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X-ray crystallographic coordinates and structure factors have been deposited in wwPDB with accession codes PDB 6Z2H (CSH module of human ACLY) and PDB 6ZNW (archaeal ACLY). Raw data for the enzymatic assays are available via the online version of this paper. Other datasets used in this study are publicly available in the wwPDB (entries PDB 6HXH, 6HXI, 6HXL, 5CTS, 6UV5, 6UI9, 6POE and 6UUW) and the Electron Microscopy Data Bank (entries EMD-20902, EMD-20904, EMD-20783 and EMD-20413).
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We thank the staff of beamlines P14 (Petra III, Deutsches Elektronen-Synchrotron) and Proxima 2A (SOLEIL) for their technical support and beamtime allocation. This project was supported by a grant from Research Foundation Flanders to K.V. (no. G0G0619) and a Ghent University grant to S.N.S. (no. BOF17-GOA-028). S.N.S. is a principal investigator of the VIB (Belgium).
K.V. and S.N.S. are listed as coinventors on a patent filed with the European Patent Office entitled ‘Crystal structure of ATP Citrate Lyase and uses thereof’ (EP19161520.2). K.H.G.V. and A.D. declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Structural basis for the retro-aldol cleavage of citryl-CoA by the CSH-module of ACLY.
a, The ACLY-catalyzed reaction proceeds via four steps. The first three steps lead up to the formation of citryl-CoA from ATP, citrate and CoA. In the fourth step, citryl-CoA undergoes retro-aldol cleavage to generate the reaction products oxaloacetate and acetyl-CoA. b, An overlay of CoA- and citrate-bound crystal structures for full-length human ACLY and its isolated CSH-module shows how the citryl-CoA intermediate can be shuttled from the ASH-active site towards the CSH-active site for retro-aldol cleavage. The CoA-binding domain of the CSH-module can rotate towards an open (white) or closed (pink) CSH-active site configuration. c, Closed active site configuration of the CSH-module of human ACLY in complex with CoA and citrate (PDB 6hxl) overlaid with the closed active site configuration of chicken citrate synthase (CS) in complex with carboxymethyl-CoA and oxaloacetate (grey carbon atoms, PDB 5cts). Residue numbers between brackets refer to the residue numbers of citrate synthase. The figures in panels b and c are adapted from Verschueren et al., (ref. 1). d, Enzymatic activity for human ACLY active site mutants reproduced from Fig. 1f in Wei et al.13 e, Overlay between chains A + C and chains B + D in PDB 6poe based on the superposition of the core of the CSH-module (residues 837–935 and residues 1055–1101; r.m.s.d.-value = 0.198 Å for 256 aligned C-alpha atoms). The superposition shows that in ACLY, bound CoA can adopt an extended conformation (white) pointing to the ASH active site, or a collapsed conformation (green) pointing to the CSH-active site. In the collapsed conformation, the CoA-binding domain (magenta) is rotated to form a closed CSH-active site configuration analogous to citrate synthase.
Extended Data Fig. 2 Ligands as modelled by Wei et al. and their corresponding experimental density.
a, Alternative conformations of acetyl-CoA (chain A, residue 1202) - as modelled by Wei et al. in PDB 6uv5 - point to ASH and CSH active sites, and oxaloacetate is modelled in both the ASH and CSH active sites. The author-recommend contour level in the EMD-databank is 0.012 (entry EMD-20904). b, Acetyl-CoA and oxaloacetate as modelled by Wei et al.13 in PDB 6ui9. Acetyl-CoA is pointing to the ASH-active site, and oxaloacetate is modelled both in the ASH- and CSH-active sites. The author-recommend contour level in the EMD-databank is 0.010 (entry EMD-20783). c, Experimental density for ligand QHD in PDB 6uuw. The black arrow indicates the poor connectivity between the phospho-citryl and CoA moieties. In panel a-c, the experimental ligand density is shown at different contour levels and carved around the ligand with a radius of 2 Å. d, The stereochemistry of the chiral carbon atom of the citryl-moiety in phosphocitryl-CoA as proposed by Wei et al.13 is incompatible with the ACLY-catalyzed reaction.
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Verstraete, K., Verschueren, K.H.G., Dansercoer, A. et al. Acetyl-CoA is produced by the citrate synthase homology module of ATP-citrate lyase. Nat Struct Mol Biol 28, 636–638 (2021). https://doi.org/10.1038/s41594-021-00624-3