SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins. The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication. The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding. The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication. Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein-protein interactions.

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.

Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclindependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.disorder ͉ dynamic complex ͉ multisite phosphorylation ͉ NMR ͉ protein interaction