Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.
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.
Orlicky
Stephen
S
Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, M5G 1X5, Toronto, Ontario, Canada.
Tang
Xiaojing
X
Willems
Andrew
A
Tyers
Mike
M
Sicheri
Frank
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019927:Cyclin E / Q000737:chemistry* / Q000378:metabolism*; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019927:Cyclin E; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; D046911:Macromolecular Substances; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D010453:Peptide Synthases