Disentangling the recognition complexity of a protein hub using a nanopore

Lauren Ashley Mayse,Liviu Movileanu,Ali Imran,Aaron James Wolfe,Motahareh Ghahari Larimi,Michael S Cosgrove

doi:10.1038/s41467-022-28465-8

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.

Highlights

WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries
The presence of WD40 repeat protein 5 (WDR5) in the cis compartment at nanomolar concentrations produced infrequent current blockades (Supplementary Fig. 2 Table 3), likely because of the entropic penalty of MLL4Win to partition into the WDR5 cavity. These current blockades occurred as WDR5 was held in the proximity of the pore opening during its reversible captures by MLL4win, obstructing a fraction of the ionic flux through the nanopore
They were not noted when an unmodified tFhuA nanopore[12,13], without the MLL4Win ligand, was exposed to WDR5 added to the cis compartment

Summary

Introduction

WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Plots of the normalized amplitude of WDR5produced current blockades as a function of WDR5-captured duration demonstrate that the short-lived events spanned the broadest range of I/I0 (Supplementary Fig. 8). The association rate constants for short-, medium-, and long-lived events, kon-1, kon-2, and kon-3, respectively, were consistent for all [WDR5] values (Supplementary Table 7).

Results

Conclusion