br Interaction between ASK and TRX Thioredoxins are small kD
Interaction between ASK1 and TRX Thioredoxins are small (∼12kDa) dithiol oxidoreductases ubiquitously expressed in species ranging from plants to archaea and mammals. They perform various biological functions, such as reducing protein disulfide bonds, supplying reducing equivalents to redox TNKS 22 receptor and regulating transcription factors and proteins, either by direct reduction of their cysteine groups or through different mechanisms (reviewed by (Hanschmann et al., 2013)). TRXs have been shown to regulate the function of several proteins, including NF-κB (Qin et al., 1995), the apoptosis inducing factor (Shelar et al., 2015), methionine sulfoxide reductases (Kumar et al., 2002) or Jab-1 (Hwang et al., 2004). The TRX molecule consists of a five-stranded β-pleated sheet that forms a hydrophobic core surrounded by four α-helices, and the highly conserved redox catalytic motif 31WCGPC35 is located between the second β-strand and the second α-helix (Fig. 1D) (Holmgren et al., 1975). The two cysteine residues Cys32 and Cys35 (in human TRX1) within the catalytic motif are responsible for the redox activity of TRX. ASK1-TBD, whose three-dimensional structure is still unknown, is located between residues 46 and 277 in the N-terminal part of ASK1 (Fig. 1) (Fujino et al., 2007). The biophysical characterization of ASK1-TBD showed that this domain is a monomeric protein with a compact shape that forms an equimolar complex with reduced TRX1 with a KD of 0.3±0.1μM (Kosek et al., 2014). Furthermore, this study indicated that ASK1-TBD interacts with TRX1 through the large binding interface without inducing any dramatic conformational change and that the interaction is not based on intermolecular disulfide bond formation. However, the molecular basis of TRX1 dissociation from the signalosome upon oxidative stress is still not fully understood. Studies have shown that the redox-inactive form (TRX1-CS mutant with both catalytic Cys residues replaced by Ser) and the oxidized form of TRX1 do not bind to ASK1 (Fujino et al., 2007, Liu and Min, 2002, Saitoh et al., 1998, Zhang et al., 2004). This suggested that the catalytic cysteine residues are involved in the interaction of TRX1 with ASK1 and that the formation of an intramolecular disulfide bond between these cysteines upon oxidation causes TRX1 dissociation from ASK1, regardless of the thiol-reductase activity of TRX1. These findings were recently corroborated by binding experiments using the isolated ASK1-TBD, which had significantly lower binding affinity for oxidized TRX1 and negligible binding affinity for the TRX1-CS mutant when compared with its binding affinity for reduced TRX1. Furthermore, of the two catalytic cysteines, the Cys32 residue is essential for the high-affinity binding of TRX1 to ASK1-TBD under reducing conditions. In addition, the preceding tryptophan residue Trp31 also directly participates in the formation of the complex, thus confirming that the catalytic site of TRX1 is involved in TRX1 binding to ASK1 (Kosek et al., 2014, Kylarova et al., 2016). The ASK1-TBD sequence has seven conserved Cys residues, thereby suggesting that oxidative stress may induce intramolecular disulfide bond formation, which may affect the structure of ASK1-TBD and its interaction with TRX1. Indeed, the oxidation of ASK1-TBD was shown to induce the formation of several intramolecular disulfide bonds, predominantly Cys185–Cys200 and Cys200–Cys206 and, to a lesser extent, Cys225/Cys226–Cys250 concurrently to conformational changes in ASK1-TBD (Kylarova et al., 2016). Whether the formation of these disulfides directly affects TRX1 binding affinity is still unclear, although the Cys200 residue has been located at the TRX binding surface of ASK1-TBD and mutating the Cys250 residue significantly affects the interaction between ASK1 and TRX1, thus suggesting that this residue is involved in TRX1 binding to and/or its importance for the integrity of ASK1-TBD (Kosek et al., 2014, Kylarova et al., 2016, Nadeau et al., 2009, Zhang et al., 2004). Therefore, ASK1-TBD oxidation most likely contributes to the dissociation of the ASK1:TRX1 complex.