Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei: photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function.
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.
Alphey
Magnus S
MS
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Gabrielsen
Mads
M
Micossi
Elena
E
Leonard
Gordon A
GA
McSweeney
Sean M
SM
Ravelli
Raimond B G
RB
Tetaud
Emmanuel
E
Fairlamb
Alan H
AH
Bond
Charles S
CS
Hunter
William N
WN
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D016831:Crithidia fasciculata / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism; D054464:Peroxiredoxins; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013095:Spermidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017356:Synchrotrons; D013879:Thioredoxins / Q000737:chemistry*; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
D004220:Disulfides; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C114110:tryparedoxin; D013879:Thioredoxins; D014364:Tryptophan; C044809:trypanothione; D010544:Peroxidases; D054464:Peroxiredoxins; D006597:Fructose-Bisphosphatase; D005978:Glutathione; D003545:Cysteine; D013095:Spermidine