期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：7960-7965
DOI：10.1073/pnas.1504376112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThioredoxin (Trx) is universally conserved thiol-oxidoreductase that regulates numerous cellular pathways under thiol-based redox control, and its activity is often upregulated in malignant cancer cells. Despite its central importance, the mechanism by which Trx recognizes its target proteins remains unknown. Herein, we address this longstanding question by investigating the noncovalent forces involved in Trx-target interactions. Using a combination of biochemical and quantitative biophysical methods, we identify favorable entropy as the major force in molecular recognition that drives target specificity. These findings, and others reported herein, afford considerable new insight into Trx-target recognition, which is critical to understanding its function in normal metabolism and represents a fundamental step toward the development of new pharmacological strategies to address redox-related disorders. Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx-protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx-target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein-protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx-target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition.
