THE SOLUTION STRUCTURE OF HUMAN THIOREDOXIN COMPLEXED WITH ITS TARGETFROM REF-1 REVEALS PEPTIDE-CHAIN REVERSAL

Background: Human thioredoxin (hTRX) is a 12 kDa cellular redox protei n that has been shown to play an important role in the activation of a number of transcriptional and translational regulators via a thiol-re dox mechanism. This activity may be direct or indirect via another red ox protein known as Ref-1. The structure of a complex of hTRX with a p eptide comprising its target from the transcription factor NF kappa B has previously been solved, To further extend our knowledge of the rec ognition by and interaction of hTRX with its various targets, we have studied a complex between hTRX and a Ref-1 peptide. This complex repre sents a kinetically stable mixed disulfide intermediate along the reac tion pathway. Results: Using multidimensional heteronuclear edited and filtered NMR spectroscopy, we have solved the solution structure of a complex between hTRX and a 13-residue peptide comprising residues 59- 71 of Ref-1. the Ref-1 peptide is located in a crescent-shaped groove on the surface of hTRX, the groove being formed by residues in the act ive-site loop (residues 32-36), helix 3, beta strands 3 and 5, and the loop between beta strands 3 and 4. The complex is stabilized by numer ous hydrogen-bonding and hydrophobic interactions that involve residue s 61-69 of the peptide and confer substrate specificity. Conclusions: The orientation of the Ref-1 peptide in the hTRX-Ref-1 complex is oppo site to that found in the previously solved complex of hTRX with the t arget peptide from the transcription factor NF kappa B. Orientation is determined by three discriminating interactions involving the nature of the residues at the P--2, P--4 and P--5 binding positions. (P-0 def ines the active cysteine of the peptide, Cys65 for Ref-1 and Cys62 for NF kappa B. Positive and negative numbers indicate residues N-termina l and C-terminal to this residue, respectively, and vice versa for NF kappa B as it binds in the opposite orientation.) The environment surr ounding the reactive Cys32 of hTRX, as well as the packing of the P-+3 to P--4 residues are essentially the same in the two complexes, despi te the opposing orientation of the peptide chains, This versatility in substrate recognition permits hTRX to act as a wide-ranging redox reg ulator for the cell.