DSBAS

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. 

Disulfide bonds in proteins are generally stable and nonreactive, acting like bolts in the structure. However, oxidized DsbA is less stable than the reduced form and its disulfide bond is very reactive. DsbA is thus a strong... 

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.)...

Martin, J.E., Bardwell, J.C.A., Kuriyan, J. Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature 365 464-468, 1993. 

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. 

Malhotra S et al. Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAOl shows induction of the disulfide bond isomerase, dsbA. J Bacterid 2000 182 6999-7006. 

Bouwman, C. W., Kohli, M., Killoran, A., Touchie, G. A., Kadner, R. J., and Martin, N. F. (2003). Characterization of SrgA, a Salmonella enterica serovar Tjrphimurium virulence plasmid-encoded paralogue of the disulfide oxidoreductase DsbA, essential for biogenesis of plasmid-encoded fimbriae. /. Bacteriol. 185, 991-1000. 

E, and K which must be assembled in the correct sequence. A chaperonin PapD is also required as is an "usher protein," PapC,50 and also the disulfide exchange protein DsbA (Chapter 10). DsbA helps PapD to form the correct disulfide bridges as it folds and PapD binds and protects the various pilus subunits as they accumulate in the periplasmic space of the host. The usher protein displaces the chaperonin PapD and "escorts" the subunits into the membrane where the extrusion occurs.50 55... 

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... 

Guddat, L. W., Bardwell,J. C., andMartin.J. L. (1998). Crystal structures of reduced and oxidized DsbA investigation of domain motion and thiolate stabilization. Structure, 6, 757-767. 

Hennecke.J., Sebbel, P., and Glockshuber, R. (1999). Random circular permutation of DsbA reveals segments that are essential for protein folding and stability. J. Mol. Biol, 286, 1197-1215. 

II. De Novo Formation of Disulfide Bonds in E. coli The Discovery of DsbA. .. 284... 

In 1991, Bardwell and co-workers reported the identification of DsbA, which they found to be involved in the formation of disulfide bonds... 

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. 1. The crystal structure of DsbA. DsbA contains a thioredoxin-like fold including the insertion of an a-helical domain. The arrow indicates the location of the active-site disulfide bond.

From the equilibrium constant with glutathione and the standard redox potential of the GSSG/GSH pair, the redox potential of DsbA can be calculated. The redox potential of DsbA is —120 mV, making it the most oxidizing disulfide bond known. For comparison, the redox potential of thioredoxin is —270 mV, and therefore much more reducing. 

The small equilibrium constant of DsbA with glutathione demonstrates that the disulfide bond formed by DsbA is highly unstable. The stability of a particular disulfide bond corresponds to the extent to which a protein is stabilized by this bond. In other words, the more stable the disulfide bond, the more stable the protein conformation. In the case of DsbA, its unstable disulfide bond should therefore destabilize the protein conformation. This is indeed observed, since the reduction of DsbA s disulfide bond leads to stabilization of its folded conformation by 4.5 kcal/mol (Zapun et al., 1993). This is unusual, since disulfide bonds normally stabilize proteins. Yet, it is in agreement with the in vivo function of DsbA as a donor of disulfide bonds. What causes the reduced form of DsbA to be more stable than its oxidized form In the CXXC motif of DsbA, the N-terminal cysteine 30 is solvent-exposed and has a very low pA/ of 3.5, in contrast to the pA, of 9.0 for cysteines commonly found in proteins (Nelson and Creighton, 1994). The pKa is the pH at which the group is half ionized. Consequently, at physiologic pH,... 

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