Thermodynamic characterization of interactions between p27(Kip1) and activated and non-activated Cdk2: intrinsically unstructured proteins as thermodynamic tethers.
The cyclin-dependent kinase inhibitor (CKI) p27Kip1 plays a critical role in cell cycle regulation by binding and inhibiting (or activating) various cyclin-dependent kinase (Cdk)/cyclin complexes. Thermal denaturation monitored by circular dichroism (CD) and isothermal titration calorimetry (ITC) were used to determine the relative stabilities and affinities of p27-KID (p27 kinase inhibitory domain) complexes with activated Cdk2 (phosphorylated at Thr160; P-Cdk2) and non-activated forms of Cdk2 and/or cyclin A. Phosphorylation of residue Thr160 only slightly increases the thermal stability of Cdk2, and its binary complexes with cyclin A and p27-KID. The p27-KID/P-Cdk2/cyclin A or p27-KID/Cdk2/cyclin A ternary complexes exhibited significantly higher thermal stabilities compared to the binary complexes (P-Cdk2/cyclin A or Cdk2/cyclin A). Differences in T(m) values between the binary and ternary complexes with P-Cdk2 and Cdk2 were +25.9 and +20.4 degrees C, respectively. These results indicate that the ternary complex with phosphorylated Cdk2 is stabilized to a larger extent than the non-phosphorylated complex. The free energy of association (deltaG(A)) for formation of the two ternary complexes was more favorable than for the binary complexes, indicating that a significantly smaller population of free components existed when all three components were present. These data indicate that p27-KID, which is intrinsically disordered in solution, acts as a thermodynamic tether when bound within the ternary complexes. It is proposed that thermodynamic tethering may be a general phenomena associated with intrinsically unstructured proteins (IUPs) which often function by binding to multiple partners in multi-protein assemblies.
Bowman
Prentice
P
Department of Structural Biology, St. Jude Children's Research Hospital, 332 North Lauderdale St., Memphis, TN 38105, USA.
Galea
Charles A
CA
Lacy
Eilyn
E
Kriwacki
Richard W
RW
D051357:Cyclin-Dependent Kinase 2 / Q000737:chemistry*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000737:chemistry*; D004789:Enzyme Activation; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry
D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D051357:Cyclin-Dependent Kinase 2