Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined pK_a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed pK_a of 5.4 ± 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine pK_a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the pK_a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its pK_a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid−cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.

Cytosine methylation at CpG dinucleotides produces m ⁵ CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m ⁵ C deamination yields mutagenic G·T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G·T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G·C pair. hTDG is inactive against normal A·T pairs, and is most effective for G·T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDG ^cat ) in complex with abasic DNA, at 2.8 Å resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDG ^cat can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo , might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.