Crystal structure of the plasma membrane proton pump

Abstract

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.

Structure and mechanism of ATP-dependent phospholipid transporters

“…Although no crystal structure is available for a P4-ATPase, these proteins are probably not very different in folding from other members of the P-type ATPase family, as suggested by the fact that (i) all P-type ATPases including P4-ATPases have conserved core sequence segments that are essential for catalysis [98,109], and (ii) all crystallized P-type ATPases show a very similar tertiary structure despite their varying TMD numbers and diverse substrates ranging from protons to heavy metals [108,110]. Thus, assuming an analogous transport mechanism for P4-ATPases as for the SERCA pump, the phospholipid would occupy the place of the counter-ion and therefore be bound in the E2P conformation at the exoplamic side of the membrane and released in the transition step to E2 on the cytosolic side.…”

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

“…Numerical estimate of membrane protein from solution 10. Calculate GFP concentration in mg ml −1 in 100 μl of solution (membranes or buffer) as follows:…”

“…Although more structures of eukaryotic membrane proteins have been determined using material isolated from heterologous overexpression in the methylotrophic yeast Pichia pastoris, this difference is likely due to the popularity of P. pastoris because of its ability to produce a large biomass during fermentation with methanol 8 . However, recent work has demonstrated that S. cerevisiae is also a suitable host for obtaining enough material that will give high-resolution membrane protein structures 9,10 .…”

Outside of the box: recent news about phospholipid translocation by P4 ATPases

“…The first model is an extrapolation from the structure of the H + (P3A) transporters [18]. These transporters also lack the anionic and polar residues that form the ion-binding sites in the Ca and Na/K transporters.…”