Aggregation, Protein
39
α
2
.
Preliminary
NMR
data
of
the
a
0
domain conFrm its structural similarity
to the a domain. The b and b
0
domains
have signiFcant sequence similarity to
each other, but no similarity with the a
domain. Nevertheless, NMR studies of
the b domain have indicated a similar
overall fold. ±rom its sequence, it could
be inferred that b
0
also has the same fold.
Neither b nor b
0
contain the active site.
The folding pathway of disulFde-bound
proteins involves isomerizations between
a number of species containing disulFde
bonds.
In vitro
experimental studies were
performed using the isolated a and a
0
do-
mains, and the results were compared with
those obtained with the holoenzyme. It
was concluded that the activity of long
length PDI is not simply the sum of
the activities of the isolated a and a
0
domains. Using a series of constructs
including nearly every linear combina-
tion of domains, the contribution of each
domain was investigated. It was deter-
mined that the thiol-disulFde chemistry
requires only the a and a
0
domains, and
that simple isomerization requires one of
these in a linear combination including b
0
,
whereas complex isomerization involving
large conformational changes requires all
the PDI domains except the C-terminal
extension. Thus, it appears that the b
0
do-
main is the principal peptide binding site,
but all domains contribute to the bind-
ing of larger polypeptide chains holding
them in a partially unfolded conformation
while the catalytic sites acts synergistically
to perform the thiol-disulFde exchange.
Since PDI has binding properties, it has
been proposed that it acts as a molecu-
lar chaperone. However, as underlined by
±reedman and coworkers, this property
does not represent a chaperone activity
and instead reflects its role as a catalyst to
accelerate the formation of native disulFde
bridges during protein folding.
Several gene products with similarity
to PDI have been identiFed in higher
eukaryotes. All are probably localized in
the endoplasmic reticulum and have thiol-
disulFde exchange activity.
In prokaryotes, the disulFde formation
occurs in the periplasm and is catalyzed by
a protein called DsbA, which exchanges
its Cys30–Cys33 to a pair of thiols in
the target protein, leaving DsbA in its
reduced state. The crystal structure of
oxidized DsbA displays a domain with a
thioredoxin-like fold and another domain,
which caps the thioredoxin-like active site
C30-P31-H32-C33, located at the domain
interface. Reoxidation of DsbA is cat-
alyzed by a cytoplasmic membrane protein
called DsbB, which contains four cys-
teine residues essential for catalysis. DsbB
transfers the electrons from the reduced
DsbA to membrane embedded quinones.
The reduced quinones are then oxidized
enzymatically either aerobically or anaero-
bically. Thus, DsbA is found in normal
cells in its oxidized state.
E. coli
also
has a complex reductive system includ-
ing another periplasmic protein DsbC,
which is a homodimer. The molecule
consists of two thioredoxin-like domains
with a CxxC motif, joined via hinged
linker helices to an N-terminal dimer-
ization domain. The hinge regions allow
movement of the active site, and a broad
hydrophobic cleft between the two do-
mains may bind the polypeptide chain.
Its function consists of reducing proteins
with incorrect disulFde bonds. DsbC is
maintained in its reduced form by a mem-
brane protein called DsbD, which contains
six essential cysteine residues. Then, the
electrons are transferred to thioredoxin
and ultimately to NADPH by thioredoxin
reductase.
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