Structure of circularly permuted DsbA_Q100T99: preserved global fold and local structural adjustments

The thiol-disulfide oxidoreductase DsbA is required for efficient formation of disulfide bonds in the Escherichia coli periplasm. The enzyme is the strongest oxidant of the family of thioredoxin-like proteins and three-dimensional structures of both oxidized and reduced forms are known. DsbA consists of a catalytic thioredoxin-like domain and a helical domain that is inserted into the thioredoxin motif. Here, the X-ray structure of a circularly permuted variant, cpDsbA_Q100T99, is reported in which the natural termini are joined by the pentapeptide linker GGGTG, leading to a continuous thioredoxin domain, and new termini that have been introduced in the helical domain by breaking the peptide bond Thr99-Gln100. cpDsbA_Q100T99 is catalytically active in vivo and in vitro. The crystal structure of oxidized cpDsbA_Q100T99, determined by molecular replacement at 2.4 Å resolution, was found to be very similar to that of wild-type DsbA. The lower thermodynamic stability of cpDsbA_Q100T99 relative to DsbA is associated with small structural changes within the molecule, especially near the new termini and the circularizing linker. The active-site helices and adjacent loops display increased flexibility compared with oxidized DsbA.