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Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1

Nature Chemical Biology volume 5, pages 758–764 (2009)Cite this article

Abstract

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

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Figure 1: Crystal structure of apo-PDK1 and PS48-bound PDK1 in the crystal form II.
Figure 2: Deuterium incorporation levels in the presence and absence of PS08.
Figure 3: Molecular insight into the activation of PDK1 by HM-polypeptides and small compounds.

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Acknowledgements

We acknowledge A. Haouz for help with the crystallization screening, F. Saul for crystallography advice, J. Imig for help setting up the conditions for the TNP-ATP assay and I. Adrian for excellent technical assistance. We give special thanks to R.W. Hartmann, R. Zimmermann and R. Bernhardt (University of Saarland), and to A. Scheidig (University of Kiel) for support for our research project. We acknowledge the financial support of the Europrofession Foundation (Saarbrücken, Germany), the Deutsche Krebshilfe (107875), Deutsche Forschungsgemeinschaft (BI 1044/2-2) and Bundesministerium für Bildung und Forschung (Go-Bio). We are grateful to N. Huntington for careful reading of the manuscript.

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Author notes

  1. Adriana Stroba and Hua Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine I, Research Group PhosphoSites, Universitätsklinikum Frankfurt, Frankfurt, Germany

    Valerie Hindie, Hua Zhang, Laura A Lopez-Garcia, Leila Idrissova, Stefan Zeuzem & Ricardo M Biondi

  2. Structural Biochemistry Unit, Pasteur Institute, Paris, France

    Valerie Hindie, Francis Schaeffer & Pedro M Alzari

  3. Department of Pharmaceutical and Medicinal Chemistry, Research Group PhosphoSites, University of Saarland, Saarbrücken, Germany

    Adriana Stroba & Matthias Engel

  4. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark

    Daniel Hirschberg & Thomas J D Jørgensen

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  1. Valerie Hindie
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Contributions

A.S. synthesized compounds under the direction of M.E. H.Z. performed deuterium exchange experiments under the direction of D.H. and T.J.D.J. A.S. and V.H. performed the ITC experiments under the supervision of F.S. V.H. performed crystallography work under the supervision of P.M.A. S.Z. gave general advice. L.I., L.A.L.-G. and V.H. performed protein purifications for crystallography work. L.A.L.-G. and V.H. performed TNP-ATP assays, purified GST fusion proteins and performed other biochemical experiments. R.M.B. supervised the protein production, general molecular biology, TNP-ATP assays, all additional biochemical work and the overall research project. The manuscript was written by R.M.B. with the support of V.H., H.Z. and P.M.A.

Corresponding author

Correspondence to Ricardo M Biondi.

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Hindie, V., Stroba, A., Zhang, H. et al. Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1. Nat Chem Biol 5, 758–764 (2009). https://doi.org/10.1038/nchembio.208

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