Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst of Escherichia coli.
BACKGROUND
DsbA, a periplasmic protein, catalyses the disulphide bond formation of other cell surface proteins in E. coli. Reoxidation of DsbA for catalytic turn over is assured by DsbB, a membrane protein with four essential cysteine residues facing the periplasm. We and others previously reported that the reactive Cys30 residue of DsbA forms a mixed disulphide with DsbB in the absence of its partner Cys33 residue.


RESULTS
Under the medium condition in which the DsbA mutant lacking Cys33 forms a mixed disulphide only with DsbB, we examined cysteine mutants of epitope-tagged DsbB for their ability to form the complex. It was shown that Cys104 of DsbB is absolutely required while other three cysteines are also required for maximum interaction. Examination of the redox states of cysteines in wild-type and mutant DsbB suggested that Cys104 and Cys130 form a disulphide bond which will be transferred to DsbA. In agreement with this notion, DsbB mutants lacking one of the N-terminally located cysteines retain weak DsbB activity in vivo. The primary role of the N-terminally located thioredoxin-like motif of DsbB is probably to reoxidize Cys104 and Cys130.


CONCLUSIONS
We propose the following reaction cycle. DsbB is initially oxidized (State A in Summary Figure). Disulphide interaction between Cys30 of DsbA and Cys104 of DsbB should then trigger the recycling reaction of DsbA (State B), allowing over all electron transfer from newly secreted protein via DsbA (Cys30/Cys33) to DsbB in which intrachain electron flow from Cys104/Cys130 (State C) to Cys41/Cys44 (State D) may occur.
Kishigami
S
S
Department of Cell Biology, Institute for Virus Research, Kyoto University, Japan.
Ito
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases
D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine