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 o f the disulfide bond that can be formed between the two cysteines at t heir active site sequence motif Cys(1)-Xaa(2)-Yaa(3)-Cys(4). This is b elieved 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 consi stent relationship between disulfide bond stability and Cys(1) thiol p K(a) value is found here for DsbA, thioredoxin, and the N-terminal thi oredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pK(a) value of 4.5. This thiolate anion is stabilize d by 5.7 kcal/mol in the dithiol form, giving rise to the correspondin g instability of the disulfide bond and the oxidizing properties of PD I 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 int eraction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial posit ive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to changes of surrounding residues.