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Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer

Nature volume 526, pages 723–727 (2015)Cite this article

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Abstract

Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases1,2,3,4,5,6. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope7,8. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250-kilodalton (kDa), multi-domain enzymes and function as homodimers and higher oligomers. They contain a unique, 80-kDa central region that shares no homology with other proteins. Although the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known1,9,10,11,12,13,14, there is currently no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500-kDa holoenzyme dimer of ScACC. The structure is remarkably different from that of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of the BC domain alone, which is a monomer. These structural changes reveal why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A15,16 and by phosphorylation of a Ser residue just before the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis.

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Figure 1: Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase (ScACC) holoenzyme dimer.
Figure 2: Interactions among the domains in the ScACC holoenzyme.
Figure 3: A dimeric BC domain in the ScACC holoenzyme.
Figure 4: Translocation of BCCP and biotin during ACC catalysis.

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Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 5CS0 for AC1–AC2, 5CS4 for AC3–AC5, 5CSA for BT–BCCP–AC1–AC5, 5CSK for unbiotinylated ACC, and 5CSL for ACC holoenzyme.

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Acknowledgements

We thank M. Bush, C.-Y. Chou, Y. Shen, L. Yu and H. Zhang for carrying out initial studies in this project; R. Jackimowicz, B. Nolan, N. Whalen, A. Heroux and H. Robinson for access to the X29A and X25 beamlines at the NSLS; S. Banerjee, K. Perry, R. Rajashankar, J. Schuermann, N. Sukumar for access to NE-CAT 24-C and 24-E beamlines at the Advanced Photon Source; W. Rice and E. Eng at New York Structural Biology Center for assistance with electron microscopy. The in-house X-ray diffraction instrument was purchased with an NIH grant to L.T. (S10OD012018). This research was supported in part by a grant from the NIH (R01DK067238) to L.T.

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Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, 10027, New York, USA

    Jia Wei & Liang Tong

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  1. Jia Wei
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Contributions

J.W. carried out protein expression, purification, crystallization, data collection, structure determination and refinement, site-directed mutagenesis and enzymatic assays. L.T. initiated the project, supervised the entire research, and analysed the results. J.W. and L.T. wrote the paper.

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Correspondence to Liang Tong.

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Extended data figures and tables

Extended Data Figure 1 Sequence alignment of the N-terminal region of eukaryotic, single-chain ACCs.

The region includes BC, BT and BCCP domains (indicated). The secondary structure elements in the ScACC holoenzyme structure are shown. The site of phosphorylation in HsACC1 (Ser80) is indicated with a star. This site does not exist in ScACC. ScACC, Saccharomyces cerevisiae ACC; TaACC, Triticum aestivum (wheat) ACC; DmACC, Drosophila melanogaster ACC; HsACC1, Homo sapiens ACC1. Modified from an output from ESPript41.

Extended Data Figure 2 Sequence alignment of the central region of eukaryotic, single-chain ACCs.

The AC1–AC5 domains are indicated. Predicted secondary structure elements in HsACC1 are also shown, and they generally match those in the structure of ScACC. The helices in AC1–AC3 are numbered consecutively. Three sites of phosphorylation in HsACC1 are indicated with stars.

Extended Data Figure 3 Sequence alignment of the C-terminal region of eukaryotic, single-chain ACCs.

The region includes N and C domains of CT (indicated).

Extended Data Figure 4 Electron density for various regions of the ScACC holoenzyme structure.

a, Schematic of the two-step reaction in the catalysis by biotin-dependent carboxylases. Biotin is carboxylated in the BC active site, and then translocates to the CT active site where the substrate is carboxylated. The longest distance from the N1′ atom of biotin to the Cα atom of the Lys residue covalently attached to biotin is ∼16 Å, giving a reach of ∼30 Å for biotin (swinging-arm model). The actual distance between the BC and CT active sites are larger than 30 Å, suggesting that BCCP needs to translocate in addition to the biotin (swinging-domain model). b, 2Fo − Fc electron density for the helical hairpin insert (α8–α9) of domain AC1 at 3.2 Å resolution, contoured at 1σ. c, 2Fo − Fc electron density for the β-sheet of domain AC5. d, 2Fo − Fc electron density for the central helix (αV) of the BT domain. e, Omit Fo − Fc electron density for CoA at 3.2 Å resolution, contoured at 2.5σ. f, Omit Fo − Fc electron density for biotin at 3.2 Å resolution.

Extended Data Figure 5 Overall structure of the ScACC holoenzyme.

a, A large channel in the centre of the ScACC holoenzyme dimer. The view is related to that of Fig. 1b by an ∼30° rotation around the vertical axis. b, Overlay of the structures of the two protomers of ScACC holoenzyme dimer. One protomer is shown in colour and other in grey. The overlay is based on the CT domain. Differences in the orientations of the other domains are indicated, as well as the r.m.s. distance for their equivalent Cα atoms. The arrow points to conformational differences in the insert domain of CT, linked to differences in the BCCP binding mode. c, Overlay of the BT domain of ScACC (in orange) and the BT domain of PCC (in grey). The ‘hook’, connecting the end of the helix (αV) to the first strand of the β-barrel (β22), is labelled. The last three strands of the β-barrel (β27–β29) are splayed further away from the central helix in ScACC compared to PCC. d, Interactions between the hook of the BT domain and the β4A–β4B loop from the C domain of CT, an inserted segment that projects away from the rest of the domain. e, The BCCP–AC1 linker has hydrophobic interactions with the top of one side of the BT domain β-barrel. The disordered segment of the linker is indicated with the blue line.

Extended Data Figure 6 Domains AC4 and AC5 share a common backbone fold.

a, Structure of AC4 domain of ScACC. b, Topological drawing of AC4 domain. c, Structure of AC5 domain. d, Topological drawing of AC5 domain. e, Overlay of the structures of AC5 domain (magenta) and formamidase (grey). Formamidase has a four-layered αββα structure, and AC5 matches only half of the structure.

Extended Data Figure 7 Structure of the BC domain dimer of ScACC.

a, Interactions in the BC dimer interface. The BC domain of protomer 1 is in red, and that of protomer 2 in pink. b, Residues in the BC dimer interface (in red) of ScACC and E. coli BC (EcBC) are weakly conserved among the biotin-dependent carboxylases. MapLCC, long-chain acyl-CoA carboxylase of Mycobacterium avium subspecies tuberculosis. c, A BC domain dimer is constructed by superposing the structure of BC domain alone (in complex with soraphen A, green) onto that of the BC dimer. Regions of steric clashes between the two monomers are highlighted in light blue. d, Close-up view of the binding site of biotin. The β19–β20 loop in the structure of BC domain alone (grey) clashes with biotin (red arrow), and cannot interact with the amide group of the biotin linkage (red dashed lines). e, Close-up view of the soraphen A (green) binding site. The movement of strands β19 and β20 from the structure of BC domain alone in complex with soraphen A (grey) is indicated with the arrows. Trp487 side chain in the holoenzyme structure clashes with soraphen A as well as the phosphorylated peptide segment containing pSer222 (light blue).

Extended Data Figure 8 Structure of the CT domain dimer of ScACC.

a, Overlay of the CT domain dimer in the ScACC holoenzyme (cyan and yellow for N and C domains of protomer 1, light cyan and light yellow for protomer 2) in complex with CoA (black) with the CT domain alone in complex with CoA (grey). Biotin is shown in black and BCCP is omitted for clarity. The bound position of the CP-640186 inhibitor (gold) is shown for reference. A conformational change for the insert domain at the top is due to the binding of BCCP in the holoenzyme, while the insert domain at the bottom shows essentially no change because the BCCP–biotin is not bound as deeply into this active site. b, Overlay of the CT active site (cyan and yellow) of ScACC holoenzyme with that of CT alone in complex with CoA (grey). The CP-640186 inhibitor (gold) clashes with the bound position of biotin (black). The thiol group of CoA in the holoenzyme complex is 4.3 Å from the N1 atom of biotin (dashed line in blue). The thiol group of CoA in the CT domain alone complex is in a different position, likely due to the absence of biotin in the active site.

Extended Data Figure 9 Comparisons of the structures of ACC central region alone with that in the holoenzyme.

a, Overlay of AC1–AC2 in the holoenzyme (colour) with AC1–AC2 alone (yellow and grey). The first helix is swapped between two monomers in the structure of AC1–AC2 alone, and the two-fold axis of that dimer is indicated with the black line. b, Overlay of AC3–AC5 in the holoenzyme (colour) with AC3–AC5 alone (grey), based on AC3–AC4. A large difference is seen for the orientation of AC5. c, Overlay of BT–BCCP–AC1–AC5 in the holoenzyme (colour) with these domains alone (grey), based on AC1–AC2. Large differences are seen for BT, BCCP and AC3–AC5. d, Overlay of BT–BCCP–AC1–AC5 in the holoenzyme (colour) with these domains alone (grey), based on AC3–AC4. Large differences are seen for BT, BCCP and AC1–AC2, although AC5 has essentially the same position.

Extended Data Table 1 Data collection and refinement statistics
Full size table

Supplementary information

Conformational changes in the BC domain near the dimer interface

Video morphing the structure of the BC domain alone (gray) to that in the holoenzyme (red), illustrating the large conformational changes in the BC dimer interface. The inhibitor soraphen A (green) and the side chain of Trp487 are shown as stick models. (MOV 916 kb)

Conformational changes in the BC domain near the active site

Video morphing the structure of the BC domain alone (gray) to that in the holoenzyme (red), illustrating the large conformational changes in the BC active site region. ADP (green) and biotin (black), observed bound to the active site of E. coli BC subunit, are shown as stick models. (MOV 908 kb)

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Wei, J., Tong, L. Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer. Nature 526, 723–727 (2015). https://doi.org/10.1038/nature15375

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