Structural characterization of the thioredoxin-binding domain of protein kinase ASK1 and its interaction with thioredoxin

Salome Kylarova^1,2, Dalibor Kosek^1,2, Katarina Psenakova¹, Lenka Rezabkova^1,2, Petr Herman³, Jaroslav Vecer³, Veronika Obsilova², Tomas Obsil^1,2

¹Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
²Institute of Physiology, Academy of Sciences of Czech Republic, 14220 Prague, Czech Rep.
³Faculty of mathematics and physics, Charles University in Prague, 12116 Prague, Czech Rep.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-Jun N-terminal kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress, endoplasmic reticulum stress, and calcium ion influx. The function of ASK1 is associated with the activation of apoptosis in various cells and plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration and cardiovascular diseases. The kinase activity of ASK1 is regulated by many factors, including binding of thioredoxin (Trx) and the 14-3-3 protein that both function as inhibitors of ASK1 [1]. However, the mechanisms by which these binding interactions inhibit ASK1 are still unclear.

To better understand the role of Trx binding in the inhibition of ASK1, we prepared the isolated Trx-binding region of ASK1 (ASK1-TBD), performed its structural and biophysical characterization and studied its interaction with Trx under both reducing and oxidative conditions. Data obtained from analytical ultracentrifugation, time-resolved fluorescence anisotropy measurements, circular dichroism and small-angle X-ray scattering suggest that: (1) ASK1-TBD is a compact monomeric and rigid domain that under reducing conditions forms with Trx a stable and well defined complex with 1:1 molar stoichiometry; (2) the structural integrity of the catalytic Trp-Cys-Gly-Pro-Cys motif of Trx is essential for its binding to ASK1-TBD; (3) Trx interacts with the region of ASK1-TBD located in the vicinity of Cys250; and (4) Trx binding does not induce significant structural change of ASK1-TBD. In addition, it seems that the interaction between ASK1-TBD and Trx does not involve the disulfide bond formation, as has been suggested in the literature [2,3].

1. Saitoh, M.; Nishitoh, H.; Fujii, M.; Takeda, K.; Tobiume, K.; Sawada, Y.; Kawabata, M.; Miyazono, K.; Ichijo, H.: EMBO J. 17, 2596 (1998).

2. Nadeau, P. J.; Charette, S. J.; Toledano, M. B.; Landry, J.: Mol. Biol. Cell 18, 3903 (2007).

3. Nadeau, P. J.; Charette, S. J.; Landry, J.: Mol. Biol. Cell 20, 3628 (2009).

This work was supported by the Czech Science Foundation (Project 14-10061S), Grant Agency of Charles University (Projects 793913 and 568912); and Academy of Sciences of the Czech Republic (Research Projects RVO: 67985823 of the Institute of Physiology).