1. Volume 26, Issue 1, Pages 28-39.e2 (January 2018)
Multivalent Interactions with Fbw7 and Pin1 Facilitate Recognition of c-Jun by the SCFFbw7 Ubiquitin Ligase 
Veronika Csizmok, Meri Montecchio, Hong Lin, Mike Tyers, Maria Sunnerhagen, Julie D. Forman-Kay 
Structure 
Volume 26, Issue 1, Pages e2 (January 2018)
DOI: /j.str
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2. Structure 2018 26, 28-39.e2DOI: (10.1016/j.str.2017.11.003)
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3. Figure 1 Primary Sequence Features of c-Jun
(A) Schematic of c-Jun indicating potential CPD sites (red circles), construct boundaries used, the DNA-binding domain (DBD), and leucine zipper domain (LZD).
(B) Disorder predictions based on IUPred (Dosztanyi et al., 2005a, 2005b) in black and PONDR (Romero et al., 2001) in red.
(C) Sequence alignment of ERK2/MEK1 phosphorylation sites in c-Jun with the optimal cyclin E CPD centered on T380 and the Cdc4 consensus CPD (Nash et al., 2001). Potential CPD sites are shaded gray; phosphorylated residues are colored red.
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4. Figure 2
Jun123 and Jun276 Are Intrinsically Disordered in Both Phosphorylated States
(A and C) 1HN-15N correlation spectra of (A) Jun123 (black) and pJun123 (red) and (C) Jun276 (black) and pJun276 (red) with the assignment of H-N peaks of phosphorylated residues. Phosphorylated residues in pJun123 (Thr2, Ser63, Ser73, Ser83, Thr91, Thr93) and pJun276 (Thr2, Ser63, Ser73, Ser83, Thr91, Thr93, Ser132, Ser160, Ser183, Thr239, Ser243) exhibit significant proton downfield shifts. Multiple resonances observed for residue Ser73 in pJun276 are indicated by a circle.
(B and D) Secondary structural propensity (SSP) values for (B) Jun123 (black) and pJun123 (red) and (D) Jun276 (black) and pJun276 (red). Red asterisks indicate phosphorylation on potential CPD sites; red squares indicate phosphorylation on other sites.
See also Figures S1 and S2.
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5. Figure 3 Multivalent c-Jun-Cdc4 and c-Jun-Fbw7 Interactions
(A–C) 1H-15N correlation spectra of phosphorylated residues of Jun123 (A) and Jun276 (B and C) in the absence (black) and presence (red) of Cdc4 (A and B) or Fbw7 (C). Multiple resonances of Ser73 are denoted with a prime.
(D–F) Intensity ratios of amide resonances of pJun123 (D) or pJun276 (E and F) upon titration with unlabeled Cdc4WD40 (D and E) and Skp1/Fbw7 (F) at pJun:Cdc4 or pJun:Fbw7 molar ratios of 1:0, 1∶0.1, 1∶0.25, 1∶0.5, 1∶0.75, 1∶1.2, and 1:2.0, represented by lines of increasing darkness. Red asterisks indicate phosphorylation on potential CPD sites; red squares indicate phosphorylation on non-CPD sites.
See also Figures S3 and S4.
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6. Figure 4 Sequence Analysis of Fbw7 Substrates
(A) Histogram bar plot of predicted disorder percentage in characterized Fbw7 substrates (blue) and in all human proteins in UniProt (green). The plot is normalized using a Kernel density estimate. Lines indicate predicted average disorder in human proteins (continuous) and in Fbw7 substrates (dashed).
(B) Histogram bar plot of the percentage of predicted long (≥30 consecutive disordered residues) disordered regions in Fbw7 substrates (blue) and in all human proteins in UniProt (green). The plot is normalized as in (A). Lines indicate the average predicted percentage of long disordered regions in human proteins (continuous) and in Fbw7 substrates (dashed).
(C) Histogram bar plot of predicted CPD sites (pSer/pThr-Pro) for Fbw7 substrates. Dashed line indicates the predicted average CPD sites in Fbw7 substrates.
(D) Histogram bar plot of average disorder scores of predicted CPD sites found in Fbw7 substrates. The continuous line indicates the threshold value of 0.5 that is used as a score characteristic of disorder in IUPred; the dashed line indicates the average disorder scores for all CPD sites found in substrates.
(E) Histogram bar plot of phosphorylation sites identified in Fbw7 substrates. Dashed line indicate the predicted average number of phosphorylation sites in Fbw7 substrates.
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7. Figure 5 Multivalent c-Jun-Pin1 Interactions
(A–C) Intensity ratios of amide resonances of pJun276 upon titration with unlabeled full-length Pin1 (A), WW domain (B), or PPIase domain (C) at pJun:Pin1 molar ratios of 1∶0, 1∶0.25, 1∶0.5, 1∶0.75, 1∶1.2, and 1:2.0, represented by lines of increasing darkness. Red asterisks indicate phosphorylation on potential CPD sites; red squares indicate phosphorylation on non-CPD sites.
(D) Multiple c-Jun phosphorylated residues (red circles) engage the binding site of the Pin1 WW domain (light gray) in a dynamic equilibrium (various blue lines representing different c-Jun conformations) (i) or PPIase domain (dark gray) with a likely simultaneous engagement of the high-affinity WW domain binding site (ii). In (iii), two snapshots of this dynamic complex are shown, with the pSer73 (yellow) binding to Pin1 during the cis-trans isomerization process.
See also Figures S5 and S6.
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8. Figure 6
Broadening and Chemical Shift Perturbation for c-Jun CPD Sites upon Pin1 Binding
For resonances of (A) pSer73, (B) pThr91, and (C) pThr93 of pJun276, the (i) intensity ratios for amide resonances of phosphorylated residues are shown upon addition of isolated WW domain (blue), PPIase domain (red) or full-length Pin1 (green). In (ii) and (iii), portions of 1H-15N spectra showing chemical shift perturbations are displayed upon addition of PPIase domain after 20 min (ii) and followed for 24 hr (iii) as colored from purple to red. In (iv), chemical shifts of carbon resonances detected in the strips of the CCC-TOCSY experiment at HN chemical shift of Pro residues adjacent to the phosphorylated residues in the presence of PPIase domain are shown. Note that the pattern of 13Cβ and 13Cγ shifts seen for Pro74 is diagnostic of a cis peptide bond.
See also Figure S7.
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9. Figure 7
Integration of Signals Generated by the Multivalent c-Jun Interactions Governing Transcription and Degradation
Multisite phosphorylated c-Jun (black line with red circles) interacts with both Pin1 and Fbw7 through a dynamic ensemble of binding events. Interaction with Pin1 results in a trans to cis (yellow circle) isomerization on the pSer73-Pro74 peptide bond that is known to increase transcriptional activity of c-Jun (Wulf et al., 2001). Engagement of Fbw7 in a dynamic equilibrium leads to c-Jun ubiquitination by SCFFbw7 and subsequent degradation by the proteasome (black rectangles). The trans to cis isomerization of c-Jun mediated by Pin1 may affect binding to Fbw7 (dashed arrow) and lead to stabilization of c-Jun in vivo.
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