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Figure 1.
PHF8 PHD domain binding of H3K4me3 enhances its jumonji
domain-mediated demethylation of H3K9me2.
(a) Schematic
representation of PHF8. (b) Effect of H3K4me3 on the
demethylation of H3K9me2 by PHF8. Top panels show progression of
demethylation as a function of reaction time. Supplementary
Figure 11a shows representative mass spectra at various time
points. Bottom panels show kinetics of PHF8 on two peptide
substrates, with calculated kinetic parameters. (c) ITC
measurement of binding of PHF8 to doubly methylated
H3[1–24]K4me3-K9me2 peptides, carried out under the conditions
of 11 μM protein concentration and 0.2 mM peptide concentration
in 100 mM NaCl and 50 mM HEPES, pH 7.0. (d) The inhibitory
effect of adding an equimolar ratio of H3[1–12]K4me3 (top) or
H3[1–21]K4me3 peptides (bottom) on the demethylation of
H3[1–24]K9me2 by PHF8. (e) The PHD (blue) and jumonji (green)
collaborate in binding the H3 peptide (magenta) containing
H3K4me3 and H3K9me2. Omit electron densities, F[o] – F[c]
(black mesh), contoured at 4σ above the mean, are shown for the
trimethlyated H3K4me3 and dimethlyated H3K9me2, respectively.
(f) The surface representation of PHF8, colored with blue (PHD),
green (jumonji) and magenta (H3 peptide). (g) H3K4me3 binding in
the cage, surrounded on four sides by Tyr14, Met20 and Trp29 of
PHD (blue) and Ser354 of jumonji (green). The carbonyl oxygen of
Ser354 is in van der Waals contact with one of the methyl
groups. Tyr7 (in thin lines) covers the top of the cage. (h)
H3K9me2 binds in the active site.
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