DsbA-DsbB interaction through their active site cysteines. Evidence from an odd cysteine mutant of DsbA.
Formation of disulfide bonds in Escherichia coli envelope proteins is facilitated by the Dsb system, which is thought to consist of at least two components, a periplasmic soluble enzyme (DsbA) and a membrane-bound factor (DsbB). Although it is believed that DsbA directly oxidizes substrate cysteines and DsbB reoxidizes DsbA to allow multiple rounds of reactions, direct evidence for the DsbA-DsbB interaction has been lacking. We examined intracellular activities of mutant forms of DsbA, DsbA30S and DsbA33S, in which one of its active site cysteines (Cys30 or Cys33, respectively) has been replaced by serine. The DsbA33S protein was found to dominantly interfere with the disulfide bonds formation and to form intermolecular disulfide bonds with numerous other proteins when cells were grown in media containing low molecular weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S protein remained specifically disulfide-bonded with DsbB. These in vivo results not only confirm the previous findings that Cys30 of DsbA is hyper-reactive in vitro but provide evidence that DsbA indeed interacts selectively with DsbB. We propose that the Cys30-mediated DsbA-DsbB complex represents an intermediate state of DsbA-DsbB recycling reaction that has been fixed because of the absence of Cys33 on DsbA.
Kishigami
S
S
Department of Cell Biology, Kyoto University, Japan.
Kanaya
E
E
Kikuchi
M
M
Ito
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007535:Isomerases / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D019704:Protein Disulfide-Isomerases; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide