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

Authors

  • Janani Prahlad,

    1. Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska, USA
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  • David N. Hauser,

    1. Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA
    2. Department of Neuroscience, Brown University/National Institutes of Health Graduate Partnership Program, Brown University, Providence, Rhode Island, USA
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  • Nicole M. Milkovic,

    1. Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska, USA
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  • Mark R. Cookson,

    Corresponding author
    1. Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA
    • Address correspondence and reprint requests to Mark R. Cookson, Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, 35 Convent Drive, Bethesda, MD 20892-3707, USA. E-mail: cookson@mail.nih.gov or Mark A. Wilson, Department of Biochemistry and the Redox Biology Center, The University of Nebraska, N118 Beadle Center, 1901 Vine Street, Lincoln, NE 68588. E-mail: mwilson13@unl.edu

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  • Mark A. Wilson

    Corresponding author
    1. Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska, USA
    • Address correspondence and reprint requests to Mark R. Cookson, Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, 35 Convent Drive, Bethesda, MD 20892-3707, USA. E-mail: cookson@mail.nih.gov or Mark A. Wilson, Department of Biochemistry and the Redox Biology Center, The University of Nebraska, N118 Beadle Center, 1901 Vine Street, Lincoln, NE 68588. E-mail: mwilson13@unl.edu

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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.

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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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