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Thermodynamic characterization of interactions between p27(Kip1) and activated and non-activated Cdk2: intrinsically

Prentice Bowman1, Charles A Galea, Eilyn Lacy

  • 1Department of Structural Biology, St. Jude Children's Research Hospital, 332 North Lauderdale St., Memphis, TN 38105, USA.

Biochimica Et Biophysica Acta
|February 7, 2006
PubMed

Insights

The cyclin-dependent kinase inhibitor p27Kip1 stabilizes Cdk2/cyclin A complexes. This stabilization is enhanced when Cdk2 is phosphorylated, suggesting a "thermodynamic tethering" mechanism for intrinsically disordered proteins in multi-protein assemblies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • p27Kip1 is a critical cyclin-dependent kinase inhibitor (CKI) regulating the cell cycle.
  • CKIs bind to cyclin-dependent kinase (Cdk)/cyclin complexes, modulating their activity.
  • Understanding the stability and binding of these complexes is crucial for cell cycle regulation research.

Purpose of the Study:

  • To determine the relative stabilities and affinities of p27-KID complexes with Cdk2 and cyclin A.
  • To investigate the impact of Cdk2 phosphorylation at Thr160 on complex stability.
  • To elucidate the role of p27-KID in ternary complex formation and stability.

Main Methods:

  • Thermal denaturation monitored by circular dichroism (CD).
  • Isothermal titration calorimetry (ITC) to measure binding affinities.
  • Analysis of p27-KID (kinase inhibitory domain) interactions with Cdk2 and cyclin A.

Main Results:

  • Phosphorylation of Cdk2 at Thr160 slightly increased the thermal stability of Cdk2 and its binary complexes.
  • Ternary complexes (p27-KID/Cdk2/cyclin A) showed significantly higher thermal stability than binary complexes.
  • The ternary complex with phosphorylated Cdk2 was more stabilized (+25.9°C) than the non-phosphorylated complex (+20.4°C).
  • Free energy of association was more favorable for ternary complexes, indicating reduced free components.

Conclusions:

  • p27-KID acts as a thermodynamic tether in ternary complexes, stabilizing the assembly.
  • Thermodynamic tethering may be a general mechanism for intrinsically unstructured proteins (IUPs) in multi-protein assemblies.
  • The phosphorylation status of Cdk2 influences the stability of the inhibitory complex.

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