The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases

Abstract

In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochemical and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative analysis of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymatic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.

Figure 1.

WDR5 binds MLL1-4, SET1A and SET1B Win motifs. ITC titration experiments with MLL1_Win (A), MLL2_Win (B), MLL3_Win (C), MLL4_Win (D), SET1A_Win (E) and SET1B_Win (F) peptides and WDR5 (upper trace) and the fitted binding curve (lower trace). The sequence of the Win motifs and the K_D are indicated as insets. Titration of WDR5 with Win peptides displays binding stoichiometries (N values) between 0.9 and 1.0.

Figure 2.

Crystal structure of WDR5 in complex with MLL2-4, SET1A and SET1B Win motifs. (A) Overall structure of WDR5 (dark gray) showing the seven blades and the relative orientations of MLL2 (yellow), MLL3 (turquoise), MLL4 (green), SET1A (firebrick) and SET1B (cyan) Win motifs. (B) Crystal structure of WDR5–MLL4_Win complex. Zoomed view of WDR5 peptidyl-arginine-binding cleft in which WDR5 and MLL4 carbon atoms are rendered in gray and green, respectively. (C) Crystal structures of WDR5–MLL2_Win and WDR5–MLL3_Win complexes. MLL2 and MLL3 carbon atoms are highlighted as in (A). (D) Crystal structures of WDR5–SET1A_Win and WDR5–SET1B_Win complexes. SET1A and SET1B carbon atoms are colored as in (A). Water molecules and key hydrogen bonds are depicted as red spheres and orange dash lines, respectively.

Figure 3.

Comparative analysis Win motif binding modes. (A) Superimposition of WDR5-bound MLL1 (beige) and MLL2 (yellow) peptides. (B) Overlay of MLL1 (beige) and MLL4 (green) peptides in complex with WDR5 (grey). (C) Structural alignment of WDR5–MLL1_Win and WDR5–SET1A_Win complex in which carbons of the peptides are highlighted as in Figure 2. Residues of WDR5 peptidyl-arginine-binding adopting divergent conformation between the complexes are rendered as pink carbon atoms. Hydrogen bonds and water molecules are rendered as in Figure 2.

Figure 4.

Stimulation of the SET1 family of KMTs activity on histone H3 by the core complex. Radiometric methyltransferase assays performed with MLL2 (A), MLL3 (B), MLL4 (C), SET1A (D) and SET1B (E) either in the absence or presence of WDR5 and the RbBP5–ASH2L complex. Methyltransferase assays were performed at a concentration of enzyme in the linear range of activity of MLL2 (1 µM), MLL3 (0.5 µM), MLL4 (1 µM), SET1A (1 µM) and SET1B (0.5 µM) complexes. Activity is represented as the average of three independent experiments performed in triplicate. Error bars indicate the standard deviation between the assays.

Figure 5.

MLL3 methylates histone H3 in the absence of the core complex subunits. Comparative analysis of MLL's enzymatic activity in the absence of the core complex subunits. Assays were performed in the presence of 5.0 µM of enzymes. Activity is represented as in Figure 4.