Structural Characterization of the Apoptosis Signal‐regulating Kinase 1 (ASK1) Domain Responsible for Thioredoxin Binding and its Complex

Abstract

Apoptosis signal‐regulating kinase 1 (ASK1) is a mitogen‐activated protein kinase kinase kinase 5 implicated in p38 mitogen‐activated protein kinase and c‐Jun N‐terminal kinase signaling cascades. ASK1 responds to a wide range of signaling cues and is dysregulated in cancer, neurodegeneration and cardiovascular diseases. The catalytic activity of ASK1 is tightly controlled through oligomerization and binding of several cofactors, among which thioredoxin stands out as the most important ASK1 inhibitor. Interestingly, only the reduced form of thioredoxin inhibits ASK1 by direct binding to its N‐terminal domain and oxidation‐driven thioredoxin dissociation is the key event in oxidative stress‐mediated ASK1 activation. Despite extensive efforts, the molecular basis of this key cellular regulatory mechanism has remained unknown until now, primarily due to the dynamic properties of the ASK1 N‐terminal domain, which has prevented its structural characterization by X‐ray crystallography. Here, we report the structural characterization of the ASK1 domain responsible for thioredoxin binding and of the ASK1: thioredoxin complex using NMR spectroscopy and chemical cross‐linking, which provides the molecular basis for complex dissociation under oxidative stress conditions. Our data reveal that the N‐terminal domain of ASK1 adopts a fold resembling the thioredoxin structure while retaining substantial conformational plasticity at the thioredoxin‐binding interface. Although oxidative stress induces relatively moderate structural changes in thioredoxin, we show that these changes lead to a substantial conformational rearrangement of the thioredoxin‐binding interface on ASK1 driven by the formation of intramolecular disulfide bridges. Lastly, we demonstrate that the cysteine residue at the position 250 of ASK1 is the key element of this molecular switch.Support or Funding InformationThis work was supported by the Czech Science Foundation (Project 19‐00121S), the Grant Agency of the Charles University (Project 368216), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and partially also by the European Regional Development Fund; OP RDE; Project: ChemBioDrug (No. CZ.02.1.01/0.0/0.0/16_019/0000729).Structural model of the ASK1‐TBD:TRX complex calculated using the program HADDOCK with the NMR‐based structural model of ASK1‐TBD, and crystal structure of reduced TRX1 (PDB ID: 1ERT).Figure 1