Isothermal titration calorimetry (ITC)

ITC was performed using a Malvern Microcal PEAQ-ITC. Proteins were equilibrated in HEPES 20 mM pH 7.97, NaCl 300 mM, TCEP 0.5 mM. The concentrations of CDK2-cyclin A or cyclin A in the cell and SKP1-SKP2N in the syringe for each experiment are provided in the accompanying Table Legends. Protein concentrations (for this and other biophysical techniques) were measured by A₂₈₀ nm on a Nanodrop2000 (Thermoscientific) using calculated extinction coefficients (http://web.expasy.org/protparam/). Experiments (including ligand dilution controls) were carried out at 25 °C. The titrations consisted of 20 injections, an initial injection of 0.5 μl and then 19 injections of 19.5 μl with 180 s between injections. The stirring speed in the reaction cell was 1000 rpm. Data were analyzed using non-linear least squares regression using MicroCal PEAQ analysis software. Drifts in the baseline were corrected during data analysis. As detailed in Table 1, for characterization of authentic interactions and mutants that were characterized further by other orthogonal techniques, K_d values are presented as the average of two independent biological replicates and SD of the population. For other mutants, K_d values were derived from one measurement where the error represents the fitting error directly derived from the thermogram. Significance was calculated at p < 0.05 using a Student’s t-test.