Published January 1, 2022 | Version v1
Journal article Open

Disentangling the Recognition Complexity of a Protein Hub Using a Nanopore

Authors/Creators

  • 1. Department of Physics, Biomedical and Chemical Engineering Syracuse University
  • 2. Department of Physics Syracuse University
  • 3. Department of Physics Syracuse university and Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health,
  • 4. Department of Biochemistry and Molecular Biology, State University of New York - Upstate Medical University,
  • 5. Department of Physics, Syracuse University, Ichor Life Sciences, Inc., Lewis School of Health Sciences, Clarkson University, Department of Chemistry, State University of New York, College of Environmental Science and Forestry
  • 6. Department of Physics, Department of Biomedical and Chemical Engineering, The BioInspired Institute Syracuse University

Description

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 proteomic 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. Surprisingly, our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions. In addition, we identify 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.

Notes

We thank our colleagues in the Movileanu and Cosgrove laboratories and at Ichor Life Sciences laboratories for their comments on the manuscript and stimulating discussions as well as for their technical assistance during the very early stage of this project. This work was supported by the National Cancer Institute of the U.S. National Institutes of Health grant R01 CA140522 (to M.S.C) and by the National Institute of General Medical Sciences of the U.S. National Institutes of Health grant R01 GM129429 (to L.M.).

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Funding

National Institutes of Health
Disentanglement of the MLL-WDR5 Protein-Protein Recognition Events 1R01GM129429-01