NMR Studies of the Interaction of Tryparedoxin with Redox-Inactive Substrate Homologues

• Published in: Biochemistry (Easton) 2003-12, Vol.42 (50), p.14720-14728
• Main Authors: Krumme, Dirk, Budde, Heike, Hecht, Hans-Jürgen, Menge, Ulrich, Ohlenschläger, Oliver, Ross, Anton, Wissing, Josef, Wray, Victor, Flohé, Leopold
• Format: Article
• Language: English
• Subjects: Animals; Computer Simulation; Crithidia; Crystallography, X-Ray; Enzyme Activation; Glutathione - analogs & derivatives; Glutathione - chemistry; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Oxidoreductases - antagonists & inhibitors; Oxidoreductases - chemistry; Protozoan Proteins - chemistry; Protozoan Proteins - metabolism; Solutions; Spermidine - analogs & derivatives; Spermidine - chemistry; Substrate Specificity; Thioredoxins - antagonists & inhibitors; Thioredoxins - chemistry; Thioredoxins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi030112d

Tryparedoxins (TXNs) are trypanothione-dependent peroxiredoxin oxidoreductases involved in hydroperoxide detoxification that have been shown to determine virulence in trypanosomatids. The structure of 15N,13C-doubly-labeled, C-terminally-His-tagged tryparedoxin 1 from Crithidia fasciculata (Cf TXN1) was elucidated by three-dimensional NMR spectroscopy. Global folding was found to be similar to the crystal structure, but regions near the active site, especially the onset of helix α1 with the redox-active Cys 43 and helix α2 relevant to substrate binding, were less well defined in solution. The redox-inactive inhibitory substrate analogue N 1,N 8-bis(ophthalmyl)spermidine was used to study the substrate/TXN interaction by two-dimensional 1H,15N NMR spectroscopy. The NMR data complemented by molecular modeling revealed several alternative modes of ligand binding. The results confirm and extend the concept of TXN action and specificity derived from X-ray analysis and site-directed mutagenesis and thus improve the rational basis for inhibitor design.