Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein

Albena T. Dinkova-Kostova, W. David Holtzclaw, Nobunao Wakabayashi

Research output: Contribution to journalArticle

Abstract

Induction of the phase 2 response, a major cellular reaction to oxidative/electrophile stress depends on a protein triad: actin-tethered Keap1 that binds to Nrf2. Inducers react with Keap1 releasing Nrf2 for nuclear translocation and activation of the antioxidant response element (ARE), which regulates phase 2 genes. The primary sensors for inducers are certain uniquely reactive cysteine thiols of Keap1. Recombinant murine Keap1 contains 0.9 zinc atoms per monomer as determined by inductively coupled plasma-optical emission spectrometry: its zinc content depends on the metal composition of the overexpression medium. Simultaneous direct measurement of bound zinc using a pyridazoresorcinol chelator and protein thiol groups using 4,4′-dipyridyl disulfide has established that (i) zinc is bound to reactive cysteine thiols of Keap1 and is displaced stoichiometrically by inducers, (ii) with these cysteines mutated to alanine, the affinity for zinc is reduced by nearly 2 orders of magnitude, and (iii) the association constant of Keap1 for zinc is 1.02 (±0.19) × 1011 M-1, consistent with a Zn 2+ metalloprotein. Co2+ substitution for Zn2+ yields an optical spectrum consistent with tetrahedral metal coordination. Coincident binding of inducers and release of zinc alters the conformation of Keap1, as shown by a profound decline of its tryptophan fluorescence and depression of fluorescence of a hydrophobicity probe. Thus, regulation of the phase 2 response involves chemical modification of critical cysteine residues of Keap1, whose reactivity is modulated by zinc binding. Keap1 is a zinc-thiol protein endowed with a delicate switch controlled by both metal-binding and thiol reactivity.

Original languageEnglish (US)
Pages (from-to)6889-6899
Number of pages11
JournalBiochemistry®
Volume44
Issue number18
DOIs
StatePublished - May 10 2005

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Metalloproteins
Oxidants
Zinc
Sensors
Sulfhydryl Compounds
Cysteine
Metals
Fluorescence
Antioxidant Response Elements
Proteins
Oxidative stress
Chemical modification
Inductively coupled plasma
Hydrophobicity
Chelating Agents
Hydrophobic and Hydrophilic Interactions
Tryptophan
Disulfides
Alanine
Spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein. / Dinkova-Kostova, Albena T.; Holtzclaw, W. David; Wakabayashi, Nobunao.

In: Biochemistry®, Vol. 44, No. 18, 10.05.2005, p. 6889-6899.

Research output: Contribution to journalArticle

Dinkova-Kostova, Albena T. ; Holtzclaw, W. David ; Wakabayashi, Nobunao. / Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein. In: Biochemistry®. 2005 ; Vol. 44, No. 18. pp. 6889-6899.
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