Evidence for Conformational Changes within DsbD: Possible Role for Membrane-Embedded Proline Residues

Abstract

The mechanism by which DsbD transports electrons across the cytoplasmic membrane is unknown. Here we provide evidence that DsbD's conformation depends on its oxidation state. Our data also suggest that four highly conserved prolines surrounding DsbD's membrane-embedded catalytic cysteines may have an important functional role, possibly conferring conformational flexibility to DsbD.Many periplasmic and secreted proteins contain disulfide bonds that are required to stabilize the protein's structure. In Escherichia coli, the well-studied DsbA-DsbB pathway catalyzes formation of disulfides in substrate proteins (2). A second, less studied, pathway performs disulfide bond rearrangement. In this pathway, the inner membrane protein DsbD maintains periplasmic disulfide isomerases DsbC and DsbG in the reduced and active form (8,14). This is necessary for DsbC and DsbG to attack incorrect disulfides and catalyze disulfide rearrangements. Genetic studies have shown that DsbD receives its electrons from cytoplasmic thioredoxin and transfers these electrons across the inner membrane to DsbC/DsbG (17). DsbD therefore connects the periplasmic isomerization pathway to the reductive power of the cytoplasm by transferring electrons from the cytoplasm to the periplasm and, correspondingly, disulfide bonds from the periplasm to the cytoplasm.DsbD is a 59-kDa protein with three domains. Two of these domains (␣ and ␥) are periplasmic while the third, ␤, is located in the inner membrane. Each domain contains a conserved pair of cysteine residues, which are essential for activity (19). In vivo and in vitro experiments suggest that electrons are transferred via a succession of disulfide bond exchange reactions, from thioredoxin to ␤, then to ␥, then to ␣, and finally to DsbC/DsbG (3,11,18). The crystal structures of both periplasmic domains ␣ and ␥ have been solved, and there is a significant amount of biochemical information available for these two domains (7,12,18). In contrast, much less is known about the membrane domain ␤. In particular, the mechanism by which the ␤ domain transports disulfides across the inner membrane remains unresolved. In fact, the question of how disulfides get across membranes has not to our knowledge been solved for any system. There are examples of systems that transport electrons across membranes, including the malateaspartate and glycerophosphate shuttles. In these systems, electrons are carried by metabolites that are transported from one compartment of the cell to the other. However, DsbD is thought to transport electrons without using a metabolite or a cofactor. If true, this makes DsbD unique among known electron transport proteins. We therefore wanted to examine this longstanding mystery of how reducing equivalents get across membranes.Conformational changes. If DsbD transports disulfides without using a cofactor, major conformational changes are likely to take place to allow the membrane-embedded cysteine residues to be alternatively exposed to the cytoplasm and to the periplasm. To test this hypoth...

Active-site Properties of the Oxidized and Reduced C-terminal Domain of DsbD Obtained by NMR Spectroscopy

“…It therefore seems that the large conformational changes that must be involved for the C-terminal domain of cDsbD to interact with the cysteine residues in the transmembrane domain, and for it then to be released to react with the N-terminal domain, occur in other regions of DsbD such as the linker regions between the domains. 43,44 pK a values of the cysteine residues in wild-type cDsbD…”

Two Snapshots of Electron Transport across the Membrane

“…The first step in the regeneration of active DsbD is the reduction of the intramembranous disulfide bond of DsbD␤ domain by cytoplasmic Trx1. It had been proposed by us and others that the two cysteines of DsbD␤ may be alternatively exposed to the cytoplasm and the periplasm by conformational changes such that the disulfide bond is more available to reduced Trx1 and, when reduced, the two cysteines are more available to oxidized periplasmic DsbD␥ (21)(22)(23). However, our findings that the shift from the oxidized to the reduced state does not result in a change in the accessibility to either side of the membrane of the cysteines and of different portions of DsbD␤ TMs suggest that those earlier proposals may not be correct.…”

“…They found that subtle changes do occur in the tertiary structure around some tryptophan residues but that they are not accompanied by any significant changes in the secondary structure. Furthermore, the experiments of Hiniker et al (23) showing different proteolytic sensitivities between the reduced and oxidized forms of DsbD were done under conditions that may not reflect the native conformations of DsbD␤ because the detergent used may not have provided the same lipid-mediated protein interactions in DsbD␤ that may be important for its structure.…”

Disulfide Bond Formation

“…Several residues that play an important role in DsbDs mechanism have been identified recently [48,49], but the precise mechanism used by DsbD to transport electrons from one side of the membrane to the other is still unclear. In vivo and in vitro experiments suggest, however, that electrons are transferred via a succession of disulfide bond exchange reactions involving all six conserved cysteine residues of DsbD [50,51].…”