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.