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

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-SO 2 − (sulfinic acid) form of DJ-1. T...

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Bibliographic Details
Published in:Journal of neurochemistry 2014-06, Vol.130 (6), p.839-853
Main Authors: Prahlad, Janani, Hauser, David N., Milkovic, Nicole M., Cookson, Mark R., Wilson, Mark A.
Format: Article
Language:English
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Summary: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-SO 2 − (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, CD spectroscopy, sedimentation equilibrium ultracentrifugation, and crosslinking. We found that all of the Glu18 DJ-1 mutants were dimeric. Thiol crosslinking indicates that these mutant dimers are more flexible than the wild-type protein and can form multiple crosslinked 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 crosslinking efficiency in cells. In addition, thiol crosslinkers may have an underappreciated value as qualitative probes of protein conformational flexibility.
ISSN:0022-3042
1471-4159
DOI:10.1111/jnc.12763