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

TY - JOUR

T1 - Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei

T2 - Photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function

AU - Alphey, Magnus S.

AU - Gabrielsen, Mads

AU - Micossi, Elena

AU - Leonard, Gordon A.

AU - McSweeney, Sean M.

AU - Ravelli, Raimond B.G.

AU - Tetaud, Emmanuel

AU - Fairlamb, Alan H.

AU - Bond, Charles S.

AU - Hunter, William N.

PY - 2003/7/11

Y1 - 2003/7/11

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/0037830694

U2 - 10.1074/jbc.M301526200

DO - 10.1074/jbc.M301526200

M3 - Article

C2 - 12707277

AN - SCOPUS:0037830694

SN - 0021-9258

VL - 278

SP - 25919

EP - 25925

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 28

ER -