Biophysical Characterization of Interaction between the Thioredoxin‐Binding Domain of Protein Kinase ASK1 and Reduced Thioredoxin

Abstract

Apoptosis signal-regulating kinase 1 (ASK1), a MAP3K, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) through unknown mechanism. In this work, we performed a detailed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1 [1]. The analytical ultracentrifugation revealed that ASK1-TBD is a monomeric domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. Residues Cys32 and Cys35 as well as Trp31 from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp31 being directly involved in binding interaction as suggested by time-resolved tryptophan fluorescence. Small angle X-ray scattering revealed a compact and slightly asymmetric shape of ASK1-TBD and suggested that reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change. Molecular modeling indicated that the TRX1 binding site is located close to the N-terminal end of a 50 residues long α-helix, which forms the C terminus of ASK1-TBD. Our results show that the isolated TRX1-binding domain of ASK1 is a valid model for studying the molecular mechanism of TRX1-dependent regulation of ASK1. This work was supported by Czech Science Foundation (Project 14-10061S).