1. Volume 26, Issue 1, Pages 131-143 (April 2007)
Structure of a Fbw7-Skp1-Cyclin E Complex: Multisite-Phosphorylated Substrate Recognition by SCF Ubiquitin Ligases 
Bing Hao, Stephanie Oehlmann, Mathew E. Sowa, J. Wade Harper, Nikola P. Pavletich 
Molecular Cell 
Volume 26, Issue 1, Pages (April 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Structure of the Skp1-Fbw7-CycEdegC Complex
(A) Overall architecture of the complex, with the secondary structure elements of Skp1, F box, and linker domains labeled. Dotted lines indicate disordered regions.
(B) CycEdegC binds across the narrow face of the Fbw7 β-propeller structure. The eight Fbw7 blades and the strands for one blade are labeled.
(C) Sequence alignment of the cyclin E peptides used in crystallization with other SCFFbw7 substrates. Arrow indicates the type II β turn, cylinder the left-handed polyproline II helix, dotted lines disordered regions, and crosses the residues of CycEdegC and CycEdegC that contact Fbw7. The substrate residues that match the structure-based degron motif (ϕ-X-ϕ-ϕ-ϕ-pT/S-P-P-X-pS/T, with ϕ representing a hydrophobic residue and X any amino acid) are highlighted in yellow.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Cyclin E-Fbw7 Contacts in the Skp1-Fbw7-CycEdegC and Skp1-Fbw7-CycEdegN Complexes
(A) Close-up view of the Fbw7-CycEdegC interface showing interacting amino acids of Fbw7 (pink) and CycEdegC (light blue). Hydrogen bonds are shown as white dotted lines. The Fbw7 blade strands that provide cyclin E contacts are labeled.
(B) Close-up view of the Fbw7-CycEdegN interface.
(C) Molecular surface representation of the WD40 domain colored according to conservation among Fbw7 13 orthologs and the Cdc4 and Pop1 homologs (Figure S1).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Phosphorylation of Ser384 Is Required for Fbw7 Binding and Cyclin E Degradation
(A) ITC-derived dissociation constants (Kd) for the binding of differentially phosphorylated cyclin E peptides to Skp1-Fbw7. Kd values were calculated as the average and standard deviation from two or three independent measurements at 25°C (Figure S4).
(B) Two Fbw7 phosphate binding sites are required for optimal cyclin E degradation in vivo. Vectors expressing the wild-type and mutant Fbw7 proteins were cotransfected with varying amounts of cyclin E-expressing vectors into 293T cells. Cell lysates were immunoblotted with anti-Myc, anti-Flag, or anti-Cul1 antibodies.
(C) Two Fbw7 phosphate binding sites are required for optimal cyclin E-Fbw7 association in vivo. Myc-cyclin E-expressing vector was cotransfected with wild-type or mutant Flag-Fbw7-expressing vectors. The cell lysates were immunoprecipitated with anti-Flag antibodies prior to immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Identification of Three Doubly Phosphorylated Sic1 Degrons that Bind to Cdc4 with High Affinity
(A) ITC measurements of the binding of Skp1-Cdc4271–779 to Sic1 peptides corresponding to the three phosphorylation-site clusters, each phosphorylated at the primary site and at additional sites. Mutation of the second phosphate binding site in Cdc4271–779 abolishes the affinity increase that results from specific second-site phosphorylation of the Sic1 degrons. Kd values were calculated as the average and standard deviation from two or three independent measurements at either 25°C or 37°C (indicated by asterisk).
(B) ITC measurements of the binding of differently phosphorylated cyclin E peptides to Skp1-Cdc4271–779.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Dimerization of Fbw7 and Cdc4
(A) Differentially tagged full-length, but not dimerization-domain mutant Fbw7 proteins, coimmunoprecipitate. Vectors expressing GFP- and Flag-tagged wild-type or DD mutant Fbw7 were cotransfected in 293T cells. Proteins were immunoprecipitated and immunoblotted with the indicated antibodies.
(B) Overlay of gel filtration chromatography profiles of Skp1-Cdc4111–779 (left) and of Skp1-Cdc4271–779 (right) that lacks the dimerization domain. The retention volumes of proteins of known mass and the void volume of the Superdex 200 column are indicated.
(C) Representative sedimentation equilibrium data for the Skp1-Cdc4271–779 (left) and Skp1-Cdc4111–779 (right) complexes, each at 64 μM.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
The In Vivo Turnover of a Weakly Bound Cyclin E Mutant and the In Vitro Ubiquitination of Sic1 Are Enhanced When Their Respective F Box Proteins Can Dimerize
(A) Coimmunoprecipitation analysis of the cyclin E with wild-type and DD mutant Fbw7 proteins in vivo (see Figure 3C legend).
(B) Turnover of wild-type (top) and S384A mutant (bottom) cyclin E by wild-type and mutant Fbw7 proteins (see Figure 3B legend).
(C) In vitro ubiquitination time course of the 32P-labeled 7p-Sic11–100 protein by the monomeric Skp1-Cdc4271–779 and dimeric Skp1-Cdc4111–779 complexes. Sic1 and Sic1-ubiquitin conjugates were separated by SDS-PAGE and quantitated by autoradiography. The ratio of high molecular weight (HMW) ubiquitinated Sic11–100 to total Sic11–100, calculated as the mean ± SD from two independent experiments, is plotted as a function of reaction time.
(D) Models of substrate crossubiquitination by a dimeric SCF. Irrespective of whether one or two multidegron substrates bind to the dimeric SCF, it is possible that the lysine(s) in the vicinity of a phosphodegron bound to one SCF is presented more optimally to the E2 active site of the adjacent SCF, thus enhancing the rate of ubiquitination. Ubiquitin chain elongation may similarly be enhanced by crossdimer ubiquitination.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions