DISULFIDE BOND FORMATION AND CORRECTION
Figure 1
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The cytoplasm of E. coli is not permissive to the formation of stable disulfide bonds due to the presence of numerous reductases which efficiently
reduce any disulfide bond formed. Disulfide-bond formation is therefore compartmentalized extra-cytoplasmically to the periplasm
(see Figure 1). A set of cell envelope proteins (named Dsb for disulfide bond) which are responsible for the formation and
correction of disulfide bonds has been studied in great detail in the last two decades and several comprehensive reviews have
been written on this subject (21–24). This section will attempt to give a brief summary of these findings.
Periplasmic disulfide bond formation
In E. coli, disulfide bond formation is catalyzed by periplasmic oxidase DsbA. DsbA is monomeric 21 kD protein containing the classic
thioredoxin fold (25) and a single catalytic disulfide bond in its active site Cys-Pro-His-Cys (26). Upon donating its active
site disulfide bond to a reduced substrate protein, DsbA becomes reduced and is re-activated to its oxidized state by the
inner membrane protein DsbB (27). DsbB transfers the electrons it has received from DsbA to the pools of quinones within the
inner membrane (28). Of the studied disulfide bond oxidases, DsbA is one of the most efficient oxidase, capable of quickly
oxidizing a protein as it enters the periplasm (29, 30). This can result in mis-oxidation of substrate proteins, especially
if the protein contains multiple non-consecutive disulfide bonds (31). A mis-oxidized and therefore misfolded protein can
be proteolytically degraded and removed by the periplasmic protease DegP [32]. However, this is an energetically expensive
solution. A more elegant solution would be to correct the mis-folded protein by isomerizing the disulfide bonds until the
protein achieves its correctly oxidized disulfide bonded state.
Disulfide bond isomerization is critical to the efficient folding of non-consecutive, multidisulfide bonded proteins. Close
to half a century has passed since the discovery of the eukaryotic protein disulfide bond isomerase (PDI) (33). Surprisingly,
the in-vivo mechanism of disulfide bond isomerization remains elusive. PDI remains to be shown as a disulfide bond isomerase in vivo and the exact mechanism of disulfide-bond isomerization by PDI or its prokaryotic functional homolog DsbC has yet to be understood
clearly.
Figure 2
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DsbC is a periplasmic homo-dimeric disulfide bond isomerase, capable of converting a mis-oxidized protein back to its correctly
oxidized state, both in vitro and in vivo [31, 34–38]. The crystal structure of DsbC shows a "V" shaped protein, where each arm of the V is a single monomer (see Figure
2) (39). Each monomer consists of a thioredoxin domain and a dimerization domain, joined together by a short alpha-helical
linker domain. Each monomer of DsbC has four cysteines. The carboxyl-terminal cysteine pair forms a structural disulfide bond
which is important for the folding and stability of DsbC (40). The amino-terminal cysteine pairs are part of the active site
CxxC motif and are maintained in their reduced state by the inner membrane protein DsbD (41).
DsbD receives its reducing potential from the cytoplasmic pool of NADPH via the thioredoxin pathway (42). The dimerization of the two monomers of DsbC results in a hydrophobic cleft 38 Å wide.
Together with the flexibility incurred by the linker domain, the uncharged cleft should be able to accommodate a large set
of proteins or mis-folded domains. It has been assumed that this hydrophobic cleft is responsible for selectively interacting
with mis-folded proteins whose hydrophobic core is exposed due to mis-oxidation. This notion is further supported by the ability
of DsbC to assist in the folding of fully denatured non-disulfide bonded D-glyceraldehyde-3-phosphate dehydrogenase(GAPDH) (43). This chaperone property of DsbC is dependent on the dimerization of
DsbC (44) and is independent of its redox active cysteines. However, no direct evidence has thus far been produced to show
the role of the hydrophobic cleft in the ability of DsbC to bind and refold mis-folded proteins.
The periplasm of E. coli is ill adapted to high-level expression of multi disulfide bonded proteins. There are two major reasons for this. First, as
the disulfide bond machinery is localized to the periplasm, the over-expressed protein needs to be efficiently exported usually
via the sec system. Thus, the expressed protein needs to be maintained in its secretion-competent unfolded state, either naturally
or with the assistance of SecB. However, the Sec apparatus is not adapted for the export of a highly-over expressed protein.
This usually results in clogging of the sec apparatus which can lead to toxicity and commonly results in low yields. Second,
the periplasmic disulfide bond forming machinery of E. coli is not adapted to folding multidisulfide bonded proteins. Of the ~1500 proteins predicted to be exported in E. coli, the significant majority (<85%) have only 0–2 cysteines with only 4% having more than 6 cysteines. Of those multi-cysteine
proteins, most of the cysteines are involved in coordinating various cofactors, such as hemes for cytochromes or iron in iron-sulfur
cluster proteins. This inability of E. coli to correctly oxidize multi-disulfide bonded proteins in the periplasm becomes apparent when eukaryotic proteins with multiple
nonconsecutive disulfide bonds are over-expressed. For example yields of tissue plasminogen activator (tPA) are only detected
when DsbC is over-expressed (45).
Furthermore, the periplasm is devoid of ATP and thus lacks ATP driven chaperones present in the cytoplasm. Thus, in comparison
to the cytoplasm the small volume and the energy poor periplasmic compartment is not ideal for expressing and folding proteins
to high yields.
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