Cysteine pK_a Depression by a Protonated Glutamic Acid in Human DJ-1

Abstract

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.Cysteine thiolates are potent nucleophiles that are used by many proteins for catalysis, metal binding, or to facilitate post-translational modification. However, the solution pK a value of cysteine (8.3) is not within the optimal physiological pH range of most proteins. To render cysteine residues reactive, the thiol pK a must be depressed by stabilization of the conjugate thiolate anion by the protein environment. Multiple types of stabilizing interactions can decrease cysteine pK a values, including electrostatic complementarity with nearby cations (1, 2), the R-helix macropole (3), and hydrogen bonding to the thiol (4).The Parkinsonism-associated protein DJ-1 contains an oxidation-sensitive cysteine residue (C106 in human DJ-1) of unknown pK a that is required for the cytoprotective activity of the protein (5, 6). DJ-1 is a homodimeric protein of 189 amino acid monomers that is found both in the cytoplasm (7) and in the mitochondria (5, 8) and protects cells from various types of oxidative stress (5,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Although C106 has been shown to be essential for the protective function of DJ-1 in cell culture (5, 16) and Drosophila melanogaster (6), the specific biochemical activity of the protein that requires this cysteine residue is uncertain. Identifying the structural determinants of C106 ionization (and hence reactivity) is an essential first step in understanding how this conserved cysteine residue contributes to the neuroprotective activity of DJ-1.The reactive cysteine in human DJ-1 is one of the most highly conserved residues in the DJ-1 superfamily (19,20). Crystal s...

Oxidizable Residues Mediating Protein Stability and Cytoprotective Interaction of DJ-1 with Apoptosis Signal-regulating Kinase 1

“…Among the three human cysteines, most previous studies identified Cys-106 as the most reactive and functionally important residue (15,17,26,28,50), except for one study, which identified Cys-53 to account for the redox-dependent chaperone activity and cytoprotection of DJ-1 (22 [C106EE]DJ-1 also had ASK1 binding and cytoprotective activity, albeit with lower efficacy most likely due to reduced protein stability because of the introduction of more bulky and charged residues in the sensible active site of DJ-1. Thus, higher order oxidation events at Cys-106 activate the cytoprotective protein DJ-1, and the C106DD mutation best mimics DJ-1 activation steps toward initial, non-covalent ASK1 binding.…”

Evaluation of Thiol Raman Activities and pKa Values Using Internally Referenced Raman-Based pH Titration

“…It requires the thiol-containing molecule to contain a chromophore that is sensitive only to the thiol's ionization. 24,31 This imposes a significant constraint on the general application of this method. Indeed, it has been recently demonstrated that the pKa values of 1,4-benzenedithiol (1,4-BDT) determined with the UV-vis method are highly erroneous.…”

Formation of a Stabilized Cysteine Sulfinic Acid Is Critical for the Mitochondrial Function of the Parkinsonism Protein DJ-1

“…Several studies have shown that of the three cysteine residues in human DJ-1, Cys 106 is the most prone to oxidative modification (8,16,17). In addition, Cys 106 has a low pK a value of 5.4 and therefore exists almost exclusively as the highly reactive cysteine thiolate anion at physiological pH (18 …”

“…Several studies have shown that of the three cysteine residues in human DJ-1, Cys 106 is the most prone to oxidative modification (8,16,17). In addition, Cys 106 has a low pK a value of 5.4 and therefore exists almost exclusively as the highly reactive cysteine thiolate anion at physiological pH (18). Replacement of Cys 106 with other amino acids in DJ-1 results in a loss of protective activity against oxidative stressors in a number of systems (15,19,20).…”

“…In reduced DJ-1, the carboxylic acid side chain of Glu 18 is protonated and donates a hydrogen bond to Cys 106 , which depresses the thiol pK a value (18). Therefore, we hypothesized that modifying the environment around the side chain of Cys 106 could decouple the oxidation propensity and pK a of Cys 106 without changing the cysteine residue itself.…”