Structures of tryparedoxins revealing interaction with trypanothione

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin-type peroxidas es by the bis-glutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. Th e 3D-structures of the following tryparedoxins are presented: authentic try paredoxin1 of Crithidia fasciculata, CfTXN1; the his-tagged recombinant pro tein, CfTXN1 H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the ac tive site motif 40-WCPPCR-45 proved to be the only solvent-exposed redox ac tive residue in CfTXN2. in reduced TXNs, its nucleophilicity is increased b y a network of hydrogen bonds. In oxidised TXNs it can be attacked by the t hiol of the N-1-glutathionyl residue of trypanothione, as evidenced by the structure of N-1-glutathionylspermidine-derivatised CfTXN2H6-Cys44Ser. Mode lling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bo nd and Arg129 (128) to be involved in the binding of trypanothione to CfTXN 2 (CfTXN1). The model of TXN-substrate interaction is consistent with funct ional characteristics of known and newly designed muteins (CfTXN2H6-Arg129A sp and Glu73Arg) and the N-1-glutathionyl-spermidine binding in the CfTXN2H 6-Cys44Ser structure.