1. Volume 25, Issue 10, Pages 1530-1539.e3 (October 2017)
A Unique pH-Dependent Recognition of Methylated Histone H3K4 by PPS and DIDO 
Adam H. Tencer, Jovylyn Gatchalian, Brianna J. Klein, Abid Khan, Yi Zhang, Brian D. Strahl, Karel H.M. van Wely, Tatiana G. Kutateladze 
Structure 
Volume 25, Issue 10, Pages e3 (October 2017)
DOI: /j.str
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2. Structure 2017 25, 1530-1539.e3DOI: (10.1016/j.str.2017.08.009)
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3. Figure 1 PPS-PHD Selects for Histone H3K4me3
(A) PPS domain composition.
(B) Snapshot of a 23 kb genomic region containing the Drosophila Sxl gene. Individual tracks for H3K4me3 (SRX331384), H3K27ac (SRX331371), H3K9ac (SRX287847), H3K79me3 (SRX331396), H3K36me3 (SRX287679), Set1 (SRX ), RNA PolII (SRX318801), and Rpll215 (SRX859009) chromatin immunoprecipitation sequencing experiments were analyzed in IGV.
(C) Superimposed 1H,15N HSQC NMR spectra of PPS-PHD recorded while indicated histone H3 peptides (1–12) were titrated in. Spectra are color coded according to the PPS-PHD:histone peptide molar ratio. Arrows indicate chemical shift changes.
(D) Binding affinities of PPS-PHD to indicated histone peptides as measured by intrinsic tryptophan fluorescence (for H3K4me3 and H3K4me2) or NMR at pH 6.9.
(E) Representative binding curves used to determine the Kd values by NMR.
(F) Western blot analysis of pull-downs using GST-PPS-PHD and indicated biotinylated histone H3 peptides at pH 7.4.
(G) Western blot analysis of pull-downs using GST-PPS-PHD and indicated biotinylated histone H3 peptides at pH 7.4.
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4. Figure 2 Structural Basis for the Recognition of H3K4me3 by PPS-PHD
(A) PPS-PHD is depicted as a solid surface colored wheat with the H3K4me3 peptide shown as green sticks.
(B) Rotated view of the structure in (A).
(C) The ribbon diagram of the PPS-PHD-H3K4me3 complex. Dashed lines represent hydrogen bonds and blue spheres represent water molecules.
(D) The electrostatic surface potential of PPS-PHD is colored blue and red for positive and negative charges, respectively.
(E) A zoom-in view of the H3K4me3-binding pocket. The distances (in Å) are indicated by dashed lines and labeled.
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5. Figure 3 Unique Binding Mechanism of PPS-PHD
(A) Structural overlay of the PPS-PHD finger in the apo-state and bound to H3K4me3 peptide.
(B) Zoom-in view of the H3K4me3-binding pocket as in (A).
(C) Binding affinities of the mutant PPS-PHD to H3K4me3 peptide; NB, no binding.
(D and E) Superimposed 1H,15N HSQC spectra of (D) the H919A mutant of PPS-PHD and (E) D928A PPS-PHD, collected upon titration with the H3K4me3 peptide. Spectra are color coded according to the protein:peptide molar ratio.
(F) Representative binding curve used to determine the Kd values by NMR.
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6. Figure 4 Binding of PPS-PHD to H3K4me3 Is pH Dependent
(A) Superimposed 1H,15N HSQC spectra of PPS-PHD recorded while the H3K4me3 peptide was titrated in at pH 6.0.
(B and F) Representative binding curves used to determine the Kd values for (B) WT PPS-PHD by fluorescence at pH 6.0 and (F) H919A PPS-PHD by fluorescence at pH 7.5.
(C and G) Summary of binding affinities of (C) WT PPS-PHD and (G) H919A PPS-PHD to H3K4me3 peptide as measured by tryptophan fluorescence.
(D) A diagram showing the effect of protonation of H919.
(E) Western blot analysis of pull-downs using GST-PPS-PHD and indicated biotinylated histone H3 peptides at pH 6.0.
(H) A titration curve used to calculate the pKa value of H919.
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7. Figure 5 pH Sensitivity Is Conserved in DIDO-PHD but Not in Bye1-PHD
(A) Alignment of amino acid sequences of the PHD fingers: absolutely, moderately, and weakly conserved residues are colored orange, pale yellow, and light green, respectively. The aromatic cage residues of the PHD fingers, for which the atomic-resolution structures of the complexes with H3K4me3 have been determined, are indicated by red ovals.
(B–D) Structural overlays of the H3K4me3-bound PHD fingers of PPS (this work) and (B) DIDO (PDB: 4L7X), (C) PHF13 (PDB: 3O7A), and (D) MLL1 (PDB: 3LQJ).
(E and I) Representative binding curves used to determine the Kd values for DIDO (E) and Bye1 (I).
(F and H) Binding affinities of DIDO-PHD (F) and Bye1-PHD (H) to H3K4me3 peptide at indicated pH.
(G) A model of the Bye1-PHD finger fold generated with Phyre2.
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8. Figure 6 Acidification Promotes DIDO Expulsion from the Nucleus
RPE-1 cells were arrested in early mitosis by inhibiting Haspin, washed, and released into control (pH 7.4) or acidic (pH 6.4) medium without inhibitor. The nuclear pH gradient was dissipated with nigericin. Mitosis was allowed to proceed for 10 min, cells were fixed and labeled for DIDO (A and B, green) and H3K4me3 (A, red) or ING1 (B, red). DNA was counterstained with DAPI (A and B, blue). Color separations are shown in Figures S1 and S2. Scale bars, 10 μm.
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