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, 112 (26), 7960-5

A Universal Entropy-Driven Mechanism for Thioredoxin-Target Recognition

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A Universal Entropy-Driven Mechanism for Thioredoxin-Target Recognition

Prakash B Palde et al. Proc Natl Acad Sci U S A.

Abstract

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.

Keywords: entropy; oxidative stress; protein–protein interactions; redox regulation; thioredoxin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Physiological function and pathological significance of Trx. (A) Mechanism for general disulfide reduction catalyzed by Trx. (B) Trx–protein interactions listed in conjunction with their physiological function and pathological implications. Trx reduces RNR with essential function in DNA synthesis and repair (5), MSrs linked to protein repair and aging (6) and Prxs, implicated in multiple redox-related disorders (7). Human Trx (Trx1) activates transcription factor Ref-1 critical in cell growth and survival (6) and inhibits proapoptotic protein ASK1 (9). Extracellular Trx1 reduces HIV envelop protein gp120 (10) and TG2, an enzyme implicated in celiac disease (11). Bacterial Trx reduces SRs with a crucial role in sulfur assimilation (8).
Fig. 2.
Fig. 2.
PAPR catalytic mechanism. The sulfur atom of PAPS undergoes nucleophilic attack by C239 of PAPR resulting in formation of E-Cys-S-SO3 intermediate, followed by release of SO32- vis-à-vis Trx-mediated thiol-disulfide exchange (8).
Fig. 3.
Fig. 3.
ITC determination of Trx binding to reduced and oxidized PAPR. (A) Pictorial representation of conformational change in PAPR going from reduced to oxidized form after the addition of saturating PAPS. (B) (Upper) Time-dependent deflection of heat signal after each injection of Trx in the microcalorimetric cell containing 70 µM/monomer of homodimeric PAPR in oxidized or reduced form. Reduced PAPR titrated with 2 mM C32A Trx (green). Oxidized PAPR titrated with 0.75 mM C32A Trx (black). ITC experiments were performed in 50 mM bis-Tris propane buffer containing 0.02% Brij-35 (pH 7.4) at 25 °C. (Lower) Integrated calorimetric data corrected for Trx dilution fit to independent binding model (NanoAnalyze software) to obtain binding and thermodynamic parameters. (C) Graphical representation of thermodynamic parameters for the C32ATrx•oxidized PAPR interaction.
Fig. 4.
Fig. 4.
Mapping molecular determinants of binding enthalpy. Crystal structure of Trx-EcPAPR covalent complex (PDB ID 208V) showing interactions between Trx (green) and PAPR (blue) at two adjacent sites (17). (A) First 800-Å binding hotspot burying PAPR residues 188–199 (α-helix 7) and 202–212 (ω-loop). Based on distance measurements, the noncovalent interactions predicted between PAPR (nonitalicized) and Trx1 residues (italicized) include: H-bonding between W205 (PAPR) and E30 (Trx); salt bridge interactions between D206 (PAPR) and K36–E30 (Trx); π-stacking interactions between Y191 (PAPR) and W31 (Trx); hydrophobic contacts of L210 (PAPR) with M37 and G33 (Trx1). (B) Second 783-Å binding hotspot burying PAPR residues 235–244 (C-terminal tail) including the catalytic cysteine, C239. An interdigitated network of salt bridges are predicted between R237, E238, and E243 (PAPR) and R73 (Trx). (C) Plot of difference in the enthalpy change (ΔHmutant − ΔHWT) obtained from two independent ITC measurements for each PAPR variant.
Fig. 5.
Fig. 5.
A proposed model for recognition of targets with disulfide bonds by Trx. Formation of disulfide bond in target proteins after oxidation induces conformational change, lowering the entropy. Trx preferentially binds to this low-entropy conformer in an interaction driven by favorable entropy, subsequently reducing it to the high-entropy native form.

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