WD repeat‐containing protein 5 (WDR5) is a conserved chromatin regulator that engages numerous binding partners via a central arginine‐binding pocket known as the WDR5‐interacting (WIN) site. Endogenous microprotein binder of WDR5 (EMBOW, also known as SCRIB overlapping open reading frame protein), recently identified as an endogenous WDR5 interactor, lacks the canonical ACR‐R‐TASCK WIN motif, and its mode of recognition remains unknown. Here, we present the 1.80 Å crystal structure of WDR5 in complex with an EMBOW‐derived peptide. Our structural analysis reveals that EMBOW engages the WIN site through a Met1‐Arg2‐Thr3 (MRT) triad. The bulky Met1 residue occupies the conserved WIN site pocket, and mutation of Thr3 to valine reduces binding affinity, while N‐terminal Gly‐Ser insertion preserves binding, indicating a degree of structural tolerance. Binding assays and mutational analysis underscore the functional importance of the MRT triad. Furthermore, structural and biochemical studies of MRT‐containing peptides from RNA‐binding protein 15 (RBM15) and zinc finger and SCAN domain‐containing protein 10 (ZSCAN10) suggest that this motif may serve as an alternative WIN site recognition signature. In summary, our findings define the molecular basis of EMBOW‐WDR5 interaction and expand the sequence space compatible with WIN site engagement.
Yang et al. (Fri,) studied this question.