Cooperative organization in a macromolecular complex.

The mechanism of assembly of multiprotein complexes and the subsequent organization of activity are not well understood. Here we report the application of biophysical tools to investigate the relationship between structure and function in protein assemblies. We used as a model system the SCF(Skp2) complex that targets p27(Kip1) for ubiquitination and subsequent degradation; this process requires an adapter protein, Cks1. By dissecting the interactions between the different subunits we show that the properties of Cks1 are highly context dependent, and its activity is acquired only when the complex is fully assembled. The results provide insights into the central role of small adapters in macromolecular assembly and explain their high sequence conservation. Simultaneous and synergistic binding of multiple subunits in a complex provides the specificity and control required before the key cell-cycle regulator p27 is committed to degradation.

Three different binding sites of Cks1 are required for p27-ubiquitin ligation.

Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound.