Influence of Peptide Dipoles and Hydrogen Bonds on Reactive Cysteine pK_a Values in Fission Yeast DJ-1

Authors

Peter Madzelan, University of Nebraska - LincolnFollow
Tetyana Labunksa, University of Nebraska - Lincoln
Mark A. Wilson, University of Nebraska-LincolnFollow

Date of this Version

2012

Citation

FEBS J. 2012 November ; 279(22): 4111–4120.

Comments

Copyright 2012 Madzelan et al.

Abstract

Cysteine residues with depressed pK_a values are critical for the functions of many proteins. Several types of interactions can stabilize cysteine thiolate anions, including hydrogen bonds between thiol(ate)s and nearby residues as well as electrostatic interactions involving charged residues or dipoles. Dipolar stabilization of thiolates by peptide groups has been suggested to play a particularly important role near the N-termini of α-helices. Using a combination of X-ray crystallography, site-directed mutagenesis, and spectroscopic methods, we show that the reactive cysteine residue (Cys111) in Schizosaccharomyces pombe DJ-1 experiences a 0.6 unit depression of its thiol pK_a as a consequence of a hydrogen bond donated by a threonine sidechain (Thr114) to a nearby peptide carbonyl oxygen at the N-terminus of an α-helix. This extended hydrogen bonded interaction is consistent with a sum of dipoles model whereby the distal hydrogen bond polarizes and strengthens the direct hydrogen bond between the proximal amide hydrogen and the cysteine thiol(ate). Therefore, our results suggest that the local dipolar enhancement of hydrogen bonds can appreciably stabilize cysteine thiolate formation. However, the substitution of a valine residue with a proline at the i+3 position has only a minor effect (0.3 units) on the pK_a of Cys111. As proline has a reduced peptide dipole moment, this small effect suggests that a more extended helix macrodipolar effect does not play a major role in this system.