Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase

Proteins with the thioredoxin fold have widely differing stabilities of the disulfide bond that can be formed between the two cysteines at their active site sequence motif Cys(1)-Xaa(2)-Yaa(3)-Cys(4). This is believed to be regulated not by varying the disulfide bond itself, but by modulating the stability of the dithiol form of the protein through interactions with the ionized form of the Cys(1) thiol group. A consistent relationship between disulfide bond stability and Cys(1) thiol pK(a) value is found here for DsbA, thioredoxin, and the N-terminal thioredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pK(a) value of 4.5. This thiolate anion is stabilized by 5.7 kcal/mol in the dithiol form, giving rise to the corresponding instability of the disulfide bond and the oxidizing properties of PDI a. Electrostatic interactions in the active site of the PDI a-domain have been characterized in order to understand the physical basis of this stabilization. Linkage with the ionization of the imidazole group of His(3) in the active site demonstrates that this charge-charge interaction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial positive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to changes of surrounding residues.