Thermodynamic characterization of interactions between p27Kip1 and activated and non-activated Cdk2: Intrinsically unstructured proteins as thermodynamic tethers

摘要: Abstract The cyclin-dependent kinase inhibitor (CKI) p27Kip1 plays a critical role in cell cycle regulation by binding and inhibiting (or activating) various (Cdk)/cyclin complexes. Thermal denaturation monitored circular dichroism (CD) isothermal titration calorimetry (ITC) were used to determine the relative stabilities affinities of p27-KID (p27 inhibitory domain) complexes with activated Cdk2 (phosphorylated at Thr160; P-Cdk2) non-activated forms and/or cyclin A. Phosphorylation residue Thr160 only slightly increases thermal stability Cdk2, its binary A p27-KID. p27-KID/P-Cdk2/cyclin or p27-KID/Cdk2/cyclin ternary exhibited significantly higher compared (P-Cdk2/cyclin Cdk2/cyclin A). Differences Tm values between P-Cdk2 + 25.9 + 20.4 °C, respectively. These results indicate that complex phosphorylated is stabilized larger extent than non-phosphorylated complex. free energy association (ΔGA) for formation two was more favorable complexes, indicating smaller population components existed when all three present. data p27-KID, which intrinsically disordered solution, acts as thermodynamic tether bound within It proposed tethering may be general phenomena associated unstructured proteins (IUPs) often function multiple partners multi-protein assemblies.