Figure 5. A divergent hub controlling allosteric coupling in Cdk2.

a,b) Metazoan sequence conservation in Cdk1/2 and Cdk4/6, determined from a set of 20 sequences (Supplementary Fig. 10), is mapped onto the structures of Cdk2 (left) and Cdk4 (right) in the Aloop-in conformation. Red represents strict conservation and white represents lower conservation. c) Divergence of the central hub residue (A151 in Cdk2, I164 in Cdk4) between Cdk1/2 and Cdk4/6 lineages. d) DEER data for the phosphorylated pCdk2^cdk4hub mutant. Data for the unphosphorylated mutant are shown in Supplementary Fig. 12. e) DEER data for the phosphorylated pCdk2^L148R mutant. The monomer data are plotted on a different y-axis scale for clarity. Data for the unphosphorylated mutant are shown in Supplementary Fig. 12. f) Conformational equilibrium values for WT Cdk2, pCdk2^L148R and pCdk2^cdk4hub. Error bars represent 75% confidence intervals, calculated from 50,000 simulations of Gaussian fits to the primary data. g) Cyclin binding affinities measured by FRET for WT Cdk2, Cdk2^L148R and Cdk2^cdk4hub mutants. Values are mean ± S.E.M; n = 3 independent experiments. h) Inhibitor K_D values measured by FRET for WT pCdk2:cyclinA and pCdk2^cdk4hub:cyclinA. Values are mean ± S.E.M; n = 3 independent experiments. Data for the monomeric kinases are shown in Supplementary Fig. 13. i) IC50 values measured in kinase assays for the 7 inhibitors for pCdk2:cyclinA (left) and pCdk4:cyclinD (right). The shaded areas represent the range of IC50 values within 10-fold of the IC50 values for palbociclib and ribociclib. Values are mean ± S.E.M; n = 3 independent experiments.