Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei

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 solventaccessible. 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.

Determination of a novel structure by a combination of long-wavelength sulfur phasing and radiation-damage-induced phasing

“…1) and in Table 4. Photoreduction of a redox-active disul®de bridge by synchrotron radiation has also been observed recently for tryparedoxins (Alphey et al, 2003). In that case, the concomitant conformational change was even implicated in the function of the protein.…”

Structural Basis for a Distinct Catalytic Mechanism in Trypanosoma brucei Tryparedoxin Peroxidase

“…Constant reducing conditions were verified by subjecting an aliquot of the solution to thiol determination with 5,5-dithiobis (2-nitrobenzoate 1O73 (36)) were used to model the complex structure. The docking was performed using HADDOCK 2.0 (37,38).…”

“…The starting structures were the lowest energy NMR structure of the oxidized Px III and the x-ray structure of oxidized T. brucei Tpx (PDB code 1O73 (36)). In the absence of a structure of reduced Tpx, this approach seems justified because reduction of Tpx from C. fasciculata has only minor effects (36,44). The expected structural change between oxidized and reduced Tpx was taken into account by defining the active site WCPPC motif fully flexible during the second stage of the docking.…”

Spatial distribution of radiation damage to crystalline proteins at 25–300 K

“…When specific damage occurs at a protein active site (Burmeister, 2000;Weik et al, 2000;Weik, Ravelli et al, 2001;Matsui et al, 2002;Alphey et al, 2003;Fioravanti et al, 2007), it can complicate biological interpretation of the structure.…”