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Conformational analysis of oxidized peptide fragments of the C-terminal redox center in thioredoxin reductases by NMR spectroscopy.

ABSTRACT: Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans - 9:91). This was determined by using the '?-gauche effect', which makes use of both (1) H-NMR and two-dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from Drosophila melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5?kJ/mol DSE and 14.3?kJ/mol for the C+ and T- conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange.

SUBMITTER: Ruggles EL

PROVIDER: S-EPMC4000577 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Conformational analysis of oxidized peptide fragments of the C-terminal redox center in thioredoxin reductases by NMR spectroscopy.

Ruggles Erik L EL Deker P Bruce PB Hondal Robert J RJ

Journal of peptide science : an official publication of the European Peptide Society 20140306 5

Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, res ...[more]

PMID: 24599608

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Project description:High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-active disulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminal redox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of a selenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineering techniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mouse mitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR from Caenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly, Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemical modifications were performed in this study: insertion of alanine residues between the cysteine residues of the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the position of the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating to insertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. In contrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues were inserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminus and a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic position by changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activity as the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate of DmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cys and Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, the results support our previous model of Sec as the leaving group during reduction of the C-terminus during the catalytic cycle.

Dataset Information

Conformational analysis of oxidized peptide fragments of the C-terminal redox center in thioredoxin reductases by NMR spectroscopy.

Publications

Conformational analysis of oxidized peptide fragments of the C-terminal redox center in thioredoxin reductases by NMR spectroscopy.

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