Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex - PubMed (original) (raw)

Figure 4

Peptide binding by WDR5. (a) Peptide injections over immobilized WDR5-biotin. Reference and blank traces were subtracted and traces from each functionalized flow cell (three traces per concentration) were averaged for ease of viewing. Injection concentrations of peptides (residues 1–14): H3 unmodified and H3K4me3, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0, 10.0 and 12.0 μM; H3K4me1, 1.0, 2.0, 4.0, 6.0, 10.0 and 12.0 μM; H3K4me2, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 2.0, 4.0 and 5.0 μM. The association/injection is from 0 to 200 s and the dissociation/buffer flow is from 200 to 460 s. The more gradual association and dissociation of H3K4me2 indicates both a slower on and slower off rate than those of the other peptides. Similar experiments with H31–9-derived peptide had slightly weaker binding but recapitulated the methyl-form binding trend shown. (b) Equilibrium _K_d analysis. Equilibrium _K_ds were determined by fitting response saturation fractions calculated by the Langmuir binding isotherm (see Methods). H3 unmodified, _K_d 3.3 ± 0.2 μM; H3K4me1, _K_d = 8.7 ± 0.3 μM; H3K4me2, _K_d = 1.02 ± 0.05 μM; H3K4me, _K_d = 7.8 ± 0.2 μM. (c) Single-protein injections of wild-type WDR5 and mutants, all at 750 nM, over immobilized H31–20-biotin peptides. Reference and blank traces were subtracted. Consistent with the importance of interactions with the N terminus of the H3 peptide, mutation of Asp107 to alanine caused the greatest decrease in peptide binding among all the mutant proteins examined. Mutation of Tyr131 to alanine leads to slightly enhanced binding of the H3 peptide, suggesting that even a methyl group is slightly larger than optimal for the A1-binding pocket. By contrast, the F149A mutant showed greatly decreased H3 peptide binding, perhaps owing to Phe149's additional role in stabilizing the R2-binding pocket.