Thioredoxin folding

The C-terminal domain of phosducin is a five-stranded mixed p sheet with a helices on both sides, similar to the thioredoxin fold of disulfide iso-merase DsbA described in Chapter 6. Despite significant sequence homology to thioredoxin, the phosducin domain, unlike other members of this family. 

Figure 13.16 Schematic diagram of the phosducin molecule. Helices are blue, p strands are red and loop regions are orange. The structure folds into two separate domains, a N-terminal helical domain and a C-terminal domain that has the thioredoxin fold. Some of the loop regions in the helical domain are not well defined. (Adapted from R. Gaudet et al.. Cell 87 577-588, 1996.)...

The 2.0-A crystal structure revealed that DsbA contains a thioredoxin-like fold (Martin et al., 1993). The thioredoxin fold includes a central /3-sheet formed by four antiparallel /3-strands. The central /3-sheet is flanked by a perpendicular helix and two helices on the opposite side (Martin, 1995). Compared to thioredoxin, DsbA contains an additional /l-strand in the central (6-sheet and the insertion of a 65-residue helical domain (Fig. 1). Such insertions are commonly observed within the thioredoxin family (Martin, 1995 McCarthy etal., 2000). Most members of the thioredoxin superfamily are involved in disulfide exchange reactions, and contain a redox-active CXXC motif in their active site. The CXXC motif participates in disulfide exchange reactions by going through reversible cycles of oxidation and reduction. In this motif, the... 

Fig. 2. Superposition of PfPDO N and C units. The active site disulfides are shown in ball-and-stick representation. The secondary structure elements belonging to the thioredoxin fold are mimbeied with Roman numerals. Adapted om B. Ren, G. Tibbelin, D. de Pascale, M. Rossi, S. Baitolucci, and R. Ladenstein, Nature Struct. Biol. 5,602 (1998).

It was found that the Pf PDO molecule can be almost equally divided into two structural units, the N-terminal one (residues 1 to 117) and the C-terminal one (residues 118 to 226). They are connected by the loop between a4 and o5 in the middle of the molecule. Either unit contains four p strands and four a helices and displays exactly the same fold (Fig. 2), despite the fact that their sequence identity is only about 20%. Their superposition reveal.s a root-mean-square (r.m.s.) deviation of 1.23 A for 66 Cot atoms. Interestingly, each of the ly PDO units is basically a thioredoxin fold motif but with an additional a helix, al or a5, inserted at the N terminus. As a result, the two units share a similar fold with other protein... 

Unlike thioredoxin, glutaredoxin, and DsbA, which possess only one thiore-doxin-like motif, P/PDO is the first protein disulfide oxidoreductase whose three-dimensional structure has been shown to contain two thioredoxin fold motifs with two active sites. Thus, P/PDO shows structural resemblance to PDI and PDI-like protein s. This structural feature suggests that P/PDO is probably not just a simple protein disulfide reductant like thioredoxin as described previously. It may belong to the growing family of PDI-like proteins. From a structural point of view, P/PDO may represent the simplest form of PDI. 

In addition to If PDO and other protein disulfide oxidoreductases, the thioredoxin fold has also been found in other proteins, including glutathione... 

The thioredoxin fold motif serves as the scaffold for the structures of these redox enzymes (Fig. 7). Various structural insertions into the thioredoxin fold lead to the diversity of these enzymes. Variations may occur at certain regions of the thioredoxin fold, including both termini and the loops between pn and all, all and pill, and piV and alll. Among the structures of these redox enzymes, the... 

Pf PDO structure is the only one that has been shown to contain two thioredoxin fold motifs with two active sites so far. Each Pf PDO unit possesses an extra a helix, al or a5, at the N terminus of the thioredoxin fold. In the crystal structure, these two helices interact with a symmetry-related moleeule and contribute to the formation of the dimer and zinc binding sites. 

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

Figure 6.8 Schematic diagram of the enzyme DsbA which catalyzes disulfide bond formation and rearrangement. The enzyme is folded into two domains, one domain comprising five a helices (green) and a second domain which has a structure similar to the disulfide-containing redox protein thioredoxin (violet). The N-terminal extension (blue) is not present in thioredoxin. (Adapted from J.L. Martin et al.. Nature 365 464-468, 1993.)...

Fig. 7.4 Reactions associated with the thioredoxin system. Thioredoxin is a redox-regulating protein with a redox-active disulfide/dithiol within the conserved active site sequence -Cys-Gly-pro-Cys-. Thioredoxin reductase, a 55 kDa flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (1) and thioredoxin oxidase (2), a flavin-dependent sulfhydry 1 oxidase that catalyzes the oxidative protein folding with the generation of disulfides...

Protein structure determinations have identified several examples of one domain inserted within another. One example is the E. coli DsbA protein, which catalyzes the formation of disulfide bonds in the periplasm. The enzyme consists of two domains a thioredoxin-like domain that contains the active site, and an inserted helical domain similar to the C-terminal domain of thermolysins (Martin et al., 1993). The inserted domain forms a cap over the active site, suggesting that it plays a role in binding to partially folded polypeptide chains before oxidation of... 

It has been noted that water molecules are involved in establishing intramolecular hydrogen-bonding interactions between protein atoms and thus serves as an integral part of the folded protein structure. The effect of the water molecules on simulated THz spectra of the thioredoxin was investigated, since water can substantially affect the dynamics of the thioredoxin. To do this, an explicit simulation of water molecules was also conducted using a more advanced model which is described below. 

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