Role of conformational heterogeneity in domain swapping and adapter function of the Cks Proteins

Cks proteins are adapter molecules that coordinate the assembly of multiprotein complexes. They share the ability to domain swap by exchanging a β-strand, β4. Here we use NMR spectroscopy and molecular dynamics simulations to investigate the dynamic properties of human Cks1 and its response on assembly with components of the SCF^Skp2 ubiquitin ligation machinery. In the NMR experiment with the free form of Cks1, a subset of residues displayed elevated R₂ values and the cross-peaks of neighboring residues were missing from the spectrum, indicating a substantial conformational exchange contribution on the microsecond to millisecond time scale. Strikingly the region of greatest conformational variability was the β4-strand that domain swaps to form the dimer. Binding of the ligand common to all Cks proteins, Cdk2, suppressed the conformational heterogeneity. This response was specific to Cdk2 binding; in contrast, binding of Skp2, a ligand unique to human Cks1, did not alter the dynamic behavior. Short time (<5 ns) molecular dynamics simulations indicate that residues of Cks1 that form the binding site for phosphorylated ligands are considerably more flexible in the free form of Cks1 than they are in the Cdk2-Cks1 complex. A cooperative interaction between Cdk2 and Cks1 is suggested, which reduces the configurational entropy of Cks1 and therefore facilitates phosphoprotein binding. Indications of an unusual dynamic behavior of strand β4 in the free form of Cks1 were obtained from longer time scale (50 ns) dynamics simulations. A spontaneous reversible unzipping of hydrogen bonds between β4 and β2 was observed, suggesting an early intermediate structure for unfolding and/or domain swapping. We propose that the dynamic properties of the β-sheet and its modification upon ligand binding underlie the domain swapping ability and the adapter function of Cks proteins.