Abstact

WD repeat-containing protein 5 (WDR5) recognizes canonical arginine-containing WDR5-interacting (WIN) motifs through a highly conserved binding pocket, in which WDR5 Phe133 and Phe263 are thought to stabilize the central arginine via cation-pi interactions. Here, we re-evaluate the contribution of these residues using the MBD3C WIN peptide, which exhibits dual-site engagement with the WIN and B pockets. Isothermal titration calorimetry reveals that WDR5 F133A and F263A variants retain robust binding affinity to MBD3C, with only an approximately twofold reduction in affinity for F133A and a modest increase for F263A compared to wild-type WDR5. Crystal structures of both variant complexes at 1.30 A and 1.57 A resolution reveal that the peptide adopts a canonical binding mode despite disruption of the aromatic cage. The cavities created by phenylalanine-to-alanine substitutions are occupied by newly recruited, well-ordered water molecules that integrate into the conserved hydration network and form compensatory hydrogen-bonding interactions with the arginine side chain. These results indicate that Phe133 and Phe263 are not strictly required for WIN motif recognition in this context and demonstrate that solvent-mediated interactions can stabilize ligand binding in the absence of canonical cation-pi contacts. Together, these findings highlight the adaptability of the WDR5 WIN binding pocket and provide a refined framework for understanding ligand recognition.