The solution structure of human thioredoxin complexed with its target from Ref-1 reveals peptide chain reversal


AbstractBackground: Human thioredoxin (hTRX) is a 12 kDa cellular redox protein that has been shown to play an important role in the activation of a number of transcriptional and translational regulators via a thiol-redox mechanism. This activity may be direct or indirect via another redox protein known as Ref-1. The structure of a complex of hTRX with a peptide comprising its target from the transcription factor NFκB has previously been solved. To further extend our knowledge of the recognition by and interaction of hTRX with its various targets, we have studied a complex between hTRX and a Ref-1 peptide. This complex represents a kinetically stable mixed disulfide intermediate along the reaction 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 active-site loop (residues 32–36), helix 3, β strands 3 and 5, and the loop between β strands 3 and 4. The complex is stabilized by numerous hydrogen-bonding and hydrophobic interactions that involve residues 61–69 of the peptide and confer substrate specificity.Conclusion The orientation of the Ref-1 peptide in the hTRX–Ref-1 complex is opposite to that found in the previously solved complex of hTRX with the target peptide from the transcription factor NFκB. Orientation is determined by three discriminating interactions involving the nature of the residues at the P−2, P−4 and P−5 binding positions. (P0 defines the active cysteine of the peptide, Cys65 for Ref-1 and Cys62 for NFκB. Positive and negative numbers indicate residues N-terminal and C-terminal to this residue, respectively, and vice versa for NFκB as it binds in the opposite orientation.) The environment surrounding 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, despite the opposing orientation of the peptide chains. This versatility in substrate recognition permits hTRX to act as a wide-ranging redox regulator for the cell

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