Guilhot C, Jander G, Martin N L, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9895-9. doi: 10.1073/pnas.92.21.9895.
Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.
二硫键的形成在大肠杆菌的周质中被催化。这个过程至少涉及两种蛋白质：DsbA和DsbB。最近的证据表明，DsbA是一种可溶性周质蛋白，可直接催化蛋白质中二硫键的形成，而内膜蛋白DsbB则参与DsbA的再氧化。在此，我们提供了DsbA与DsbB之间相互作用的直接证据。（岸上文等[岸上文，金谷英，菊池正，伊藤克（1995年）《生物化学杂志》270卷，第17072 - 17074页]已描述了类似的发现。）我们分离出了dsbA的显性负突变体dsbAd，其中DsbA活性位点的半胱氨酸 - 33被替换为酪氨酸。DsbAd和DsbA都能够与DsbB形成混合二硫键，这可能是DsbA再氧化过程中的一个中间体。这种复合物与DsbAd结合更稳定。通过增加DsbB的产量可以抑制显性作用。通过使用其中一个或两个半胱氨酸已被替换为丙氨酸的DsbB突变体，我们表明只有半胱氨酸 - 104对于复合物的形成很重要。因此，我们认为在体内，还原态的DsbA与DsbB形成复合物，其中DsbA的半胱氨酸 - 30与DsbB的半胱氨酸 - 104形成二硫键。半胱氨酸 - 104会迅速被DsbA的半胱氨酸 - 33取代，从而产生该蛋白质的氧化形式。
