SOLUTION STRUCTURE OF HUMAN THIOREDOXIN IN A MIXED DISULFIDE INTERMEDIATE COMPLEX WITH ITS TARGET PEPTIDE FROM THE TRANSCRIPTION FACTOR NF-KAPPA-B

Background: Human thioredoxin is a 12 kDa cellular redox protein that plays a key role in maintaining the redox environment of the cell. It has recently been shown to be responsible for activating the DNA-bindi ng properties of the cellular transcription factor, NF kappa B, by red ucing a disulfide bond involving Cys62 of the p50 subunit. Using multi dimensional heteronuclear-edited and heteronuclear-filtered NMR spectr oscopy, we have solved the solution structure of a complex of human th ioredoxin and a 13-residue peptide extending from residues 56-68 of p5 0, representing a kinetically stable mixed disulfide intermediate alon g the reaction pathway. Results: The NF kappa B peptide is located in a long boot-shaped cleft on the surface of human thioredoxin delineate d by the active-site loop, helices alpha 2, alpha 3 and alpha 4, and s trands beta 3 and beta 4. The peptide adopts a crescent-like conformat ion with a smooth 110 degrees bend centered around residue 60 which pe rmits it to follow the path of the cleft. Conclusions: In addition to the intermolecular disulfide bridge between Cys32 of human thioredoxin and Cys62 of the peptide, the complex is stabilized by numerous hydro gen-bonding, electrostatic and hydrophobic interactions which involve residues 57-65 of the NF kappa B peptide and confer substrate specific ity. These structural features permit one to suggest the specificity r equirements for human thioredoxin-catalyzed disulfide bond reduction o f proteins.