Sensing Mixed Lineage Leukemia Win Motif Peptide-Wd Repeat Protein-5 Association With An Engineered Biosensor

Mixed Lineage Leukemia-1 (MLL1) protein is a member of the SET1 family of enzymes. All SET1 lysine methyltransferases must be bound to a multi subunit enzyme called WRAD in order to be fully activated. The MLL1 enzyme binds specifically to the WD Repeat Protein-5 (WDR5) subunit via a conserved arginine-containing recognition site, referred to as the WDR5 interaction motif (Win motif). The activated MLL1-WDR5 complex is associated with Mixed Lineage Leukemia (MLL). MLL is a high-risk blood cancer, which has both myeloid and lymphocytic characteristics. This complex cancer predominately effects pediatric patients and sparks great clinical concern. This clinical connection lead to the identification of six Win motif peptides capable of inhibiting MLL1-WRAD association and provide promising therapeutic potential as highly specific treatments for MLL. Unfortunately, details about the interaction between these Win motif peptides and WDR5 remain unknown. In order to study this critical peptide-protein interaction (PPI), each Win motif peptide was genetically engineered onto the robust and monomeric t-FhuA protein pore, an extensive truncation of ferric hydroxamate uptake component A (FhuA) of Escherichia coli. This protein fusion was facilitated through either a flexible or a rigid peptide linker at the N-terminus of the t-FhuA protein pore. Then, this engineered MLL-t-FhuA protein pore was functionally reconstituted into a planar lipid membrane for single-molecule electrophysiology measurements. Results of this study include outcomes acquired with various Win motifs and linkers that modulate PPI in solution. We conclude that the flexibility of the peptide tether is a critical factor for obtaining binding affinities from single-molecule measurements in accord with results from other bulk-phase approaches.