Disentangling the recognition complexity of a protein hub using a nanopore

Abstract

WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiological and disease-like conditions, their reversible interactions with other proteins remain modestly examined. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-associated hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 derivative and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.

Application of Nanopore Sensors for Biomolecular Interactions and Drug Discovery

“…[53] Mayse et al also used engineered t-FhuA nanopores to detect of WD40 repeat protein 5 (WDR5), a chromatin-associated hub involved in the epigenetic regulation of histone methylation. [45] For WDR5 detection using engineered t-FhuA, the authors fused the N-terminus of t-FhuA with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand (Figure 3F). These nanopores exhibit an open state before WDR5 binds to the Win motif of t-FhuA; however, the WDR5-MLL4Win interaction can be detected by the change to a closed state when WDR5 binds to the Win motif.…”

“…(F) Architecture of MLL4WintFhuA protein nanopores. Reproduced with permission [45] . Copyright 2022, The Author(s).…”

“…Mayse et al. also used engineered t‐FhuA nanopores to detect of WD40 repeat protein 5 (WDR5), a chromatin‐associated hub involved in the epigenetic regulation of histone methylation [45] . For WDR5 detection using engineered t‐FhuA, the authors fused the N‐terminus of t‐FhuA with a 14‐residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand (Figure 3F).…”

Convergent Alterations of a Protein Hub Produce Divergent Effects within a Binding Site

“… 37 Moreover, WDR5 is overexpressed under various oncogenic conditions and its upregulation catalyzes cancer development. 38 − 42 In recent years, the multitasking Win binding site has received a lot of interest 21 , 43 − 45 because it is a promising target for anti-cancer drug discovery. 46 − 53 Therefore, a quantitative understanding of WDR5’s interactions with other Win motif partners has wide-ranging fundamental significance.…”

“…The Win binding site is located within a central cavity and facilitates high-affinity interactions of WDR5 with each of the six human histone methyltransferases (HMTs; MLL1-4 and SETd1A-B), participating in the formation of corresponding six SET1 enzymatic complexes. , Rearrangements in the MLL1 gene lead to solid tumors and aggressive lymphocytic leukemias in humans . Moreover, WDR5 is overexpressed under various oncogenic conditions and its upregulation catalyzes cancer development. − In recent years, the multitasking Win binding site has received a lot of interest ,− because it is a promising target for anti-cancer drug discovery. − Therefore, a quantitative understanding of WDR5’s interactions with other Win motif partners has wide-ranging fundamental significance. ,− For example, the kinetic fingerprints and affinities of the interactions of WDR5 with Win motif peptides of SET1 family members (SET1 Win ) have been previously reported. , …”