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The ionization of cysteine to form a thiolate anion greatly increases the nucleophilicity of this residue and is critical for the functions of many proteins that contain cysteine in their active sites. The unperturbed solution pKa value of the cysteine thiol is 8–9, which is higher than the pH of the cytosol. Consequently, proteins whose functions require cysteine thiolates must decrease the thiol pKa value in order to render these cysteine residues reactive. The best established structural mechanism for depressing cysteine pKa values is the donation of hydrogen bonds to the thiol(ate) . Electrostatic stabilization of the thiolate using proximal cationic groups such as lysine, arginine or protonated histidine side chains can also contribute , although this is thought to be of secondary importance in most systems due to the flexibility of these side chains [3, 4]. Despite well-understood general physical principles of cysteine pKa depression, the contribution of other protein structural features to cysteine ionization is more poorly understood.
Backbone peptide bonds could play an important role in cysteine pKa depression, as these abundant groups have a permanent dipole moment and can also donate a hydrogen bond to the thiolate. Therefore, correctly oriented peptide groups can lower the pKa value of a cysteine, as has been observed in the thioredoxin family . In the case of an α-helix, the vector sum of the partially aligned peptide dipoles along the helical axis has been proposed to result in a cumulative helical ‘macrodipole’ moment, with partial positive charge (~ +0.5e) at the N-terminus and partial negative charge (~ −0.5e) at the C-terminus [6, 7]. The α-helix macrodipole has been suggested as the explanation for pKa perturbation of residues near helical termini , particularly the frequent occurrence of reactive cysteine residues near the N-termini of α-helices . However, the magnitude and relevance of this effect have been called into question by computational work indicating that donation of hydrogen bonds by amide hydrogen atoms, rather than an electrostatic helix macrodipolar effect, is primarily responsible for the lowered pKa values of residues at the N-termini of helices [4, 10]. Furthermore, solvent exposure of one or both ends of the helix can substantially reduce its effective macrodipole moment, although well-shielded helical termini can accumulate significant partial charges .
Experimentally addressing the influence of peptide groups on cysteine pKa values in proteins has been hampered by the difficulty of creating mutations that alter peptide dipoles or protein backbone hydrogen bonds but do not perturb other aspects of the protein structure. Unlike simple amino acid substitutions that can be used to alter the charge or the hydrogen bonding potential of side chains, no comparable experimental strategy exists for easily modifying peptide groups in proteins. Despite these difficulties, a detailed experimental study of the helical macrodipole effect in sperm whale myoglobin has been performed, suggesting that both peptide hydrogen bonding and the helical macrodipole effect contribute to lowering cysteine pKa values . Other experimental studies, however, have found little support for a contribution from the helical macrodipole, even when local interactions with peptides are thought to stabilize charged groups .
The DJ-1 superfamily is a functionally diverse collection of proteins containing a highly conserved cysteine residue that can function as a catalytic nucleophile [14-16] or a potential redox sensor  in various members. This cysteine residue is functionally critical in most characterized DJ-1 superfamily proteins, although rare exceptions that lack the cysteine exist . Human DJ-1 is a disease-associated protein with multiple proposed functions in cytoprotection and mitochondrial function [19, 20]. The oxidation-sensitive cysteine (Cys106) in human DJ-1 has a depressed pKa value of 5.4 due, in part, to an unusual hydrogen bond formed between the Cys106 thiolate and a protonated glutamic acid side chain (Glu18) . However, mutagenesis has shown that this hydrogen bond accounts for only ~ 1 unit of Cys106 pKa depression, with the remaining ~ 2 units unexplained . The reactive cysteine residue is located at the N-terminus of an α-helix, suggesting that hydrogen bonding or dipolar contributions from the helix may contribute to its low pKa value. Problematically, an empirically based computational method fails to accurately predict either the direction or the magnitude of the reactive cysteine pKa perturbation in DJ-1 , complicating the computational analysis of the structural determinants of cysteine reactivity in this superfamily. Because the DJ-1 superfamily is a varied group of proteins with a conserved reactive cysteine residue, it is an ideal system in which to investigate the significance of various structural contributions to cysteine pKa modulation.
We have investigated the contribution of peptide groups to the lowering of cysteine pKa values using a combination of structural and biochemical methods applied to a DJ-1 homologue from the fission yeast Schizosaccharomyces pombe (SPAC22E12.03c; SpDJ-1 hereafter). The pKa value of the reactive cysteine (Cys111) in SpDJ-1 is ~ 1 unit lower than that of the highly homologous human protein due to subtle changes in the dipolar environment of the Cys111 thiol resulting from a rare Pro/Thr substitution near the N-terminal region of the α-helix that contains Cys111. Site-directed mutation of Thr114 in SpDJ-1 indicates that hydrogen bond donation by the threonine side chain to the proximal peptide depresses the pKa of Cys111, suggesting that the interplay of hydrogen bonding and local dipolar effects can significantly modulate the pKa of reactive cysteine residues. In contrast, the helical macrodipole appears to exert little influence on the pKa of Cys111.
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- Materials and methods
The contribution of peptide groups to protein electrostatics has long been appreciated; however, their specific contribution to the pKa values of ionizable groups is disputed [9, 10, 12, 29, 30]. Peptide groups can influence cysteine thiol pKa values either by electrostatic interaction with the partial charges on peptide atoms or through hydrogen bonding interactions, which are restricted to shorter scales (3–5 Å) and have partially covalent character. In the dipolar approximation, the peptide group's electrostatic field possesses a directional dependence that is described by its dipole moment, ~ 3.5 D. Consequently, electrostatic interactions between peptides and thiolates have favored geometries that are similar to those expected for hydrogen bonded interactions. We note that the dipolar approximation requires that the distance between the charges in the dipole be infinitesimally small compared with the distance between the dipole and other interacting groups, which is not strictly valid on hydrogen bond length scales. However, if the dipole approximation is applied to hydrogen bonds involving peptide groups, then dipole-charge interactions could contribute to the strength of hydrogen bonds between peptides and thiolates. Due to these considerations, formally separating dipolar and hydrogen bond contributions to thiolate stabilization on shorter length scales (3–5 Å) is not straightforward.
The critical distinction between dipolar and hydrogen bonded contributions to thiolate stabilization occurs on longer length scales (d > 5 Å), where hydrogen bonding is no longer possible but electrostatic interactions persist. The observation that reactive cysteine residues are frequently located near the N-termini of α-helices has led to the suggestion that a cumulative helical macrodipole [6, 7], comprising the vector sum of the individual peptide dipoles aligned along the helical axis, may electrostatically stabilize thiol ionization [9, 30]. However, some computational studies have suggested that only peptide dipoles in the first turn of the α-helix contribute significantly to pKa depression  and that hydrogen bonds between the thiol(ate) and the peptide backbone may be more significant contributors to the helical effect [1, 3, 10]. Other computational studies, however, find more evidence for a macrodipolar effect . Performing experiments capable of directly testing these hypotheses has been stymied by the inherent problem of altering peptide moieties, which are fundamental to protein structure and thus cannot be changed using standard mutagenesis approaches.
In the light of these limitations, SpDJ-1 provides an ideal system to study the impact of dipolar contributions to cysteine pKa values, as it contains both a reactive cysteine residue (Cys111) at the N-terminus of an α-helix and a hydrogen bond between an amino acid side chain (Thr114) and a backbone peptide group that is collinear with the helical axis. Therefore, we were able to alter this hydrogen bond by standard site-directed mutagenesis of Thr114 and then study the impact of the lost hydrogen bond–peptide interaction on the thiol pKa value of Cys111. Our findings are broadly consistent with the proposal that only the first few amino acids of an α-helix contribute significantly to modulating cysteine pKa values . The helix containing Cys111 is significantly solvent exposed at both termini, suggesting that solvent reaction field screening probably minimizes any helical macrodipolar effect . Furthermore, there was little difference between the Cys111 pKa value for a proline or valine substitution at the i + 3 position, despite the fact that these amino acids have different peptide dipole moments, indicating that the helix macrodipole is not a significant contributor to the low pKa of Cys111.
Despite the absence of strong evidence for a helical macrodipolar effect, a surprising result of our study is that a hydrogen bond donated to a nearby peptide can have an appreciable effect on thiol ionization even though the affected hydrogen bond is not made directly with the thiolate and does not alter the orientation of the peptide group. We found that side-chain hydrogen bonding between the hydroxyl group of Thr114 and the carbonyl oxygen of Cys111 depresses the pKa of the thiol by ~ 0.6 units, which is a considerable decrease for an indirect interaction. For comparison, the established direct hydrogen bond between the protonated Glu18 side chain and the thiol(ate) of Cys106 of human DJ-1 decreases the Cys106 pKa value by 1.0 unit . As there are no other observed structural changes that can explain the elevated Cys111 pKa value in T114V SpDJ-1, we propose that this is a bona fide dipolar effect, supporting the view that the alignment of dipoles proximal to the thiol can significantly influence its ionization. In the case of SpDJ-1, we propose that the hydrogen bond between Thr114 and the Cys111-Ala112 peptide results in partial charge cancellation of the peptide carbonyl oxygen and the Hγ atom of Thr114 (Fig. 5B). This partial charge cancellation effect at the peptide carbonyl oxygen atom may enhance the strength of the direct hydrogen bond between the amide hydrogen and the thiol(ate) (Fig. 5B), thereby resulting in a more substantial depression of Cys111 pKa than expected for a purely electrostatic phenomenon involving partial charges.
Although our experiments are confined to a single example from the DJ-1 superfamily, there is strong evidence that dipolar enhancement of thiolate formation is a general phenomenon. For example, local peptide dipolar interactions have been proposed to contribute to the low catalytic cysteine pKa values in protein tyrosine phosphatase 1B and the RNA triphosphatase domain of mRNA capping enzyme . Interestingly, the same study  suggested that the threonine side chain has an under-appreciated role in modulating cysteine pKa values in various systems, which is strongly supported by our results. Therefore, we propose that dipolar interactions, particularly when combined with hydrogen bonding, are a potentially common mechanism by which peptide groups can increase the reactivity of functionally important cysteine residues.