The disulfide bond formation (Dsb) system
Introduction
Formation and breakage of a protein disulfide bond are simple (at least superficially) chemical reactions, in which two electrons are either donated to or abstracted from a cysteine pair. Nevertheless, the reactions in vivo are controlled by a range of disulfide–dithiol oxidoreductases. Stable/structural disulfide bonds are introduced into extracytoplasmic proteins [1, 2, 3] in most organisms except some thermophiles [4, 5] and virus-infected cells [6] that contain cytosolic oxidation systems. Chlamydias appear to have reducing periplasm [7].
This review focuses on recent developments and concepts gained on disulfide bond formation machineries in mesophilic eubacteria, in particular Escherichia coli. In the bacterial periplasm, oxidative protein folding is supported by the cooperation of different oxidoreductases that are kept in different redox states by specific membrane-integrated redox regulators, through which oxidizing or reducing equivalents come from ubiquinone/menaquinone or NADPH, respectively. Comprehensive recent reviews on this subject can be found in [3, 8, 9, 10•, 11, 12].
Section snippets
TRX proteins
Thioredoxin (TRX) superfamily proteins, having one or more TRX folds [13], participate in disulfide bond formation, along with other redox proteins. Figure 1 and Table 1 summarize major E. coli components that support oxidative folding of proteins. A TRX fold contains characteristic α-helices that are juxtaposed with β-strands as well as a Cys–XX–Cys motif in front of the first helix. Many of them contain additional domain insertions with implicated importance in substrate recognition and
DsbA, the disulfide bond introducer
DsbA is a major disulfide bond introducer in the periplasm and has the second highest redox potential (Table 1) among the known TRX-related proteins. DsbL, recently identified in a uropathogenic E. coli strain, is even more oxidizing and dedicated to the oxidation of arylsulfate sulfotransferase, a pathogenicity-contributing enzyme, but not of general unfolded proteins [19•]. DsbA contains a helical insertion that forms a hydrophobic patch on the molecular surface as well as a hydrophobic
DsbC
DsbC is kept in the reduced form by DsbD. It facilitates isomerization of disulfide bonds formed between incorrect pairs in the three known native substrates [38] or cloned eukaryotic substrates. DsbC thus assists in the acquisition of nonconsecutive disulfide bonds. The DsbC protomer has a C-terminally located TRX fold that is connected via an α-helical linker to the N-terminal dimerization domain [39]. It forms a V-shaped dimer. Substrates are thought to be captured in the V-shaped cavity,
The reducing and the oxidizing pathways are insulated
Although the oxidation and the reduction pathways take place in the same cellular compartment, their crosstalk should be minimized to avoid futile consumption of electrons and metabolic energy. In vitro kinetic measurements of reactions between various combinations of DsbA, DsbB, DsbC, DsbDα, and DsbBγ showed that only the reactions of ‘physiological’ combinations and direction proceed rapidly. Compared with them, nonphysiological disulfide exchange reactions are mostly 103-fold to 107-fold
Conclusions
It is remarkable that, for oxidative protein folding, intrinsically ubiquitous reactions of oxidation and reduction occur in precisely regulated fashions in the cell through sophisticated and specific protein interaction network. Now remarkable progress has been achieved in our understanding of how redox proteins communicate with each other, but precise nature of their substrate selection is still obscure. Also, much should be learned about the conformational changes that accompany molecular
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Rudi Glockshuber and Jim Bardwell for communicating the preprint information. The work from the authors’ groups was supported by PRESTO (to K Inaba) and CREST (to K Ito) from the Japan Science and Technology Agency and grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 19687005 to K Inaba and No. 14037231 to K Ito) and from the Special Coordination Funds for Promoting Science and Technology of MEXT (to K Inaba).
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