Abstact

WD repeat-containing protein 5 (WDR5) is a core scaffolding component of multiple chromatin-modifying complexes that engages diverse partner proteins through a conserved arginine-binding cavity known as the WDR5-interacting (WIN) site. Dysregulation of WDR5 has been implicated in oncogenesis, making the WIN site a promising therapeutic target. Current inhibitor development has primarily focused on mimicking canonical WIN motif interactions, thereby limiting exploration of alternative recognition modes. Here, we present high-resolution crystal structures of two arginine-containing peptide probes that reveal previously unrecognized binding geometries at the WIN pocket. One peptide adopts an extended linear conformation that bridges both the WIN pocket and the adjacent S7 site. The other binds in a reversed, or "trans-WIN," orientation, in which a C-terminal arginine anchors the WIN site while an upstream proline residue occupies the S7 pocket. Isothermal titration calorimetry confirmed moderate and specific affinities for both peptides. These findings reveal unexpected conformational adaptability of the WIN site and demonstrate that its recognition capacity extends beyond the canonical mode defined by histone H3 and other partner proteins. Collectively, our results expand the structural repertoire of WIN-site recognition and establish a framework for rational design of next-generation WDR5 inhibitors that exploit multi-site engagement and alternative binding topologies.