Use of cysteine-reactive cross-linkers 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-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.
Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska, USA.
Janani
J
Prahlad
David N
DN
Hauser
Nicole M
NM
Milkovic
Mark R
MR
Cookson
Mark A
MA
Wilson
D000465:Algorithms; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003460:Crystallization; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D009587:Nitrogen Isotopes; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D000071617:Protein Deglycase DJ-1; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012997:Solvents; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes; D015513:Oncogene Proteins; D011994:Recombinant Proteins; D012997:Solvents; D018698:Glutamic Acid; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1; D003545:Cysteine
DJ-1; PARK7; Parkinson disease; cysteine oxidation; parkinsonism; structure