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-bo und 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forms of DsbA, DsbA30S and DsbA33S, in which one of its active site cysteines (Cys(3 0) or Cys(33), respectively) has been replaced by serine, The DsbA33S protein was found to dominantly interfere with the disulfide bond form ation and to form intermolecular disulfide bonds with numerous other p roteins when cells were grown in media containing low molecular weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S protei n remained specifically disulfide-bonded with DsbB, These in vivo resu lts not only confirm the previous findings that Cys(30) of DsbA is hyp er-reactive in vitro but provide evidence that DsbA indeed interacts s electively with DsbB. We propose that the Cys(30)-mediated DsbA-DsbB c omplex represents an intermediate state of DsbA-DsbB recycling reactio n that has been fixed because of the absence of Cys(33) on DsbA.