Volume 20, Issue 1, Pages 9-19 (October 2005) Structural Basis of the Cks1-Dependent Recognition of p27Kip1 by the SCFSkp2 Ubiquitin Ligase  Bing Hao, Ning Zheng, Brenda A. Schulman, Geng Wu, Julie J. Miller, Michele Pagano, Nikola P. Pavletich  Molecular Cell  Volume 20, Issue 1, Pages 9-19 (October 2005) DOI: 10.1016/j.molcel.2005.09.003 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Summary of Sequence Conservation, Secondary Structure Elements, and Intermolecular Contacts for the Skp1-Skp2-Cks1-p27Kip1 Complex The LRR domain of Skp2 (A), Cks1 (B), and the p27Kip1 peptide used in crystallization (C) are shown. Sequence conservation is shown as a bar graph, with black bars indicating identity among the Skp2 and Cks1 orthologs (see also Figures S2 and S3). For the p27Kip1 peptide, residues conserved in >50% of the shown orthologs are highlighted in yellow, and the corresponding region of the p57 paralog is aligned based on the phospho-threonine. Cylinders indicate α helices, and arrows indicate β strands. Residues that interact with Skp2 are indicated by red crosses, with Cks1 by blue dots, p27Kip1 by yellow stars, and Cdk2 (Bourne et al., 1996) by purple triangles. The p27Kip1 residues that have clear electron density in the crystals are underlined. Molecular Cell 2005 20, 9-19DOI: (10.1016/j.molcel.2005.09.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Structure of the Skp1-Skp2-Cks1-p27Kip1 Complex Ribbon diagrams of the complex are shown in two views related by a 90° rotation. Skp1, Skp2, Cks1, and the p27Kip1 peptide are showed in blue, red, cyan, and yellow, respectively. Molecular Cell 2005 20, 9-19DOI: (10.1016/j.molcel.2005.09.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Intermolecular Contacts in the Skp1-Skp2-Cks1-p27Kip1 Complex (A) Close-up view of the Skp2-Cks1 interface showing interacting amino acids of Skp2 (pink) and Cks1 (light blue). Hydrogen bonds are indicated by white dotted lines. The portion of the Cks1 structure not involved in Skp2 binding is omitted for clarity. (B) Molecular surface of Skp2 colored according to orthologous conservation. Skp1, Cks1, and p27Kip1 are shown as backbone worms, colored as in Figure 2 except for p27Kip1, which is green. (C) Close-up view of the interface between p27Kip1 and Skp2-Cks1 showing interacting amino acids. (D) Molecular surface representation of the Skp2-Cks1 region involved in p27Kip1 binding, colored according to the local electrostatic potential. Molecular Cell 2005 20, 9-19DOI: (10.1016/j.molcel.2005.09.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 In Vitro Ubiquitination of Wt and Mutant Full-Length p27Kip1 Proteins (A) The top panel shows p27Kip1 and p27Kip1-ubiquitin conjugates detected by immunoblotting with an anti-p27Kip1 antibody. The bottom panel shows phosphorylation of Thr187 detected by immunoblotting with anti-p27Kip1 phospho-Thr187 antibody. (B) Ubiquitination time course of the 32P-labeled p27Kip1 proteins. 32P-labeled p27Kip1 and p27Kip1-ubiquitin conjugates were analyzed by SDS-PAGE followed by autoradiography. (C) Quantitive phosphorimager analysis of the remaining 32P-labeled p27 proteins from (B). The ratios of the remaining p27/total p27 are plotted as a function of reaction time. Data represent mean ± SD for two independent experiments, except three experiments for the V184A mutant. Molecular Cell 2005 20, 9-19DOI: (10.1016/j.molcel.2005.09.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Putative Macromolecular Binding Sites in Fbxl2 and Its Close Homolog Fbxl20, and in Fbxl4 Sequence conservation among orthologs of Fbxl2/20 and Fbxl4 is mapped onto the molecular surface of the Skp2 LRR domain structure consisting of repeats three to ten with the side chains truncated to the Cγ atoms. Complete alignments are shown in Figures S5 and S6. Molecular Cell 2005 20, 9-19DOI: (10.1016/j.molcel.2005.09.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 The Regions of Cks1 Involved in Skp2-p27Kip1 Binding Are Distinct from Its Cdk2 Binding Site (A) Molecular surface of Cks1 highlighting the binding sites for p27Kip1 (yellow), Skp2 (magenta), and Cdk2 (purple). (B) Model of the SCFSkp2-Cks1-p27Kip1-Cdk2-cyclin A complex indicates that the pairwise interactions that have been structurally characterized can coexist in one complex. Also shown is the model of a docked E2 (orange) and its active site cysteine (cyan) (Zheng et al., 2002). A middle region of p27Kip1 (residues 94–180) for which no structural information exists is shown as a dashed line. (C) Gel-filtration chromatography (top) showing the coelution of the eight proteins of the SCFSkp2-Cks1-p27Kip1-Cdk2-cyclin A complex. The retention volumes of various other complexes are indicated on the chromatogram. Cul1 is expressed as two noncovalently associated polypeptides (NTD and CTD) as previously described (Zheng et al., 2002). Fractions were analyzed by SDS-PAGE and Coomassie staining (bottom). Molecular Cell 2005 20, 9-19DOI: (10.1016/j.molcel.2005.09.003) Copyright © 2005 Elsevier Inc. Terms and Conditions