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PDBsum entry 2fwf
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Oxidoreductase
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PDB id
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2fwf
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References listed in PDB file
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Key reference
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Title
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High-Resolution structures of escherichia coli cdsbd in different redox states: a combined crystallographic, Biochemical and computational study.
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Authors
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C.U.Stirnimann,
A.Rozhkova,
U.Grauschopf,
R.A.Böckmann,
R.Glockshuber,
G.Capitani,
M.G.Grütter.
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Ref.
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J Mol Biol, 2006,
358,
829-845.
[DOI no: ]
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PubMed id
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Abstract
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Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to
periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a
C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane
domain. Each domain possesses two cysteine residues essential for electron
transport. The transport proceeds via disulfide exchange reactions from
cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to
nDsbD, which then reduces the periplasmic target proteins. We determined four
high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically
reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased
from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the
highest achieved so far for a thioredoxin superfamily member. The data reveal
unprecedented structural details of cDsbD, demonstrating that the domain is very
rigid and undergoes hardly any conformational change upon disulfide reduction or
interaction with nDsbD. In full agreement with the crystallographic results,
guanidinium chloride-induced unfolding and refolding experiments indicate that
oxidized and reduced cDsbD are equally stable. We confirmed the structural
rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of
cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using
two different experimental methods, surprisingly was around 2.5 units higher
than expected on the basis of the redox potential. Additionally, taking
advantage of the very high quality of the cDsbD structures, we carried out pKa
calculations, which gave results in agreement with the experimental findings. In
conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution
crystallography, computational chemistry and biophysical measurements,
highlighted two so far unrecognized key aspects of this domain: its unusual
redox properties and extreme rigidity. Both are likely to be correlated to the
role of cDsbD as a covalently linked electron shuttle between the membrane
domain and the N-terminal periplasmic domain of DsbD.
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Figure 2.
Figure 2. Mechanism of disulfide opening in crystals
induced by synchrotron radiation (adapted from Weik et al.37 and
Fauvodon et al.40).
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Figure 6.
Figure 6. Reduced DsbA active site (green and atom colors)
superimposed onto the active site of cDsbD[red] (magenta and
atom colors).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
358,
829-845)
copyright 2006.
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