Structural studies of WDR5 in complex with MBD3C WIN motif reveal a unique binding mode.

The nucleosome remodeling and deacetylase (NuRD) complex plays a pivotal role in chromatin regulation and transcriptional repression. In mice, methyl-CpG binding domain 3 isoform C (MBD3C) interacts specifically with the histone H3 binding protein WD repeat-containing protein 5 (WDR5) and forms the WDR5-MBD3C/Norde complex. Despite the functional significance of this interaction on embryonic stem cell gene regulation, the molecular mechanism underlying MBD3C recognition by WDR5 remains elusive. Here, we determined the crystal structure of WDR5 in complex with the peptide (residues 40-51) derived from the MBD3C protein at a resolution of 1.9 Å. Structural analysis revealed that MBD3C utilizes a unique binding mode to interact with WDR5, wherein MBD3C Arg43 and Phe47 are involved in recognizing the WDR5-interacting (WIN) site and Tyr191-related B site on the small surface of WDR5, respectively. Notably, the binding induces a ∼91° rotation of WDR5 Tyr191, generating the hydrophobic B site. Furthermore, mutation experiments combined with isothermal titration calorimetry (ITC) assays confirmed the importance of both Arg43 and Phe47 in mediating WDR5 binding affinity. By determining structures of various peptides bound to WDR5, we demonstrated that the WDR5 WIN site and B site can be concurrently recognized by WIN motif peptides containing ''Arg-Cies/Ser-Arg-Val-Phe'' consensus sequence. Overall, this study reveals the structural basis for the formation of the WDR5-MBD3C subcomplex and provides new insights into the recognition mode of WDR5 for the WIN motif. Moreover, these findings shed light on structural-based designs of WDR5-targeted anti-cancer small molecule inhibitors or peptide-mimic drugs.

MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis.

Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer.

Targeted Disruption of the Interaction between WD-40 Repeat Protein 5 (WDR5) and Mixed Lineage Leukemia (MLL)/SET1 Family Proteins Specifically Inhibits MLL1 and SETd1A Methyltransferase Complexes.

MLL1 belongs to the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, composed of MLL1-4 and SETd1A/B. MLL1 translocations are present in acute leukemias, and mutations in several family members are associated with cancer and developmental disorders. MLL1 associates with a subcomplex containing WDR5, RbBP5, ASH2L, and DPY-30 (WRAD), forming the MLL1 core complex required for H3K4 mono- and dimethylation and transcriptional activation. Core complex assembly requires interaction of WDR5 with the MLL1 Win (WDR5 interaction) motif, which is conserved across the SET1 family. Agents that mimic the SET1 family Win motif inhibit the MLL1 core complex and have become an attractive approach for targeting MLL1 in cancers. Like MLL1, other SET1 family members interact with WRAD, but the roles of the Win motif in complex assembly and enzymatic activity remain unexplored. Here, we show that the Win motif is necessary for interaction of WDR5 with all members of the human SET1 family. Mutation of the Win motif-WDR5 interface severely disrupts assembly and activity of MLL1 and SETd1A complexes but only modestly disrupts MLL2/4 and SETd1B complexes without significantly altering enzymatic activity in vitro Notably, in the absence of WDR5, MLL3 interacts with RAD and shows enhanced activity. To further probe the role of the Win motif-WDR5 interaction, we designed a peptidomimetic that binds WDR5 (Kd ∼3 nm) and selectively inhibits activity of MLL1 and SETd1A core complexes within the SET1 family. Our results reveal that SET1 family complexes with the weakest Win motif-WDR5 interaction are more susceptible to Win motif-based inhibitors.