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

Magnus S. Alphey, Mads Gabrielsen, Elena Micossi, Gordon A. Leonard, Sean M. McSweeney, Raimond B.G. Ravelli, Emmanuel Tetaud, Alan H. Fairlamb, Charles S. Bond, William N. Hunter

Research output: Contribution to journalArticlepeer-review

43 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)25919-25925
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number28
DOIs
Publication statusPublished - 11 Jul 2003

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