Abstract
The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro.
Highlights
Tein (Fig. 5e, and Table 2), suggesting that the overall conformation of the S91K and F133A mutant proteins is similar to that of wild-type WDR5. These results suggest that the central arginine binding cavity of WDR5 is essential for the interaction with MLL1
The WDR5F133ARbBP5-Ash2L subcomplex in the absence of MLL3745 sediments with an s* value similar to that of the wild-type subcomplex at 4 s* (Fig. 7e, dotted line). These results are consistent with a system in which the dissociation rate constant is rapid when compared with the time scale of sedimentation [45]. These results suggest that the interaction between MLL1 and WDR5 within the MLL1 core complex is significantly weakened when Phe-133 of WDR5 is replaced with alanine
Previous research suggests that WDR5 functions as a structural platform that bridges the interaction between the catalytic and regulatory subunits of the MLL1 core complex [39, 40]
Summary
We individually replaced Ser-3763, Arg-3765, and Glu-3767 of MLL1 with alanine and compared the wild-type and mutant MLL3745 proteins for their ability to interact with WDR5 in sedimentation velocity experiments. Enzymatic assays reveal that wild-type MLL3745 is a slow monomethyltransferase in the absence of interacting proteins but becomes a fast dimethyltransferase in the presence of WDR5, RbBP5, and Ash2L.4 in the current investigation, when the core components were mixed with the R3765A MLL3745 protein, the 5 s* complex was not detected in sedimentation velocity experiments (compare Fig. 7, a and b).
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