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 NF k appa B in vivo. Thioredoxin can also act as a growth factor, and is ov erexpressed and secreted in certain tumor cells. Results: Crystal stru ctures were determined for reduced and oxidized wild type human thiore doxin (at 1.7 and 2.1 Angstrom nominal resolution, respectively), and for reduced mutant proteins Cys73-->Ser and Cys32-->Ser/Cys35-->Ser (a t 1.65 and 1.8 Angstrom, respectively). Surprisingly, thioredoxin is d imeric in all four structures; the dimer is linked through a disulfide bond between Cys73 of each monomer, except in Cys73-->Ser where a hyd rogen bond occurs. The thioredoxin active site is blocked by dimer for mation. Conformational changes in the active site and dimer interface accompany oxidation of the active-site cysteines, Cys32 and Cys35. Con clusions: It has been suggested that a reduced pK(a) in the first cyst eine (Cys32 in human thioredoxin) of the active-site sequence is impor tant for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pK(a) o f Cys32 and this pK(a) depression probably results in increased nucleo philicity of the Cys32 thiolate group. This nucleophilicity, in turn, is thought to be necessary for the role of thioredoxin in disulfide-bo nd reduction. The physiological role, if any, of thioredoxin dimer for mation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidat ive stress.