Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers

Janani Prahlad, David N. Hauser, Nicole M. Milkovic, Mark R. Cookson, Mark A. Wilson

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The oxidation of a key cysteine residue (Cys106) in the parkinsonism-associated protein DJ-1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106-SO2- (sulfinic acid) form of DJ-1. To study this important post-translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ-1 dimerization, which would severely compromise the protein's function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X-ray crystallography, NMR spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross-linking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol cross-linking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple cross-linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild-type protein. The enhanced flexibility of Glu18 DJ-1 mutants provides a parsimonious explanation for their lower observed cross-linking efficiency in cells. In addition, thiol cross-linkers may have an underappreciated value as qualitative probes of protein conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility.

Original languageEnglish (US)
Pages (from-to)839-853
Number of pages15
JournalJournal of Neurochemistry
Volume130
Issue number6
DOIs
StatePublished - Sep 1 2014

Fingerprint

Dimers
Cysteine
Mutation
Dimerization
Oxidative stress
Proteins
Oxidative Stress
Sulfhydryl Compounds
Oxidation
Sulfinic Acids
Circular dichroism spectroscopy
Ultracentrifugation
X Ray Crystallography
Parkinsonian Disorders
Oligomerization
Post Translational Protein Processing
Circular Dichroism
X ray crystallography
Sedimentation
Spectrum Analysis

Keywords

  • DJ-1
  • PARK7
  • Parkinson disease
  • cysteine oxidation
  • parkinsonism
  • structure

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers. / Prahlad, Janani; Hauser, David N.; Milkovic, Nicole M.; Cookson, Mark R.; Wilson, Mark A.

In: Journal of Neurochemistry, Vol. 130, No. 6, 01.09.2014, p. 839-853.

Research output: Contribution to journalArticle

Prahlad, Janani ; Hauser, David N. ; Milkovic, Nicole M. ; Cookson, Mark R. ; Wilson, Mark A. / Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers. In: Journal of Neurochemistry. 2014 ; Vol. 130, No. 6. pp. 839-853.
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N2 - The oxidation of a key cysteine residue (Cys106) in the parkinsonism-associated protein DJ-1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106-SO2- (sulfinic acid) form of DJ-1. To study this important post-translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ-1 dimerization, which would severely compromise the protein's function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X-ray crystallography, NMR spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross-linking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol cross-linking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple cross-linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild-type protein. The enhanced flexibility of Glu18 DJ-1 mutants provides a parsimonious explanation for their lower observed cross-linking efficiency in cells. In addition, thiol cross-linkers may have an underappreciated value as qualitative probes of protein conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility.

AB - The oxidation of a key cysteine residue (Cys106) in the parkinsonism-associated protein DJ-1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106-SO2- (sulfinic acid) form of DJ-1. To study this important post-translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ-1 dimerization, which would severely compromise the protein's function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X-ray crystallography, NMR spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross-linking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol cross-linking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple cross-linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild-type protein. The enhanced flexibility of Glu18 DJ-1 mutants provides a parsimonious explanation for their lower observed cross-linking efficiency in cells. In addition, thiol cross-linkers may have an underappreciated value as qualitative probes of protein conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility. DJ-1 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ-1 dimerization. We use cysteine cross-linking and various biophysical techniques to show that these DJ-1 mutants form dimers with increased conformational flexibility.

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