Cysteine pKa depression by a protonated glutamic acid in human DJ-1.
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 p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K 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 p K 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 p K 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 p K 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.
Department of Biochemistry and Redox Biology Center, The University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Anna C
AC
Witt
Mahadevan
M
Lakshminarasimhan
Benjamin C
BC
Remington
Sahar
S
Hasim
Edwin
E
Pozharski
Mark A
MA
Wilson
D003460:Crystallization; D003545:Cysteine / Q000378:metabolism*; D030541:Databases, Genetic; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018384:Oxidative Stress; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D000071617:Protein Deglycase DJ-1; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
D047908:Intracellular Signaling Peptides and Proteins; D015513:Oncogene Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1; D003545:Cysteine