence alignment between the C-terminal regions of ACLY, CCL and CS. Active-site residues are highlighted according to the numbering scheme in chicken CS. His274, highlighted in yellow, is not conserved in ACLY sequences. b, Comparison between crystal structures for the CCL module of hACLY and chicken CS (PDB accessions 5CSC and 5CTS) in open and closed states. For clarity, only the helical secondary structure elements are shown. Bound substrates are shown as coloured spheres. c, Overlay of the CCL active site of hACLY (blue) in complex with citrate and CoA, with the active site of chicken CS (orange) in complex with oxaloacetate and carboxymethyl-CoA (PDB accession 5CTS). The interaction between the carboxylate group of hACLY(Asp1026) and citrate, and the interaction between the carboxylate group of Asp375 of CS and carboxymethyl-CoA are indicated. d, By analogy to the aldol condensation of acetyl-CoA and oxaloacetate to citryl-CoA as catalysed by CS (top), citryl-CoA may undergo retro-aldol cleavage catalysed by ACLY as indicated by the chemical reaction arrows (bottom). Dashed lines indicate polar interactions.

Extended Data Fig. 9 Flowchart showing negative-stain electron microscopy data processing for human ACLY.

Starting from a final dataset of 27,293 particles, initial models were made using the stochastic gradient descent method in RELION2.1, applying C1, C2 or D2 symmetry. Subsequent 3D classification was performed using the C1, C2 or D2 starting models as an input, again applying C1, C2 or D2 symmetry, respectively. 3D classification using C1 and C2 symmetry clearly shows well-defined CCS modules in one-half of the hACLY molecule (C1: class 3, C2: class 3 and 4). Although 3D classification using D2 symmetry results in two classes displaying all four CCS modules (class 1 and 4), subsequent 3D refinement in C2 using an averaged map of these two classes resulted in a disappearance of two CCS modules in the lower half of hACLY, pointing to flexibility of the peripheral domains of hACLY.

Extended Data Table 1 Crystallographic data and refinement statistics
Full size table
Extended Data Table 2 SAXS data collection and scattering-derived parameters
Full size table

Supplementary information

Supplementary Figure

Supplementary Figure 1 - Uncropped scans with size marker indications. Uncropped SDS-PAGE gel for Extended Data Figure 1c.

Reporting Summary

Video 1: The CCL module of hACLY cycles between open and closed states.

The video shows a morph between the CCL core module of hACLY in the open state - as observed in the hACLY-A/B crystal structure (Fig. 1b) – and the crystal structure of the CCL module of hACLY in the closed state (Extended Data Fig. 3c).

Video 2: Shuttling of the citryl-CoA intermediate between the citryl-CoA synthetase and lyase modules.

The video shows a morph between the hACLY-A/B crystal structure (Fig. 1b) and the crystal structure of the CCL core module of hACLY in the closed state (Extended Data Fig. 3c).

Video 3: Proposed conformational switching of hACLY.

The video shows a morph between the crystal structure of hACLY-A/B in the open state (Fig. 1b) with a model for hACLY-A/B in the closed state. The latter model was generated by merging a model of the human CCL module with all four protomers in the closed state, with four CCS modules reoriented to match the CCS modules in the C. limicola ACLY-A/B crystal structure (Extended Data Fig. 4 and Extended Data Fig. 6d).

Video 4: Proposed conformational switching of hACLY (viewed from top).

As in Supplementary Video 3, but viewed from top.

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Verschueren, K.H.G., Blanchet, C., Felix, J. et al. Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle. Nature 568, 571–575 (2019). https://doi.org/10.1038/s41586-019-1095-5

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