Crystal structures of reduced, oxidized, and mutated human thioredoxins: Evidence for a regulatory homodimer

Background: Human thioredoxin reduces the disulfide bonds of numerous proteins in vitro, and can activate transcription factors such as NFκB in vivo. Thioredoxin can also act as a growth factor, and is overexpressed and secreted in certain tumor cells. Results: Crystal structures were determined for reduced and oxidized wild type human thioredoxm (at 1.7 and 2.1 Å nominal resolution, respectively), and for reduced mutant proteins Cys73→Ser and Cys32→Ser/Cys35→Ser (at 1.65 and 1.8 Å, respectively), Surprisingly, thioedoxin is dimeric in all four structures; the dimer is linked through a disulfide bond between Cys73 of each monomer, except in Cys73→Ser where a hydrogen bond occurs. The thioredoxin active site is blocked by dimer formation. Conformational changes in the active site and dimer interface accompany oxidation of the active-site cysteines, Cys32 and Cys35.ts Conclusions: It has been suggested that a reduced K_a in the first cysteine (Cys32 in human thioredoxin) of the active-site sequence is important for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pK_a of Cys32 and this pK_a depression probably results in increased nucleophilicity of the Cys32 thiolate group. This nucleophilicity, in turn, is thought to be necessary for the role of thioredoxin in disufide-bond reduction. The physiological role, if any of thioredoxin dimer formation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidative stress.