Kinetic Analyses of Keap1–Nrf2 Interaction and Determination of the Minimal Nrf2 Peptide Sequence Required for Keap1 Binding Using Surface Plasmon Resonance

Authors

  • Yu Chen,

    1. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
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  • Daigo Inoyama,

    1. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
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  • Ah-Ng Tony Kong,

    1. Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
    2. The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
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  • Lesa J. Beamer,

    1. Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA
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  • Longqin Hu

    Corresponding author
    1. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
    2. The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
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Corresponding author: Longqin Hu, longhu@rutgers.edu

Abstract

The Keap1–Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for Keap1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of Keap1 in solution using a surface plasmon resonance-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and Keap1 Kelch domain in solution (inline image) was found to be 23.9 nm, which is 10× lower than the surface binding constant (inline image) of 252 nm obtained for the direct binding of Keap1 Kelch domain to the immobilized 16mer Nrf2 peptide on a surface plasmon resonance sensor chip surface. The binding affinity of Nrf2 peptides to Keap1 Kelch domain was not lost until after deletion of eight residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a inline image of 352 nm and the affinity was increased 15× upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for Keap1 binding is the 9mer sequence of LDEETGEFL.

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