Disentanglement of the MLL-WDR5 Protein-Protein Recognition Events - Project summary
Mixed lineage leukemia protein-1 (MLL1) is a member of the human SET1 family of histone H3 lysine 4
(H3K4) methyltransferases, which include MLL1-4 and SETd1A/B. Rearrangements of MLL1 are
frequently present in acute leukemia, whereas genetic alterations in other SET1 family members are
associated with developmental disorders as well as a number of cancers. A minimal evolutionarily
conserved complex, which is formed by MLL1 and four additional proteins, is required for the sequential
mono- and dimethylation of H3K4. WD40 repeat protein-5 (WDR5), one of the MLL1 core complex
proteins, specifically interacts with a conserved WDR5 interaction motif of the SET1 proteins, also named
the Win motif. Targeting the Win motif-WDR5 interaction with small-molecule drugs and Win-based
peptidomimetics has emerged as a strategic approach for treatment of acute leukemia that harbors the
MLL1 protein, because the MLL1-WDR5 interaction is a key regulatory mechanism of the MLL1
enzymatic activity. However, progress in identifying inhibitors of the MLL1-WDR5 interactions remains
modest due to: (i) the lack of proteomics technologies for the quantitative evaluation of the transient
protein-protein interactions (PPI) at the MLL1-WDR5 interface; (ii) the lack of a mechanistic knowledge
pertaining to the MLL1-WDR5 recognition system. To address these scientific and technological gaps, we
will develop monomeric protein-pore based sensors for sampling transient PPI at single-recognition event
resolution. The central player of these sensors will be the t-FhuA protein pore, a heavily truncated
derivative of ferric hydroxamate uptake component A (FhuA) of E. coli. t-FhuA will be fused to a water-
soluble MLL1 SET binding domain via a short peptide tether. Such a MLL1 binding polypeptide-containing
t-FhuA-based sensor will rely on precise protein engineering, along with biomolecular recognition,
scalable high-resolution electrical recordings, and single-protein channel reconstitution. The presence of
WDR5 will produce a specific, sensitive, and quantitative readout that encompasses reversible current
blockades, the nature of which depends on the PPI strength and WDR5 concentration. The expected
immediate outcomes will be the following: (i) the design, creation, and optimization of the next-generation
t-FhuA-based sensors equipped with single receptor elements for the real-time, selective sampling of
transient PPI in aqueous phase; (ii) the development of a mechanistic and quantitative information on the
Win motif-WDR5 interactions for each SET1 family member; (iii) the multiplexed screening of Win motif-
based inhibitors with improved translational potential. These research studies will ultimately lead to a
fundamental basis for accelerated discoveries in clinical molecular diagnostics, proteomics, and biosensor
technology.
