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Use of a Fluorescent ATP Analog to Probe the Allosteric Conformational Change in the Active Site of the Protein Kinase PDK1

  • Valerie Hindie
  • Laura A. Lopez-Garcia
  • Ricardo M. BiondiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 928)

Abstract

There is growing interest in exploring allosteric sites on proteins for drug discovery. At the center of the regulation of many protein kinases from the AGC family there is an allosteric site termed “PIF-pocket.” The regulated binding of a C-terminal region of the kinase to the PIF-pocket, within the small lobe of the catalytic core, modulates the activity of AGC kinases. Small compounds that bind to the PIF-pocket can mimic its physiological mechanism of regulation and modulate the kinase activity in vitro, e.g., small compounds can activate the phosphoinositide-dependent protein kinase 1 (PDK1). Compounds binding to an allosteric site on a protein kinase may produce conformational changes at the ATP-binding site within the active site of the kinase domain. We here describe a fluorescent method using the ATP analog TNP-ATP that allows evaluating the allosteric conformational changes at the ATP-binding site of PDK1 triggered by small compounds binding to the PIF-pocket.

Key words

PDK1 AGC protein kinase Conformational change Allosteric site ATP-binding site 

References

  1. 1.
    Cohen P (2002) Protein kinases–the major drug targets of the twenty-first century? Nat Rev Drug Discov 1:309–315PubMedCrossRefGoogle Scholar
  2. 2.
    Bogoyevitch MA, Fairlie DP (2007) A new paradigm for protein kinase inhibition: blocking phosphorylation without directly targeting ATP binding. Drug Discov Today 12:622–633PubMedCrossRefGoogle Scholar
  3. 3.
    Kirkland LO, McInnes C (2009) Non-ATP competitive protein kinase inhibitors as anti-tumor therapeutics. Biochem Pharmacol 77:1561–1571PubMedCrossRefGoogle Scholar
  4. 4.
    Lewis JA, Lebois EP, Lindsley CW (2008) Allosteric modulation of kinases and GPCRs: design principles and structural diversity. Curr Opin Chem Biol 12:269–280PubMedCrossRefGoogle Scholar
  5. 5.
    Hindie V, Stroba A, Zhang H, Lopez-Garcia LA, Idrissova L, Zeuzem S, Hirschberg D, Schaeffer F, Jorgensen TJD, Engel M, Alzari PM, Biondi RM (2009) Structure and allosteric effects of low molecular weight activators on the protein kinase PDK1. Nat Chem Biol 5:758–764PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Valerie Hindie
    • 1
  • Laura A. Lopez-Garcia
    • 1
  • Ricardo M. Biondi
    • 1
    Email author
  1. 1.Department of Internal Medicine I, Research Group PhosphoSitesUniversitätsklinikum FrankfurtFrankfurtGermany

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