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

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.

A Glutathione-independent Glyoxalase of the DJ-1 Superfamily Plays an Important Role in Managing Metabolically Generated Methylglyoxal in Candida albicans

“…Interestingly, significant consumption of MG was also measured for Sz. pombe DJ-1 (SPAC22E12.03c), which is a close homolog of human DJ-1 and not a member of the Hsp31 clade (52,53). However, unlike C. albicans Glx3 and S. cerevisiae YDR533C, no D-lactate production was detected for the human or Schizosaccharomyces pombe enzymes, suggesting either that another product is generated or that the D-lactate assay was not sensitive enough to detect the small quantity of product formed.…”

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA

“…Whether a direct and close interaction between Arg11 and Cys18 of wild-type β1 exists remains to be elucidated. The side chain of free cysteine has a pK a value of 8-9 (37) but it can be significantly lower in some protein environments, especially near the N termini of α-helices, so that the side chain may bear a partial negative charge (38,39). The side chain of Cys18, located near the N-terminal end of TM1 in β1, could be ionized (thionate -) and may interact directly with the positively charged side chain of Arg11.…”

A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast

“…FoldX is an empirical energy function that employs an all-atom representation of the protein and it has been tested on a dataset of more than 1000 sequence variants from more than 20 different proteins. FoldX calculations were carried out for each of the monomer structures included in the PDB entry 4QYT (33) to assess the reproducibility of the results, and the average is reported here. The RepairPDB function of FoldX was first applied to the wild type structures, during which we also replaced the oxidized (cysteine-sulfinate) Cys-111 present in the crystal structure with its standard thiol form.…”

“…The RepairPDB function of FoldX was first applied to the wild type structures, during which we also replaced the oxidized (cysteine-sulfinate) Cys-111 present in the crystal structure with its standard thiol form. The known high structural similarity between the reduced and oxidized forms of the human protein (33) suggests that this is a valid approach.…”