Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution.

Abstract

The three-dimensional structure of the electron transport protein thioredoxin-S2 from E. coli has been determined from a 2.8 A resolution electron density map. The molecule is built up of a central core of three parallel and two antiparallel strands of pleated sheet surrounded by four helices. The residues involved in the active center 14-membered disulfide ring of thioredoxin form a protrusion between one of the helices and the middle strand of the pleated sheet. This region of the molecule, comprising two parallel strands joined by the protrusion and a helix, is structurally very similar to corresponding functionally important regions in the nucleotide-binding domains of flavodoxin and the dehydrogenases. The molecule has about 75% of the residues in well-defined secondary structures. The structure indicates that the carboxy-terminal third of the molecule forms an independent folding unit consisting of two strands of antiparallel pleated sheet and a terminal ahelix. This agrees with the noncovalent reconstitution experiments from thioredoxin peptide fragments. Thioredoxin is an example of a protein with the active center located on a protrusion rather than in a cleft, thus demonstrating the existence of male proteins.Thioredoxin is a low-molecular-weight protein which transports electrons via an oxidation-reduction active disulfide (1,2). Together with the FAD-containing enzyme, thioredoxin reductase, it forms a cyclic electron transport system (thioredoxin system) which makes the reducing power of NADPH available for reductions (3) (see Fig. 1.). Thioredoxin seems to be a universal component of all living cells capable of replicating DNA, since it is involved in the synthesis of deoxyribonucleotides (3, 4).Thioredoxin from Escherichia coli B has the following properties. It is an acidic protein, (isoelectric point 4.5) containing 108 amino-acid residues of known sequence (2) and is devoid of metals and cofactors. The oxidation-reduction disulfide bridge is formed from Cys-32 and Cys-35 spaced by Gly-33 and Pro-34, thereby forming a 14-membered disulfide ring (2). A localized conformational change accompanies reduction of thioredoxin-S2 to thioredoxin-(SH)2 (5) which drastically changes the fluorescence emission of 7). Noncovalent reconstitution of the thioredoxin molecule from two sets of peptide fragments obtained by selective cleavage at the single arginine or methionine (_Met-37) residues has been obtained (8,9). This suggests a favorable equilibrium of nucleated folding of the peptide fragments into their native format as found in thioredoxin (9).Crystallization of E. coli thioredoxin-S2 from alcoholic solution was found to be absolutely dependent upon the extraneous addition of cupric ions (10).

Sensitivity of Hemoglobin Thiol Groups within Red Blood Cells of Rat during Oxidation of Glutathione

“…Thus, the thiol groups of rat hemoglobin may be as reactive as the thiol groups of GSH [18]. Some other proteins have been shown to be reactive towards diazene oxidants : Thioredoxin, a dithiol protein, with 'highly exposed' thiol groups [19], is very reactive towards diamide (Holmgren, personal communication). Formation of protein glutathione mixed disulfide has been found by Flohe et al in isolated liver cells treated with diamide [20].…”

The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin

“…These include the structure of oxidized E. coli thioredoxin by X-ray crystallography [25,26], and the solution structures of reduced E. coli [27] and human [28] thioredoxins determined by two-dimensional NMR. The E. coli and human proteins exhibit very similar three-dimensional folds despite the large variation in amino acid sequence (25 % sequence identity with some deletions and insertions).…”

Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects

“…Thioredoxins are small (-12 kDa) heat-stable oxidoreductases implicated in many biological processes (reviewed by Holmgren [1985]). Reduced by thioredoxin reductase via NADPH, Escherichia coli thioredoxin serves as a hydrogen donor for enzymes such as ribonucleotide reductase and methionine sulfoxide reductase.…”