Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase.
The ubiquitin ligase SCF(Cdc4) (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.
Center for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5.
Xiaojing
X
Tang
Stephen
S
Orlicky
Tanja
T
Mittag
Veronika
V
Csizmok
Tony
T
Pawson
Julie D
JD
Forman-Kay
Frank
F
Sicheri
Mike
M
Tyers
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D044783:F-Box Proteins; D011993:Recombinant Fusion Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases