Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

Abstract

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.The thioredoxin fold is the core scaffold of numerous proteins that control disulfide redox activity in the cell (1-3). These redox proteins share very little sequence homology, but all of them incorporate the four-stranded ␤-sheet, three flanking ␣-helices, and the redox-active CXXC motif of the TRX 5 fold (Fig. 1A). The archetype of the family is thioredoxin (4), a disulfide reductase that maintains a reducing cytosolic environment. Other TRX fold redox proteins include the Dsb proteins (1), which regulate the formation of disulfide bonds in prokaryotes, and protein-disulfide isomerase (5), which catalyzes the oxidation and shuffling of disulfides in the endoplasmic reticulum of eukaryotic cells.This wide range of redox activities of TRX fold proteins is thought to be a consequence of modifications to the common scaffold, which result in different redox properties. Thus, the redox potential of Escherichia coli thioredoxin is very reducing, at Ϫ271 mV (6, 7), whereas that of the oxidizing periplasmic protein E. coli DsbA is Ϫ120 mV (8). Thioredoxin fold proteins that participate in a wide range of thiol disulfide exchange reactions, such as the eukaryotic protein-disulfide isomerases, have intermediate redox potentials (around Ϫ160 mV (9)).Thioredoxin-related proteins provide an attractive model for the study of how protein function is dictated by sequence and three-dimensional structure; this is because their functions are, in part, determined by their redox properties, which in turn, are easy to quantify. For example, mutations in thioredoxin that make its redox potential more oxidative complement null mutations in the oxidase DsbA (10, 11). A detailed understanding of how thioredoxin fold sequence affects redox properties provides an excellent opportunity to relate sequence and function. Previous work has focused on the role of the CXXC "redox rheostat" active site in determining the properties of thioredoxin-related proteins (3,12,13). Experiments that exchange the X-X dipeptide of one thiol-disulfide oxidoreductase with that of another generally result in an oxidoreductase with a redox potential partially shifted in the direction of the oxidoreductase protein that served as the source of the dipepti...

Dissecting Molecular Interactions Involved in Recognition of Target Disulfides by the Barley Thioredoxin System

“…21,36 The position of M88 HvTrxh2 is occupied by an isoleucine residue in EcTrx1 (Table 1), and a comprehensive mutational analysis, including determination of the structure of EcTrx1 I75T, demonstrated that this "cisPro minus 1 residue" is a general activity regulator of Trx fold proteins. 38 EcTrx1 I75P, however, was not generally more negatively affected than mutants with charged side chains (K, R, H, E, and D), and I75P had ∼10% residual activity toward insulin with either DTT or NTR as the reductant, which is similar to the results with HvTrxh2 M88P in the case of DTT but different from the results of the NTR recycling assay using DTNB as the final electron acceptor (Table 3). In the two different NTRindependent single-turnover BASI reduction assays, mutant M88P performed like A106P ( Figure S4 of the Supporting Information and Figure 7).…”

Sulfur Denitrosylation by an Engineered Trx-like DsbG Enzyme Identifies Nucleophilic Cysteine Hydrogen Bonds as Key Functional Determinant

“…This has also been shown in other studies where the XX dipeptide is changed (14,18 (Fig. 6), which has been shown to destabilize the oxidized form of the protein (14,17).…”

“…This has also been shown in other studies where the XX dipeptide is changed (14,18 (Fig. 6), which has been shown to destabilize the oxidized form of the protein (14,17). The lack of this interaction from Met 200 in the DsbG CGPC-T200M mutant could lead to the observed stabilization of the oxidized form.…”

“…DsbG CGPC-T200M has a redox potential of Ϫ280 mV, even more reducing than WT Trx-1, Ϫ271 mV (15), and a nucleophilic Cys pK a of 6.1 (Table 1). A similar result was obtained for E. coli Trx-1, where combining the CPHC active site of DsbA with an I75T (the residue before the cis-Pro in E. coli Trx) mutation shifted the redox potential to Ϫ180 mV, 91 mV more oxidizing than the WT Trx (14). Even a single mutation, P31T, in the active site of Staphylococcus aureus Trx, in which the surrounding of the nucleophilic cysteine becomes more hydrophilic, resulted in a decrease of the pK a value of the cysteine by approximately 1 pH unit, whereas its redox potential increased by 30 mV (24).…”

“…However, the XX active site dipeptide is not the only important feature determining the function of Trx family proteins. The loop containing the conserved cisproline, which is in close proximity to the active site CXXC motif, also affects the function and the redox properties (14). A DsbG mutant where the residue located N-terminally from the proline in the cis-Pro loop was mutated, T200M, showed a more reducing redox potential of Ϫ181 mV and was shown to behave more like the disulfide isomerase DsbC (13).…”