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Kinetic Analyses of Keap1–Nrf2 Interaction and Determination of the Minimal Nrf2 Peptide Sequence Required for Keap1 Binding Using Surface Plasmon Resonance
Author(s) -
Chen Yu,
Inoyama Daigo,
Kong AhNg Tony,
Beamer Lesa J.,
Hu Longqin
Publication year - 2011
Publication title -
chemical biology and drug design
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.59
H-Index - 77
eISSN - 1747-0285
pISSN - 1747-0277
DOI - 10.1111/j.1747-0285.2011.01240.x
Subject(s) - surface plasmon resonance , peptide , chemistry , dissociation constant , keap1 , binding site , affinities , stereochemistry , biochemistry , receptor , nanotechnology , materials science , gene , transcription factor , nanoparticle
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 () was found to be 23.9 n m , which is 10× lower than the surface binding constant () of 252 n m 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 of 352 n m 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.