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Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase

Nature volume 465, pages 961–965 (2010)Cite this article

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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.

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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.

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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.

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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

Authors and Affiliations

  1. Department of Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada,

    Jimin Zheng & Zongchao Jia

  2. College of Chemistry, Beijing Normal University, Beijing 100875, China

    Jimin Zheng

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  1. Jimin Zheng
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  2. Zongchao Jia
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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.

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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)

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)

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

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