Structural analysis of the ASK1-CD:14-3-3 complex by small angle X-ray scattering

Olivia Petrvalska1,2,  Dalibor Kosek1,2, Tomas Obsil1,2

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


Apoptosis signal-regulating kinase 1(ASK1) is a Ser/Thr protein kinase that plays an essential role in stress and immune responses. Its increased activity has been linked to the development of several diseases such as cancer, cardiovascular and neurodegenerative diseases. The whole enzyme consists of almost 1400 amino acids. Catalytically active is only a kinase domain located approximately in the middle of the molecule and this is the only domain that has been crystallized so far [1]. 14-3-3 form a family of evolutionary conserved regulatory proteins that participate in a variety of intracellular processes through binding interactions with hundreds of diverse cellular proteins. In an inactive state ASK1 kinase is bound to the 14-3-3 protein, which is a negative regulator of ASK1 [2]. However, the mechanism whereby the 14-3-3 protein inhibits the kinase activity of ASK1 remains unknown.

To elucidate the mechanism of this inhibition, the catalytic domain of ASK1 (ASK1-CD) and the C-terminally truncated form of the 14-3-3 protein (14-3-3ΔC) were purified and a structural characterization of the ASK1-CD:14-3-3ΔC complex was performed using analytical ultracentrifugation and small-angle X-ray scattering. The analytical ultracentrifugation measurements revealed that ASK1-CD interacts with the 14-3-3ΔC protein in 2:2 stoichiometry with KD in micromolar range. Small angle X-ray scattering showed that molecular envelope of the complex is highly asymmetric suggesting that the 14-3-3ΔC dimer binds only one chain of ASK1 dimer.

1.         G. Bunkoczi, E. Salah, P. Filippakopoulos, O. Fedorov, S. Müller, F. Sobott, S.A. Parker, H. Zhang, W. Min, B.E. Turk, S. Knapp, Structural and functional characterization of the human protein kinase ASK1, Structure, 15 (2007) 1215–26.

2.         L. Zhang, J. Chen, H. Fu, Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins, Proc. Natl. Acad. Sci. U. S. A., 96 (1999) 8511–5.

This work was supported by the Czech Science Foundation project #14-10061S.