Influence of peptide dipoles and hydrogen bonds on reactive cysteine pKa values in fission yeast DJ-1

Abstract

Cysteine residues with depressed pKa 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 pKa as a consequence of a hydrogen bond donated by a threonine side chain (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 pKa 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. Database Structural models and diffraction data are available in the Protein Data Bank under the accession number(s) 4GDH (wild-type SpDJ-1), 4GE0 (T114P SpDJ-1) and 4GE3 (T114V SpDJ-1) Structured digital abstract SpDJ-1 and SpDJ-1 bind by x-ray crystallography (View interaction) The structural determinants of cysteine pKa depression are incompletely understood. We show that a hydrogen bond between a threonine side chain and a peptide group lowers the pKa value of a proximal reactive cysteine in Schizosaccharomyces pombe DJ-1. This result provides experimental support for the role of peptide dipoles in modulating cysteine pKa values. © 2012 The Authors Journal compilation © 2012 FEBS.