1. Phospho-Pon Binding-Mediated Fine-Tuning of Plk1 Activity
Kang Zhu, Zelin Shan, Lu Zhang, Wenyu Wen 
Structure 
Volume 24, Issue 7, Pages (July 2016)
DOI: /j.str
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2. Structure 2016 24, 1110-1119DOI: (10.1016/j.str.2016.04.012)
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3. Figure 1
Cdk1 Phosphorylation of Pon Creates a Docking Site for Plk1 PBD
(A) Amino acid sequence of the Pon fragment. The potential Cdk1 phosphorylation site is indicated by a triangle.
(B) Cdk1 specifically phosphorylates Pon1-116 on Thr63.
(C) Priming Cdk1 phosphorylation promotes the binding of Pon1-116 to Plk1 PBD.
(D) ITC-based measurements of the binding between pPon or Pon52-66 and Plk1 PBD.
Structure  , DOI: ( /j.str )
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4. Figure 2 The Overall Structure of the Plk1 PBD/pPon Complex
(A) Ribbon diagram representation of the Plk1 PBD/pPon complex as viewed from the side. The dimeric Plk1 PBD domains are shown in green and purple, respectively. The pPon peptides are shown in yellow, with the phosphate group in the ball-and-stick model.
(B) Cylinder representation of the Plk1 PBD/pPon complex structure as viewed from the top.
(C) Combined ribbon and surface presentations of the Plk1 PBD/pPon complex with its orientation corresponding to that shown in (A). The side chains of L505E′ from PBD′ and Phe60 from the pPon peptide are shown in ball-and-stick model.
See also Table S1.
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5. Figure 3 The Interfaces of Plk1 PBD/pPon and PBD Dimer
(A) Superimposition of Plk1 PBD/pPon complex (green) with the optimal phosphopeptide-bound PBD (orange) (PDB: 1UMW).
(B) The interaction details between the representative Plk1 PBD and pPon with the same color scheme and orientation as in Figure 2A. Charge-charge and hydrogen-bonding interactions are highlighted by dashed lines in yellow.
(C) Summary of the quantitative binding constants between various Plk1 PBD and Pon fragments derived from the ITC-based titration assays shown in Figure S2.
(D) Open-book view of the Plk1 PBD dimer showing the surface complementation. The aromatic ring of Phe60pPon fills in a gap to integrate the hydrophobic pocket on PBD for accommodating L505E′ from PBD′. In the surface presentation, the hydrophobic residues are in yellow, the positively charged residues are in blue, the negatively charged residues are in red, and the rest of the amino acids are in gray. The side chain of Phe60 from the pPon peptide is shown as both dots and spheres.
(E) A close-up view of the Plk1 PBD dimer and pPon peptide with the same color scheme as in Figure 2C.
See also Figures S1, S2, S3, and S5.
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6. Figure 4
pPon Binding-Induced Dimerization of Plk1 PBD and the Full-Length Protein in Solution
(A and B) SLS measurements of the molecular weights of 60 μM His-tagged Plk1 PBD WT and L505E in the absence or presence of two molar ratios of pPon (A) or pPonF60A (B) peptide. (A) The elution profile of Plk1 PBD (magenta line) displays as a major peak corresponding to a monomer (30.6 kDa) and a broad minor peak (indicated by the magenta arrow) corresponding to a dimer or monomer-to-dimer transition state. The pPon-bound PBD elutes as a heterogeneous sample containing both monomeric and dimeric forms, but the majority of the proteins are dimers (54.2 kDa, black line). The pPon binding-induced formation of PBD oligomers is indicated by the black arrow. In either the apo (blue line) or holo (red line) form, PBDL505E elutes as a homogeneous sample of 30.8 or 31.1 kDa, respectively, consistent with a monomeric PBD. The theoretical molecular weights of the 1:1 and 2:2 Plk1 PBD/pPon complexes are 31.9 and 63.8 kDa, respectively. (B) The pPonF60A-bound PBD WT and L505E elutes as a monomer. The elution profiles are represented according to retention volume (in milliliters), with the molar mass (in grams per mole) indicated on the left axis and the normalized refractive index indicated on the right axis.
(C) GST pulldown assay showing that pPon binding leads to dimerization of the full-length Plk1.
(D) HEK293T cells were transfected with full-length HA-Plk1 and either FLAG-Plk1 or FLAG-IC. Lysates were further incubated with or without 0.8 mg of pPon.
See also Figure S3.
Structure  , DOI: ( /j.str )
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7. Figure 5
Phospho-Pon Binding-Induced PBD Dimerization Partially Relieves the Autoinhibition of Plk1
(A) Superposition analysis of the structures of the pPon-bound human Plk1 PBD (pink and green)- and the KD (orange)-bound zebrafish Plk1 PBD (gray, PDB: 4J7B) to PBD′.
(B and C) The commercial Plk1 kinase activity was assayed by phosphorylation of Trx-tagged Pon in the absence or presence of the pPon (B) or pPonF60A (C) peptide. The molar ratios of Plk1 to pPon or pPonF60A were 1:5, 1:10, and 1:20 in (B) and 1:10, 1:20, and 1:40 in (C). The ratio of the 32P-labeled Trx-Pon band intensities with and without the addition of Pon peptides was quantified. Error bars denote SD for triplicate experiments.
(D) pPon but not pPonF60A binding induced the dimerization and activation of Plk1, thus resulting in its autophosphorylation.
(E) Two-step kinase assay.
(F) Relative kinase activity of Plk1 on Pon Ser611 phosphorylation as quantified by the band densitometry in (E).
(G) Model of pPon binding-mediated Plk1 activation. To simplify the model we omitted the Plk1 activation by phosphorylation on KD, and the active KD was indicated by phosphorylating the same substrate with a PBD docking site.
See also Figures S4 and S5.
Structure  , DOI: ( /j.str )
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