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. 2016 Nov 29:2:16044.
doi: 10.1038/celldisc.2016.44. eCollection 2016.

A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

Jia Wei  1 , Yixiao Zhang  2 , Tai-Yuan Yu  3 , Kianoush Sadre-Bazzaz  1 , Michael J Rudolph  1 , Gabriele A Amodeo  1 , Lorraine S Symington  3 , Thomas Walz  2 , Liang Tong  1
Affiliations

A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

Jia Wei et al. Cell Discov. .

Erratum in

Abstract

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3-AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

Keywords: enzyme phosphorylation; enzyme regulation; fatty acid metabolism; metabolic syndrome.

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Figures

Figure 1
Figure 1
Overall structure of yeast acetyl-CoA carboxylase (ACC) (ScACC). (a) Domain organization of ScACC. The domains are labeled and given different colors. The five domains of ACC Central (AC1–AC5) are labeled 1–5. The phosphorylation site in the central region is indicated. The phosphorylation site before the biotin carboxylase (BC) domain core is indicated with the dashed lines, as it is absent in ScACC. (b) Structure of the ScACC holoenzyme dimer [7]. One protomer is shown as ribbons, while the other as a surface. The domains in the monomers are colored according to panel (a) and labeled. Ser1157 (red star) is located in a loop missing in the structure (dashed lines), and its distances to the BC and carboxyltransferase (CT) active sites (black asterisks) and the BC dimer interface (black rectangle) in the holoenzyme are indicated. (c) Sequence conservation near the phosphorylation site before the BC domain core in animal ACCs. Sc: Saccharomyces cerevisiae, Rn: Rattus novegicus, Hs: Homo sapiens. (d) Sequence conservation near the phosphorylation site in the central region. The structure figures were produced with PyMOL (www.pymol.org).
Figure 2
Figure 2
SNF1 directly phosphorylates Ser1157 of yeast acetyl-CoA carboxylase (ScACC). (a) Schematic drawing of the in vitro phosphorylation system. SNF1 is activated by the upstream protein kinase Tos3, which in turn phosphorylates ScACC. (b) Sodium dodecyl sulfate gel shift assay for ScACC phosphorylation, showing a clear shift for the migrating position of domains AC3–AC5 after treatment with SNF1 and Tos3. (c) Activity assay for ScACC phosphorylation, showing ~80% loss of the catalytic activity of full-length ScACC after 10 min incubation with SNF1 and Tos3. (d) Activity assay showing that the ScACC mutant in which residues 1137–1170 are replaced with a (Gly)4 linker is insensitive to activated SNF1. (e) Activity assay showing that the S1157A mutant is insensitive to activated SNF1, even after 75 min incubation.
Figure 3
Figure 3
Crystal structure of phosphorylated domains AC3–AC5 of yeast acetyl-CoA carboxylase (ScACC). (a) Overlay of the structure of phosphorylated AC3–AC5 (in color) with that of unphosphorylated AC3–AC5 alone (gray). The pSer1157 side chain is shown as stick models. (b) Molecular surface of ScACC near the loop containing the pSer1157 residue, colored by electrostatic potential (blue: positive; red: negative). (c) The binding site for pSer1157 in domain AC4. Interactions with the phosphate are indicated with dashed lines (red). Omit Fo–Fc electron density for the phosphate group is shown in light blue, contoured at 3σ. (d) Activity assay showing that the R1173A/R1260A double mutant is only mildly inhibited by activated SNF1. (e) Overlay of the structure of phosphorylated AC3–AC5 (in color) with that of AC3–AC5 in the holoenzyme (gray). Domains AC3–AC4 were used for the overlay, and the large conformational difference for domain AC5 corresponds to a rotation of 40° and is indicated. (f) The position of Ser1157 moves by ~16 Å upon phosphorylation. Ser1157 interacts with different residues at the AC4–AC5 interface in the unphosphorylated ScACC holoenzyme structure.
Figure 4
Figure 4
Conformational variability of the yeast acetyl-CoA carboxylase (ScACC) holoenzyme dimer. (a) Electron microscopic (EM) image of negatively stained ScACC holoenzyme in the regular protein buffer (20 mM Tris (pH 7.5) and 300 mM NaCl). Predominantly elongated shapes were observed, both straight (black arrowhead) and bent (white arrowhead). A few compact shapes (red arrowhead) are likely similar to the structure observed in the crystal. (b) EM image of negatively stained ScACC holoenzyme in a buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl and 100 mM sodium citrate. Mostly, compact shapes were observed, corresponding to front (red arrowhead) and side (magenta arrowhead) views of the structure observed in the crystal. (c) Three class averages of negatively stained ScACC in the presence of citrate. The crystal structure of ScACC was overlaid manually to indicate that the EM images are in good agreement with the crystal structure. (d) Negative-stain EM image of phosphorylated ScACC holoenzyme in a buffer containing 100 mM citrate. Predominantly elongated shapes were observed. Smaller particles (blue arrowhead) are likely the protein kinases used for phosphorylation (SNF1 and/or Tos3). (e) Negative-stain EM image of ScACC holoenzyme with soraphen A in a buffer containing 100 mM citrate. Predominantly elongated shapes were observed.
Figure 5
Figure 5
The S1157A mutant has no obvious effect on yeast cell growth under glucose-replete and glucose-limiting conditions. WT1 and WT2: two wild-type strains, SA1 and SA2: two S1157A mutant strains.
Figure 6
Figure 6
A unified molecular mechanism for the inhibition of eukaryotic acetyl-CoA carboxylases (ACCs) by phosphorylation and soraphen A binding. Phosphorylation at the site before the biotin carboxylase (BC) domain core in animal ACCs (indicated with Ser80 and Ser222 in human ACC1 and ACC2, respectively, labeled P), soraphen A binding (labeled Sor) and phosphorylation in the central region (indicated with Ser1157 in yeast ACC (ScACC)) all stabilize the monomeric form of the BC domain. The monomeric BC domain has large conformational changes in the dimer interface and in the active site region, which blocks biotin binding and thereby catalysis. Biotin is indicated with the fused pentagons in black. Once the BC domain dimer dissociates, the holoenzyme can assume a continuum of elongated conformations, from bent to straight shapes, as observed by electron microscope.
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References

    1. Tong L. Structure and function of biotin-dependent carboxylases. Cell Mol Life Sci 2013; 70: 863–891. - PMC - PubMed
    1. Waldrop GL, Holden HM, St. Maurice M. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms. Prot Sci 2012; 21: 1597–1619. - PMC - PubMed
    1. Cronan JE Jr., Waldrop GL. Multi-subunit acetyl-CoA carboxylases. Prog Lipid Res 2002; 41: 407–435. - PubMed
    1. Polyak SW, Abell AD, Wilce MCJ, Zhang L, Booker GW. Structure, function and selective inhibition of bacterial acetyl-CoA carboxylase. Appl Microbiol Biotechnol 2012; 93: 983–992. - PubMed
    1. Abramson HN. The lipogenesis pathway as a cancer target. J Med Chem 2011; 54: 5615–5638. - PubMed

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