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

Authors

Janani Prahlad, University of Nebraska - Lincoln
David N. Hauser, National Institute on Aging
Nicole M. Milkovic, University of Nebraska-Lincoln
Mark R. Cookson, National Institute on AgingFollow
Mark A. Wilson, University of Nebraska - LincolnFollow

Date of this Version

9-2014

Citation

J Neurochem. 2014 September ; 130(6): 839–853. doi:10.1111/jnc.12763.

Comments

Copyright 2014 Authors. Used by permission.

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, 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.