Institute for Virus Research, Kyoto University^* Institute for Scientific and Industrial Research, Osaka University^** Institute for Protein Research, Osaka University^*** Nara Institute of Science and Technology^****
○Kenji Inaba^* Satoshi Murakami^** Mamoru Suzuki^*** Atsushi Nakagawa^*** Eiki Yamashita^*** Kengo Okada^**** Koreaki Ito^*

Kenji InabaKenji InabaDsbB is an inner membrane protein that transforms oxidizing equivalents of ubiquinone into protein disulfides in E. coli. Disulfide bonds generated by DsbB are relayed via DsbA to newly exported proteins. To understand structural basis of this disulfide generating system, we determined the crystal structure of a disulfide-bonded DsbB-DsbA complex having endogenous ubiquinone at 3.7 angstrom resolution. The relatively low-resolution structure was complemented with engineered methionine/selenomethionine signals to enable structural refinement and consequent illumination of the reaction mechanisms. The DsbB portion of the complex was found to possess a transmembrane four-helix bundle and a mobile periplasmic region, which includes a Cys104-containing segment captured by the hydrophobic groove of DsbA and a short horizontal helix juxtaposed with Cys130. While DsbB in resting state contains the Cys104-Cys130 disulfide bond, the intermediate-like DsbB-DsbA complex has Cys104 engaged in the intermolecular disulfide bond with the hyper-reactive Cys30 of DsbA. Remarkably, the residue 130 now proves to be relocated away from Cys104 to the vicinity of Cys41, whose resting partner is Cys44 that we revealed previously to form a thiolate-ubiquinone charge transfer complex during DsbB catalysis. Such a geometrical separation of Cys104 and Cys130 leads to prevention of the backward attack by Cys130 against the Cys30 (DsbA)-Cys104 (DsbB) intermolecular disulfide. Thus, the dynamic repositioning of the periplasmic region is induced when DsbB encounters reduced DsbA, as a mechanism that enables DsbB to oxidize the extremely oxidizing DsbA protein.