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Structural insight into the autoinhibition mechanism of AMP-activated protein kinase

Nature volume 459, pages 11461149 (25 June 2009) | Download Citation


The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells1,2. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity3. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments4. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK α-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the ‘backside’, to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix αC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation5,6. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

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Data deposits

The structural and atomic coordinates have been deposited in the Protein Data Bank under accession codes 3H4J for S. pombe KD-AID and 3DAE for S. cerevisiae Snf1-pKD.


  1. 1.

    The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem. Sci. 29, 18–24 (2004)

  2. 2.

    , , & AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1, 15–25 (2005)

  3. 3.

    AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nature Rev. Mol. Cell Biol. 8, 774–785 (2007)

  4. 4.

    AMP-activated protein kinase as a drug target. Annu. Rev. Pharmacol. Toxicol. 47, 185–210 (2007)

  5. 5.

    , & Regulation of rat liver acetyl-CoA carboxylase. Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge. J. Biol. Chem. 255, 2308–2314 (1980)

  6. 6.

    , , & 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2AC. FEBS Lett. 377, 421–425 (1995)

  7. 7.

    , , , & Functional domains of the α1 catalytic subunit of the AMP-activated protein kinase. J. Biol. Chem. 273, 35347–35354 (1998)

  8. 8.

    et al. Conserved α-helix acts as autoinhibitory sequence in AMP-activated protein kinase α subunits. J. Biol. Chem. 282, 495–506 (2007)

  9. 9.

    et al. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J. Biol. Chem. 271, 27879–27887 (1996)

  10. 10.

    et al. CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J. Clin. Invest. 113, 274–284 (2004)

  11. 11.

    et al. AMP-activated protein kinase β subunit tethers α and γ subunits via its C-terminal sequence (186–270). J. Biol. Chem. 280, 13395–13400 (2005)

  12. 12.

    , , & The glycogen-binding domain on the AMPK β subunit allows the kinase to act as a glycogen sensor. Cell Metab. 9, 23–34 (2009)

  13. 13.

    et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 3, 403–416 (2006)

  14. 14.

    et al. Defining the mechanism of activation of AMP-activated protein kinase by the small molecule A-769662, a member of the thienopyridone family. J. Biol. Chem. 282, 32539–32548 (2007)

  15. 15.

    et al. Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase. J. Biol. Chem. 282, 32549–32560 (2007)

  16. 16.

    et al. Thienopyridone drugs are selective activators of AMP-activated protein kinase β1-containing complexes. Chem. Biol. 15, 1220–1230 (2008)

  17. 17.

    & Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase. Science 315, 1726–1729 (2007)

  18. 18.

    , & Crystal structure of the heterotrimer core of Saccharomyces cerevisiae AMPK homologue SNF1. Nature 449, 492–495 (2007)

  19. 19.

    et al. Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature 449, 496–500 (2007)

  20. 20.

    , , & Crystal structure of the protein kinase domain of yeast AMP-activated protein kinase Snf1. Biochem. Biophys. Res. Commun. 337, 1224–1228 (2005)

  21. 21.

    et al. Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family. Structure 14, 477–485 (2006)

  22. 22.

    , & Regulation of protein kinases: controlling activity through activation segment conformation. Mol. Cell 15, 661–675 (2004)

  23. 23.

    & Protein–protein interactions in the allosteric regulation of protein kinases. Curr. Opin. Struct. Biol. 16, 702–709 (2006)

  24. 24.

    et al. Structural variations in the catalytic and ubiquitin-associated domains of microtubule-associated protein/microtubule affinity regulating kinase (MARK) 1 and MARK2. J. Biol. Chem. 281, 27586–27599 (2006)

  25. 25.

    et al. The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation. Biochem. J. 394, 545–555 (2006)

  26. 26.

    & The conformational plasticity of protein kinases. Cell 109, 275–282 (2002)

  27. 27.

    et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell 129, 735–746 (2007)

  28. 28.

    , , & Regulation of AMP-activated protein kinase by a pseudosubstrate sequence on the gamma subunit. EMBO J. 26, 806–815 (2007)

  29. 29.

    et al. Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells. J. Biol. Chem. 283, 16051–16060 (2008)

  30. 30.

    et al. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem. J. 403, 139–148 (2007)

  31. 31.

    , , , & Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr. Purif. 30, 230–237 (2003)

  32. 32.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  33. 33.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  34. 34.

    et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

  35. 35.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  36. 36.

    et al. Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis. J. Biol. Chem. 278, 28434–28442 (2003)

  37. 37.

    et al. Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab. 2, 9–19 (2005)

  38. 38.

    Assays of protein kinase. Methods Enzymol. 99, 3–6 (1983)

  39. 39.

    , & Tissue distribution of the AMP-activated protein protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive protein assay. Eur. J. Biochem. 186, 123–128 (1989)

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We thank Y. Shi and S. C. Lin for critical discussions and reading of the manuscript; J. Chai and Y. Dong for help with data collection and processing; L. Gu and J. Wang for advice on structure determination. This work is supported in part by MOST grants 2006CB503900 and 2007CB914400, and by NSFC grants 30425005 and 30770476.

Author Contributions L.C., Z.-H.J. and L.-S.Z. designed, performed and analysed most of the experiments. Y.-Y.Z. and S.-T.X. provided technical assistance. Z.-X.W. contributed to discussions. J.-W.W. led the team and wrote the paper.

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Author notes

    • Lei Chen
    • , Zhi-Hao Jiao
    •  & Li-Sha Zheng

    These authors contributed equally to this work.


  1. MOE Key Laboratory of Bioinformatics, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

    • Lei Chen
    • , Zhi-Hao Jiao
    • , Yuan-Yuan Zhang
    • , Shu-Tao Xie
    • , Zhi-Xin Wang
    •  & Jia-Wei Wu
  2. Institute of Biophysics and Graduate University, Chinese Academy of Sciences, Beijing 100101, China

    • Li-Sha Zheng
    •  & Zhi-Xin Wang


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Corresponding author

Correspondence to Jia-Wei Wu.

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    Supplementary Information

    This file contains Supplementary Tables 1-2, Supplementary Figures 1-9 with extended Legends and Supplementary References.

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