Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase

Abstract

The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH1. ICDH inactivation short-circuits the Krebs cycle by enabling the glyoxlate bypass2,3. It was the discovery of AceK and ICDH that established the existence of protein phosphorylation regulation in prokaryotes1,4. As a 65-kDa protein, AceK is significantly larger than typical eukaryotic protein kinases. Apart from the ATP-binding motif, AceK does not share sequence homology with any eukaryotic protein kinase or phosphatase5,6. Most intriguingly, AceK possesses the two opposing activities of protein kinase and phosphatase within one protein, and specifically recognizes only intact ICDH7,8. Additionally, AceK has strong ATPase activity9. It has been shown that AceK kinase, phosphatase and ATPase activities reside at the same site6,10, although the molecular basis of such multifunctionality and its regulation remains completely unknown. Here we report the structures of AceK and its complex with ICDH. The AceK structure reveals a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold. As an AceK phosphatase activator and kinase inhibitor, AMP is found to bind in an allosteric site between the two AceK domains. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and ICDH-binding induces further conformational change for AceK activation. The substrate recognition loop of AceK binds to the ICDH dimer, allowing higher-order substrate recognition and interaction, and inducing critical conformational change at the phosphorylation site of ICDH.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The overall structure of AceK.
Figure 2: The ATP-binding site and the allosteric AMP-binding site in AceK.
Figure 3: The AceK–ICDH complex structure.
Figure 4: Higher-order interaction between the AceK SRL and the ICDH dimer, and AceK mutant activity.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for the structures reported here have been deposited in the Protein Data Bank under accession codes 3EPS, 3lC6 and 3lCB.

References

  1. 1

    LaPorte, D. C. & Koshland, D. E. Jr. A protein with kinase and phosphatase activities involved in regulation of tricarboxylic acid cycle. Nature 300, 458–460 (1982)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Garnak, M. & Reeves, H. C. Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli . J. Biol. Chem. 254, 7915–7920 (1979)

    CAS  PubMed  Google Scholar 

  3. 3

    Hoyt, J. C. & Reeves, H. C. In vivo phosphorylation of isocitrate lyase from Escherichia coli D5H3G7. Biochem. Biophys. Res. Commun. 153, 875–880 (1988)

    CAS  Article  Google Scholar 

  4. 4

    Garnak, M. & Reeves, H. C. Phosphorylation of isocitrate dehydrogenase of Escherichia coli . Science 203, 1111–1112 (1979)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Klumpp, D. J. et al. Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase. J. Bacteriol. 170, 2763–2769 (1988)

    CAS  Article  Google Scholar 

  6. 6

    Stueland, C. S., Ikeda, T. P. & LaPorte, D. C. Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem. 264, 13775–13779 (1989)

    CAS  PubMed  Google Scholar 

  7. 7

    LaPorte, D. C. & Koshland, D. E. Jr. Phosphorylation of isocitrate dehydrogenase as a demonstration of enhanced sensitivity in covalent regulation. Nature 305, 286–290 (1983)

    ADS  CAS  Article  Google Scholar 

  8. 8

    McKee, J. S., Hlodan, R. & Nimmo, H. G. Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase: recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties. Biochimie 71, 1059–1064 (1989)

    CAS  Article  Google Scholar 

  9. 9

    Stueland, C. S., Eck, K. R., Stieglbauer, K. T. & LaPorte, D. C. Isocitrate dehydrogenase kinase/phosphatase exhibits an intrinsic adenosine triphosphatase activity. J. Biol. Chem. 262, 16095–16099 (1987)

    CAS  PubMed  Google Scholar 

  10. 10

    Miller, S. P., Karschnia, E. J., Ikeda, T. P. & LaPorte, D. C. Isocitrate dehydrogenase kinase/phosphatase: kinetic characteristics of the wild-type and two mutant proteins. J. Biol. Chem. 271, 19124–19128 (1996)

    CAS  Article  Google Scholar 

  11. 11

    Kornberg, H. L. The role and control of the glyoxylate cycle in Escherichia coli . Biochem. J. 99, 1–11 (1966)

    CAS  Article  Google Scholar 

  12. 12

    LaPorte, D. C. The isocitrate dehydrogenase phosphorylation cycle: regulation and enzymology. J. Cell. Biochem. 51, 14–18 (1993)

    CAS  Article  Google Scholar 

  13. 13

    Miller, S. P. et al. Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem. 275, 833–839 (2000)

    CAS  Article  Google Scholar 

  14. 14

    LaPorte, D. C., Stueland, C. S. & Ikeda, T. P. Isocitrate dehydrogenase kinase/phosphatase. Biochimie 71, 1051–1057 (1989)

    CAS  Article  Google Scholar 

  15. 15

    Takio, K. et al. Guanosine cyclic 3′,5′-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families. Biochemistry 23, 4207–4218 (1984)

    CAS  Article  Google Scholar 

  16. 16

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

    ADS  CAS  Article  Google Scholar 

  17. 17

    Kim, C., Xuong, N. H. & Taylor, S. S. Crystal structure of a complex between the catalytic and regulatory (RIα) subunits of PKA. Science 307, 690–696 (2005)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Chen, H. et al. A crystallographic snapshot of tyrosine trans-phosphorylation in action. Proc. Natl Acad. Sci. USA 105, 19660–19665 (2008)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Hurley, J. H. et al. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc. Natl Acad. Sci. USA 86, 8635–8639 (1989)

    ADS  CAS  Article  Google Scholar 

  20. 20

    Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, D. E. Jr & Stroud, R. M. Regulation of an enzyme by phosphorylation at the active site. Science 249, 1012–1016 (1990)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Finer-Moore, J. et al. Access to phosphorylation in isocitrate dehydrogenase may occur by domain shifting. Biochemistry 36, 13890–13896 (1997)

    CAS  Article  Google Scholar 

  22. 22

    Bennett, P. M. & Holms, W. H. Reversible inactivation of the isocitrate dehydrogenase of Escherichia coli ML308 during growth on acetate. J. Gen. Microbiol. 87, 37–51 (1975)

    CAS  Article  Google Scholar 

  23. 23

    Ubersax, J. A. & Ferrell, J. E. Jr. Mechanisms of specificity in protein phosphorylation. Nature Rev. Mol. Cell Biol. 8, 530–541 (2007)

    CAS  Article  Google Scholar 

  24. 24

    Dar, A. C., Dever, T. E. & Sicheri, F. Higher-order substrate recognition of eIF2α by the RNA-dependent protein kinase PKR. Cell 122, 887–900 (2005)

    CAS  Article  Google Scholar 

  25. 25

    Singh, S. K., Matsuno, K., LaPorte, D. C. & Banaszak, L. J. Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 Å: insights into the nature of substrate specificity exhibited by Escherichia coli isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem. 276, 26154–26163 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Singh, S. K., Miller, S. P., Dean, A., Banaszak, L. J. & LaPorte, D. C. Bacillus subtilis isocitrate dehydrogenase: a substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem. 277, 7567–7573 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Ikeda, T. & LaPorte, D. C. Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity. J. Bacteriol. 173, 1801–1806 (1991)

    CAS  Article  Google Scholar 

  28. 28

    Zheng, J., Lee, D. C. & Jia, Z. Purification, crystallization and preliminary X-ray analysis of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli . Acta Crystallogr. F 65, 536–539 (2009)

    CAS  Article  Google Scholar 

  29. 29

    Zheng, J., Ji, A. X. & Jia, Z. Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coli . Acta Crystallogr. F 65, 1153–1156 (2009)

    CAS  Article  Google Scholar 

  30. 30

    Stueland, C. S., Gorden, K. & LaPorte, D. C. The isocitrate dehydrogenase phosphorylation cycle: identification of the primary rate-limiting step. J. Biol. Chem. 263, 19475–19479 (1988)

    CAS  PubMed  Google Scholar 

  31. 31

    Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    CAS  Article  Google Scholar 

  32. 32

    Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  Google Scholar 

  33. 33

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

    Article  Google Scholar 

  34. 34

    McRee, D. E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999)

    CAS  Article  Google Scholar 

  35. 35

    Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  36. 36

    Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D 60, 2184–2195 (2004)

    Article  Google Scholar 

  37. 37

    Collaborative. Computation Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  38. 38

    Storoni, L. C., McCoy, A. J. & Read, R. J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D 60, 432–438 (2004)

    Article  Google Scholar 

  39. 39

    Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank M. Cygler and A. Matte for help in cloning, L. Li and R. Theiss for help in mutagenesis and activity measurements, and J. Allingham and G. Cote for critical reading of the manuscript. We would also like to thank the staff of the Cornell High Energy Synchrotron Source for helping with the collection of synchrotron X-ray data. This work was supported by the Canadian Institutes of Health Research (Z.J.). Z.J. is a Canada Research Chair in Structural Biology.

Author information

Affiliations

Authors

Contributions

Experiments were performed by J.Z. Data were analysed by J.Z. and Z.J. The manuscript was prepared by J.Z. and Z.J.

Corresponding author

Correspondence to Zongchao Jia.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Results and Discussion, Supplementary Tables 1-5, Supplementary Figures 1-6 with legends and References. (PDF 2205 kb)

41586_2010_BFnature09088_MOESM279_ESM.mov

This movie shows the interaction of AceK with ICDH. ICHD dimer is coloured in marine blue. AceK is coloured in green. The phosphorylation site is coloured in gold. (MOV 8108 kb)

Supplementary Movie 1

This movie shows the interaction of AceK with ICDH. ICHD dimer is coloured in marine blue. AceK is coloured in green. The phosphorylation site is coloured in gold. (MOV 8108 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zheng, J., Jia, Z. Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase. Nature 465, 961–965 (2010). https://doi.org/10.1038/nature09088

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.