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Biophysical and structural characterization of the thioredoxin-binding domain of protein kinase ASK1 and its
Dalibor Kosek1, Salome Kylarova1, Katarina Psenakova1
1From the Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, 12843 Prague, the Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague, and.
Abstract:
Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.
Insights
Thioredoxin 1 (TRX1) binding to apoptosis signal-regulating kinase 1 (ASK1) is crucial for disease pathways. Structural analysis reveals a stable 1:1 complex, highlighting key interactions without disulfide bonds.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Medicine
Background:
- Apoptosis signal-regulating kinase 1 (ASK1) is implicated in various diseases.
- Thioredoxin 1 (TRX1) regulates ASK1 activity, but the mechanism remains structurally undefined.
Purpose of the Study:
- To elucidate the structural basis of TRX1 regulation of ASK1.
- To characterize the ASK1-TBD and TRX1 complex formation.
Main Methods:
- Biophysical characterization
- Structural analysis (Small-angle X-ray Scattering)
- Time-resolved fluorescence spectroscopy
Main Results:
- ASK1-TBD is a monomeric, rigid domain.
- A stable 1:1 complex forms between ASK1-TBD and reduced TRX1.
- Trp(31) of TRX1 is critical for binding, with no disulfide bonds formed.
Conclusions:
- The study provides the first structural insights into the ASK1-TRX1 interaction.
- Understanding this complex is vital for developing therapeutics targeting ASK1-mediated diseases.
