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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).

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The three-dimensional structure of thioredoxin from bacteriophage T4 has been determined from a 2.8-A resolution electron density map. Phase angles for this map were determined from one heavy atom derivative and anomalous differences from cadmium in the native crystals. The molecule of 87 amino acid residues is built up from two sim le folding units; a " unit from the amino end of the chain anJ a #a unit from the carboxyl end. This structure is similar to that of thioredoxin from Escherichia coli in spite of their completely different amino acid sequences. The redox-active S-S bridge is part of a protrusion of the molecule as in E coli thioredoxin, but with quite different surroundings. The structural differences in this region have been correlated to differences in specificity towards the enzyme ribonucleotide reductase from different species. Thioredoxins are small proteins, containing one redoxactive disulfide bridge, which can function as electron carriers in the synthesis of deoxyribonucleotides from the corresponding ribonucleotides (1). This enzymatic reduction is catalyzed by ribonucleotide reductase. The electrons needed for the reduction are provided by NADPH and transferred via the flavoprotein thioredoxin reductase to the oxidized form of thioredoxin.When Escherichia colh cells are infected by bacteriophage T4, a phage-coded thioredoxin, T4 thioredoxin, is produced. In the phage-infected cells T4 thioredoxin brings about an electron flow from NADPH via the bacterial thioredoxin reductase to a phage-specific ribonucleotide reductase (2).Apart from the thioredoxin system there exists another recently discovered hydrogen donor system for ribonucleotide reductase in E. coli composed of NADPH, glutathione reductase, glutathione, and a small protein called glutaredoxin (3). Glutaredoxin seems to have certain features in common with thioredoxins. It interacts with both E. cob and T4 ribonucleotide reductases, but does not show any interaction with thioredoxin reductase from E. coli (A. Holmgren, personal communication).The molecular properties of thioredoxins from several species,--have been studied (4-8). The amino acid sequence of the 108 residues in thioredoxin from E. coli is known (4) as well as the tertiary structure of the molecule (5).The polypeptide chain of T4 thioredoxin consists of 87 amino acid residues of known sequence (6). No sequence homology can be observed between E. colh and T4 thioredoxin. Crystals of T4 thioredoxin were recently obtained from Cd2+-containing solutions (9). Here we report the three dimensional structure to 2.8-A resolution of these crystals.We initiated this study in order to answer several questions: Is there a similarity between T4 and E. coli thioredoxins in tertiary structure, although no obvious similarity in primary structure is seen? Can the strict specificity of T4 thioredoxin towards the phage-induced ribonucleotide reductase be explained in terms of three-dimensional structure? Is it possible to locate an interaction site with E. coli thioredoxin redu...