Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei: photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function

J Biol Chem. 2003 Jul 11;278(28):25919-25. doi: 10.1074/jbc.M301526200. Epub 2003 Apr 21.


Tryparedoxin (TryX) is a member of the thioredoxin (TrX) fold family involved in the regulation of oxidative stress in parasitic trypanosomatids. Like TrX, TryX carries a characteristic Trp-Cys-Xaa-Xaa-Cys motif, which positions a redox-active disulfide underneath a tryptophan lid. We report the structure of a Crithidia fasciculata tryparedoxin isoform (CfTryX2) in two crystal forms and compare them with structures determined previously. Efforts to chemically generate crystals of reduced TryX1 were unsuccessful, and we carried out a novel experiment to break the redox-active disulfide, formed between Cys-40 and Cys-43, utilizing the intense x-radiation from a third generation synchrotron undulator beamline. A time course study of the S-S bond cleavage is reported with the structure of a TryX1 C43A mutant as the control. When freed from the constraints of a disulfide link to Cys-43, Cys-40 pivots to become slightly more solvent-accessible. In addition, we have determined the structure of Trypanosoma brucei TryX, which, influenced by the molecular packing in the crystal lattice, displays a significantly different orientation of the active site tryptophan lid. This structural change may be of functional significance when TryX interacts with tryparedoxin peroxidase, the final protein in the trypanothione-dependent peroxidase pathway. Comparisons with chloroplast TrX and its substrate fructose 1,6-bisphosphate phosphatase suggest that this movement may represent a general feature of redox regulation in the trypanothione and thioredoxin peroxidase pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Chloroplasts / enzymology
  • Cloning, Molecular
  • Crithidia fasciculata / metabolism*
  • Cysteine / chemistry
  • Disulfides
  • Escherichia coli / metabolism
  • Fructose-Bisphosphatase / metabolism
  • Glutathione / analogs & derivatives*
  • Glutathione / chemistry
  • Glutathione / metabolism
  • Light
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Neoplasm Proteins*
  • Oxidation-Reduction*
  • Peroxidases / chemistry
  • Peroxidases / metabolism
  • Peroxiredoxins
  • Protein Conformation
  • Protein Isoforms
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Spermidine / analogs & derivatives*
  • Spermidine / chemistry
  • Spermidine / metabolism
  • Synchrotrons
  • Thioredoxins / chemistry*
  • Time Factors
  • Trypanosoma brucei brucei / metabolism*
  • Tryptophan / chemistry


  • Disulfides
  • Neoplasm Proteins
  • Protein Isoforms
  • Recombinant Proteins
  • tryparedoxin
  • Thioredoxins
  • Tryptophan
  • trypanothione
  • Peroxidases
  • Peroxiredoxins
  • Fructose-Bisphosphatase
  • Glutathione
  • Cysteine
  • Spermidine

Associated data

  • PDB/1O73
  • PDB/1O7U
  • PDB/1O85
  • PDB/1O8W
  • PDB/1O8X
  • PDB/1OC8
  • PDB/1OC9