Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

Abstract

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

FIG. 1

Motility assay for various chromosomal dsbA mutant alleles. The identity of the DsbA active-site dipeptide is indicated. Strains used were PB401 (dsbA-null mutant), SF100 (wild type), PB406 (CPPC dipeptide), PB410 (CSFC dipeptide), PB416 (CPSC dipeptide), and PB419 (CPLC dipeptide).

FIG. 2

Yields of active mouse urokinase obtained in strains with altered chromosomal dsbA alleles. Strains SF110, PB350, and PB420 to PB433 were grown in rich media as described in Materials and Methods, and the activation of human plasminogen by cell lysates was determined using a coupled assay. Where known, the in vitro standard redox potential of the DsbA variant as calculated from Grauschopf et al. (15) is indicated (in millivolts). Error bars, standard deviations of four experiments.

FIG. 3

Yields of active mouse urokinase obtained in PB351/pRDB8-A with coexpression of DsbC active-site mutants. Error bars, standard deviations of four experiments.