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Article

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

Supporting Info

Anna C. Witt^§, Mahadevan Lakshminarasimhan^§, Benjamin C. Remington^§, Sahar Hasim^§, Edwin Pozharski and Mark A. Wilson*^§

Department of Biochemistry and Redox Biology Center, The University of Nebraska, Lincoln, Nebraska 68588-0664, and Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201

Biochemistry, 2008, 47 (28), pp 7430–7440

DOI: 10.1021/bi800282d

Publication Date (Web): June 21, 2008

†

This work was supported in part by a grant to M.A.W. from the American Parkinson’s Disease Association as well as a grant from the National Institutes of Health (P 20RR-17675). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract W-31-109-Eng-38. Use of BioCARS Sector 14 was supported by the National Institutes of Health, National Center for Research Resources, under Grant R07707.

‡

Refined model coordinates and experimental structure factors have been deposited in the Protein Data Bank as entries 2OR3 (orthorhombic wtDJ-1), 3CY6 (E18Q), 3CYF (E18N), 3CZ9 (E18L), and 3CZA (E18D).

The University of Nebraska.

University of Maryland School of Pharmacy.

* To whom correspondence should be addressed: N164 Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. E-mail: mwilson13@unl.edu. Phone: (402) 472-3626. Fax: (402) 472-4961.

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.

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