Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the
Transmembrane Oxidoreductase
DsbD |
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Authors: | Despoina A I Mavridou Julie M Stevens Alan D Goddard Antony C Willis Stuart J Ferguson and Christina Redfield |
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Institution: | ‡Department of Biochemistry and §Medical Research Council Immunochemistry Unit, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom |
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Abstract: | The bacterial protein DsbD transfers reductant from the cytoplasm to the
otherwise oxidizing environment of the periplasm. This reducing power is
required for several essential pathways, including disulfide bond formation
and cytochrome c maturation. DsbD includes a transmembrane domain
(tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each
contains a cysteine pair involved in electron transfer via a disulfide
exchange cascade. The final step in the cascade involves reduction of the
Cys103-Cys109 disulfide of nDsbD by Cys461 of
cDsbD. Here we show that a complex between the globular periplasmic domains is
trapped in vivo only when both are linked by tmDsbD. We have found
previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J.,
& Redfield, C. (2007) J. Mol. Biol. 370
,643
-658) that the attacking
cysteine (Cys461) in isolated cDsbD has a high
pKa value (10.5) that makes this thiol relatively
unreactive toward the target disulfide in nDsbD. Here we show using NMR that
active-site pKa values change significantly when cDsbD
forms a complex with nDsbD. This modulation of pKa values
is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a
poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in
complex with nDsbD, the nucleophilicity of cDsbD increases permitting
reductant transfer. The observation of significant changes in active-site
pKa values upon complex formation has wider implications
for understanding reactivity in thiol:disulfide oxidoreductases.DsbD is a unique protein that transfers reductant across the cytoplasmic
membrane to the periplasm in many Gram-negative bacteria
(1,
2). Provision of reductant to
the periplasm is required because this compartment is otherwise considered to
be an oxidizing environment
(2). DsbD includes three
domains, each containing a pair of cysteine residues that perform a series of
disulfide exchange reactions (). In the first step, the transmembrane domain (tmDsbD)
accepts electrons from thioredoxin in the cytoplasm; these are then
transferred to the periplasmic C-terminal domain (cDsbD) and finally to the
N-terminal domain (nDsbD), which is also located in the periplasm
(3-5).
nDsbD acts as a junction point for several pathways that require reductant,
including the general disulfide isomerase system and the pathway that is
thought to reduce the cysteine thiols of apocytochromes in the cytochrome
c biogenesis pathway
(6). In Gram-positive bacteria,
CcdA, an integral membrane protein, and ResA, which has a thioredoxin fold,
provide the reductant required for cytochrome c maturation
(7).Open in a separate windowSchematic representation of DsbD.
A, proposed pathway of
electron flow from thioredoxin (TrxA) in the cytoplasm, via the three
domains of DsbD, to the cytochrome c maturation (Ccm) and
disulfide bond isomerization pathways in the periplasm is shown. The crystal
structure of nDsbD is from Protein Data Bank code 1L6P
(8), cDsbD from Protein Data
Bank code 1UC7 (11), and the
nDsbD-cDsbD complex from Protein Data Bank code 1VRS
(12). The cyan boxes
indicate the thrombin cleavage sites introduced into full-length DsbD to allow
detection of the nDsbD-cDsbD complex following its formation in vivo.
The cysteine residues are shown in yellow. B, schematic
representation of the active site of cDsbD in the covalent complex with nDsbD
(12). Some active-site
residues of cDsbD are indicated in stick representation and the
inter-domain disulfide (Cys461-SS-Cys109) is shown in
yellow.Structural studies have sought to explain how DsbD functions and interacts
with its various partners. The structures of the two soluble periplasmic
domains have been determined (, left). nDsbD has an immunoglobulin-like
structure (8,
9) and is the only known
thiol:disulfide oxidoreductase with this fold. cDsbD has the more typical
thioredoxin fold found in many oxidoreductases; this has the characteristic
active-site CXXC motif
(10,
11). A covalent complex
between single-cysteine variants of each of these two domains was produced
in vitro and its x-ray structure solved
(12), revealing the interface
between the two domains (, right). Although this mixed disulfide is
accepted as a physiological intermediate in the function of DsbD, an in
vivo complex between the two soluble domains has not been reported
previously (3). Further
complexes between nDsbD and its other physiological partners have also been
trapped and their structures examined
(9,
13). Interestingly, all of the
interaction partners of nDsbD are thioredoxin-like proteins; similarities in
their folds are congruous with common interaction interfaces
(14). However, only cDsbD will
reduce nDsbD, whereas nDsbD will reduce several partners. This raises
questions about how the direction of reductant flow is maintained and
controlled within the series of disulfide-exchange reactions.As part of our structural and mechanistic characterization of DsbD and its
domains in solution, we have previously measured by NMR the
pKa values of the active-site cysteine pair,
Cys461 and Cys464, of cDsbD (numbered according to the
full-length Escherichia coli DsbD sequence)
(15). An unusually high
pKa value of 10.5 was measured for the N-terminal cysteine
of the CXXC motif, Cys461, and the pKa
value of the second cysteine, Cys464, was significantly higher than
the maximum pH value that was studied (pH 12.2). The pKa
value of 10.5 is the highest reported for the N-terminal cysteine of the
CXXC motif in a thioredoxin fold. The striking consequence of the
elevated pKa value is that the active-site cysteine of
cDsbD, Cys461, is not strongly nucleophilic, raising critical
questions about how this cysteine reacts with the disulfide in nDsbD. It was
demonstrated using site-directed mutagenesis that the negatively charged side
chains of Asp455 and Glu468, which are located close to
the CXXC motif (), are responsible for the unusually high
pKa value of Cys461; mutation of one or both of
these residues to Asn and Gln, respectively, resulted in decreases in the
pKa value of Cys461 from 10.5 to 9.9 (E468Q),
to 9.3 (D455N), and to 8.6 (D455N/E468Q). The pKa values
for Asp455 were found to be 5.9 and 6.6 in oxidized and reduced
cDsbD; these values are significantly higher than the value of ~4 for an
unperturbed aspartic acid. We postulated that the properties of the amino acid
side chains in the immediate environment of the cysteines in cDsbD would
change upon complex formation with nDsbD, changing the reactivity of the
cysteines and explaining how the reaction between the two domains is initiated
(15). Specifically, we
proposed that an increase in the pKa value of
Asp455 upon complex formation would lead to a decrease in the
pKa value of Cys461, thereby making it a better
nucleophile. Stirnimann et al.
(10) previously presented
pKa calculations suggesting an increase in the
Asp455 pKa value upon complex formation.The aim of this work has been to determine the molecular basis of the
control of the reactivity of the active-site cysteine residues in cDsbD, using
NMR to compare the active-site properties of cDsbD alone and in its
physiological complex with nDsbD. We demonstrate that the
pKa value of Asp455 is elevated by at least 1.1
pH units when cDsbD forms a complex with nDsbD. This modulation of the
pKa value is critical for the specificity and function of
cDsbD. These in vitro studies are complemented by in vivo
studies on complex formation, in which we have trapped the nDsbD-cDsbD complex
for the first time. The results of our experiments explain how the
intramolecular disulfide cascade within the soluble domains of DsbD functions,
and demonstrate the importance of the transmembrane domain in controlling and
facilitating complex formation between the soluble domains. |
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