Bacterial multi-drug efflux transporter AcrB – the pumping mechanism

DsbB is an E. coli membrane protein that oxidizes DsbA, the primary protein disulfide donor present in the periplasm.To understand how disulfide bonds are generated and introduced into secreted proteins, we determined the crystal structure of DsbB in a complex with DsbA and endogenous ubiquinone at 3.7 Å resolution.The first structure of DsbB revealed that DsbB contains the four-helix bundle scaffold in the transmembrane region and one short membrane-parallel α-helix in the long periplasmic loop.Strikingly, the disulfide-generating reaction center composed of Cys41, Cys44, Arg48 and ubiquinone is located near the N-terminus of the transmembrane helix 2, where oxidizing equivalents of ubiquinone are converted to a protein disulfide bond de novo.Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the ternary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in its separation from Cys130.This DsbA-induced conformational change in DsbB seems to prevent the backward resolution of the complex and thereby promote the physiological electron flow from DsbA to DsbB.Recently, I examined functional roles of the membrane-parallel α-helix with strong amphiphilicity by systematic mutation analyses.Introduction of charged or helix-breaking residues into this region not only disrupted the peripheral membrane-association of this helix but also impaired DsbA oxidation activity of DsbB.On the basis of structural and biochemical data so far obtained, I propose the "cysteine relocation mechanism", by which DsbB oxidizes the extremely oxidizing (reduction-prone) dithiol oxidase, DsbA, efficiently.