Báo cáo khoa học: A structural overview of the PDI family of proteins docx

REVIEW ARTICLE
A structural overview of the PDI family of proteins
Guennadi Kozlov, Pekka Ma
¨
a
¨
tta
¨
nen, David Y. Thomas and Kalle Gehring
Department of Biochemistry, Groupe de Recherche Axe
´
sur la Structure des Prote
´
ines, McGill University, Montre
´
al, Que
´
bec, Canada
Introduction
Protein disulfide isomerase (PDI) was the first protein
folding catalyst discovered [1]. Since its discovery more
than 40 years ago, studies of this remarkable enzyme
have shown that PDI acts as a dithiol–disulfide oxido-
reductase that is capable of reducing, oxidizing and
isomerizing disulfide bonds. Independently of its redox
activity, PDI can also act as a chaperone both in vitro
[2] and in vivo [3]. PDI is the founding member of a
family of 20 related mammalian proteins that are
chiefly located and function in the endoplasmic reticu-
lum (ER) (Fig. 1). PDI family members are abundant
and play a significant role in protein folding and qual-

ity control in the calcium-rich oxidative environment
of the ER [4]. The members vary in length and domain
arrangement, but share the common structural feature
of having at least one domain with a thioredoxin-like
structural fold, babababba. Most PDI family members
contain both catalytic and non-catalytic thioredoxin-
like domains that are identified as either a or b based
on the presence or absence of a catalytic motif, with
use of the prime symbol to indicate their position in
the protein. PDI has four such domains, a, b, b¢ and a¢
[5]. The a and a¢ domains functionally resemble thiore-
doxin, and each contains catalytic Cys-x-x-Cys motifs
that react with thiols of newly synthesized proteins to
confer disulfide oxidoreductase activity. The b and b¢
domains, although structurally similar to thioredoxin,
do not contain catalytically active cysteines. Instead,
the b and b¢ domains appear to act as spacers, and are
often responsible for substrate recruitment [6–9]. The
non-catalytic domains have lower sequence identity
than the catalytic domains across PDI family members,
and show more structural variability. For instance, the
b domain of ERp44 (ER protein 44 kDa) has an
unorthodox arrangement of the secondary structure
elements, bbabbba [10]. The family members most
Keywords
disulfide; endoplasmic reticulum; ERp44;
ERp57; ERp72; PDI; protein folding; protein
structure; thioredoxin-like; X-ray
crystallography
Correspondence

K. Gehring, Department of Biochemistry,
McGill University, 3649 Promenade Sir
William Osler, Montre
´
al, Que
´
bec, H3G 0B1,
Canada
Fax: +1 514 398 2983
Tel: +1 514 398 7287
E-mail:
(Received 8 April 2010, revised 11 July
2010, accepted 27 July 2010)
doi:10.1111/j.1742-4658.2010.07793.x
Protein disulfide isomerases (PDIs) are enzymes that mediate oxidative pro-
tein folding in the endoplasmic reticulum. Understanding of PDIs has
historically been hampered by lack of structural information. Over the last
several years, partial and full-length PDI structures have been solved at an
increasing rate. Analysis of the structures reveals common features shared
by several of the best known PDI family members, and also unique
features related to substrate and partner binding sites. These exciting
breakthroughs provide a deeper understanding of the mechanisms of oxida-
tive protein folding in cells.
Abbreviations
CNX, calnexin; CRT, calreticulin; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; SAXS, small-angle X-ray scattering.
3924 FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS
similar to PDI have a short interdomain region between
the b¢ and a¢ domains that is termed the x-linker [11].
Recently determined structures of several PDI family
members have revealed their detailed architecture and

led to mechanistic insights into their function. The
most exciting breakthrough came when the full-length
crystal structure was solved. Determination of the
structure of yeast Pdi1p (yeast PDI) showed that its
four domains form an overall ‘U’ shape, suggesting
how substrates may be positioned relative to the two
catalytic domains [12]. The structure of ERp44 showed
that its three thioredoxin-like domains (abb¢) are
arranged like a cloverleaf. A long C-terminal tail folds
back and makes contacts with the a and b¢ domains
[10]. This capping function of the C-terminal tail may
have assisted the successful crystallization and struc-
ture determination. Recently, the crystal structure of
full-length human ERp57 (ER protein 57 kDa, also
known as protein disulfide-isomerase A3 or 58 kDa
glucose-regulated protein) in complex with tapasin was
also solved [13]. The ERp57 structure provides the first
structural insight into protein binding by the catalytic
domains. Two other crystal structures of PDI-like pro-
teins have been solved: human ERp29 (ER protein 29
kDa) [14] and yeast Mpd1p (member of the protein
disulfide isomerase family 1) [15]. Additionally, the
structures of the non-catalytic fragments of human
ERp57 [16], human PDI [8,17] and rat ERp72 (ER
protein 72 kDa, also known as protein disulfide-iso-
merase A4) [18] have been determined.
Here we review these structural studies, with special
focus on mammalian PDIs and what can be learnt
from their similarities and differences. Excellent
reviews of the process of disulfide bond formation in

the ER and the biology of PDIs are also available
[19,20].
What constitutes a PDI family member?
The PDI family contains both thiol-reactive and
thiol non-reactive members, and this has led to some
confusion. A thioredoxin-like domain has been loosely
Fig. 1. Domain architectures of human
disulfide isomerases (PDIs). Catalytic thiore-
doxin-like domains (a and a¢) are colored
pink, and non-catalytic domains (b and b¢)
are blue. The first domain of PDILT, which
does not contain active site cysteines, is
hatched to indicate its strong similarity to
the a domains of other PDIs. Yellow boxes
correspond to the linker between the b¢ and
a¢ domains (x-linker). The DnaJ domain of
ERdj5 and the C-terminal helical domain of
ERp29 are shown in green. White boxes
indicate transmembrane domains. The sec-
ond column lists available structures of
mammalian PDIs. Among notable non-mam-
malian structures are two structures of
yeast PDI crystallized at different tempera-
tures (PDB accession numbers 2b5e and
3boa) and yeast Mpd1p (PDB accession
number 3ed3). The structure of full-length
ERp57 (PDB accession number 3f8u) was
determined in complex with tapasin.
G. Kozlov et al. PDI family of proteins
FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS 3925

defined as anything with a predicted thioredoxin-like
structure (based on sequence). However, some authors
have used a more specific definition of a thioredoxin-
like domain as one capable of reacting with cysteines
[21]. This definition appears to make the most sense,
as members of a disulfide isomerase family should be
capable of reacting with cysteine side chains. However,
the founding member of this family, PDI, also exhibits
non-specific polypeptide-binding chaperone activity
[22]. Thus, a more inclusive definition of PDI family
members would comprise proteins that contain
non-thiol-reactive thioredoxin-like domains with chap-
erone-like activities for ER folding and secretion of
proteins. This includes ERp29 [23] and ERp27 (ER
protein 27 kDa) [24], two PDI family members that do
not contain thiol-reactive active sites. The proteins
PDILT (protein disulfide isomerase-like protein of the
testis) and TMX2 (thioredoxin-related transmembrane
protein 2) have catalytic motifs, Ser-x-x-Cys, that lack
the N-terminal cysteine required for full oxidoreduc-
tase activity. However, because they contain C-termi-
nal cysteines, and PDILT has been shown to form
mixed disulfides with partners and substrates in vivo
[25], we categorize these as thiol-reactive. Their contri-
butions to oxidative protein folding in cells remain
unclear, and because of their inability to act as oxido-
reductases, their chaperone functions are probably
more important [26]. Similar considerations apply to
ERp44, AGR2 (anterior gradient protein 2 homolog),
AGR3 (anterior gradient protein 3 homolog) and

TMX5 (thioredoxin-related transmembrane protein 5),
which c ontain Cys-x-x-Ser motifs that lack the C-terminal
cysteine.
PDIs are a diverse family
PDI family members have functions as diverse as their
sequences and domain arrangements. In Fig. 1, we dis-
tinguish between thiol-reactive and non-reactive PDI
family members. Most PDIs contain more than one
active site, and usually contain a combination of active
and inactive thioredoxin-like domains. The inactive
domains perform functions such as substrate or part-
ner recruitment. Importantly, although in vitro activi-
ties have been demonstrated for most PDIs, their
function in vivo is more difficult to determine, and may
be intrinsically more complex, involving other partners
or specific conditions. For example, while PDI gener-
ally promotes protein folding, it can act as an unfol-
dase, favoring ER exit of cholera toxin [27]. The
specific function of ERp57 is unclear, but its gene
knockout is embryo-lethal at day 13.5 [28] for reasons
that may relate to its modulation of STAT3 (signal
transducer and activator of transcription 3) signaling
[29]. A conditional B-cell knockout has adverse effects
on folding of glycosylated influenza virus hemaggluti-
nin, but little effect on folding and secretion of the
Semliki Forest virus coat spike proteins p62 and E1.
ERp57 knockout did not change ER morphology or
function drastically, and ER stress levels were not
affected, suggesting more functional overlap between
PDIs than previously appreciated. Remarkably, treat-

ment with castanospermine rescued the folding of viral
hemagglutinin in ERp57
) ⁄ )
mouse fibroblasts [30], pre-
sumably by preventing its entry into the calnexin cycle,
and thereby allowing other disulfide isomerases to act
on it. These results highlight the need for in vivo stud-
ies to clarify the functions of the various PDIs, and
the difficulty in assigning functions to PDIs based on
their in vitro activities or structures alone. With nota-
ble exceptions such as ERp57, ERdj5 [31], and AGR2,
which is involved in the production of intestinal mucin
[32,33], use of knockouts to address the in vivo
functions of mammalian PDIs has not been reported.
Catalytic sites
In thioredoxin-like domains, the conserved catalytic
Cys-x-x-Cys motif is found at the N-terminus of a long
helix, a2 (Fig. 2). Within the catalytic motif, the two
cysteines play distinct roles. The N-terminal cysteine
forms a mixed heterodimer with a protein substrate,
while the C-terminal cysteine is involved in substrate
release [34]. Several other residues in the catalytic site
contribute to the reaction mechanism. A conserved
glutamate positioned below the C-terminal cysteine
functions in proton transfer during substrate release
[35]. A neighboring arginine modulates the pK
a
of this
catalytic cysteine by its placement in the active site
[36,37]. The recent determination of the structure of

the ERp72 catalytic domains allowed a glimpse into
the effects of local rearrangements of the N-terminal
part of helix a2 on positioning of the conserved argi-
nine residue [38]. In the a
0
domain, the arginine side
chain is surface-accessible, but in the a domain, the
equivalent arginine points towards the catalytic site
and forms a salt bridge with the glutamate residue,
Glu200, that is implicated in substrate release (Fig. 2).
This suggests that coordinated arginine–glutamate
interactions may serve to modulate the catalytic activ-
ity of protein disulfide isomerases.
Substrate binding sites
One major area of interest has been how PDI family
members recognize and bind substrate molecules. In
PDI family of proteins G. Kozlov et al.
3926 FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS
pioneering studies, the substrate binding site of
mammalian PDI was identified by cross-linking with
radiolabeled model hydrophobic peptides that mimic
unfolded proteins [6]. The major binding site for
unfolded proteins was shown to be the b¢ domain, and
this was confirmed by structural studies that used
NMR chemical shifts to map the binding site of
unfolded RNase A and peptide ligands onto the struc-
ture of the bb¢ fragment of PDI [8,9]. The binding site
consists of a large hydrophobic cavity between helices
a1 and a3, comprising Phe223, Ala228, Phe232, Ile284,
Phe287, Phe288, Leu303 and Met307 side chains

(Fig. 3A). Determination of the crystal structure of the
b¢x domain of human PDI provided a more detailed
insight into substrate binding, as the x-linker is folded
back and mimics how hydrophobic stretches bind to
the b¢ domain [17]. Specifically, the side chains of
Leu343 and Trp347, which are part of the linker
between the b¢ and a¢ domains, are inserted into the
hydrophobic cavity of the b¢ domain. The binding sur-
face appears to be conserved across species, as the
same pocket is fully accessible and well positioned for
protein substrate binding in the structure of yeast PDI
[12]. In human PDI, the b¢a¢ fragment is required for
efficient binding of non-native protein substrates [6].
A recent study of Humicola insolens PDI also demon-
strated that the a¢ domain assists in substrate binding,
as the b¢a¢ fragment shows extensive contacts with the
hydrophobic peptide mastoparan [39].
ERp44 contains three thioredoxin-like domains, a, b
and b¢, in addition to a C-terminal regulatory domain
[10]. There are obvious structural similarities between
the b¢ domains of ERp44 and PDI, and most of the
substrate binding PDI residues are conserved in
ERp44 (Fig. 3). The b¢ domain of ERp44 also has a
hydrophobic pocket. As observed with the PDI b¢x
fragment, a hydrophobic stretch C-terminal to the b¢
domain of ERp44 folds back and binds to the a1–a3
cavity as a short helical segment using the side chains
of Phe358 and Leu361 (Fig. 3B). The C-terminal tail
also partly shields a hydrophobic patch of the a
domain, and its removal increases the in vitro activities

of ERp44 as an oxidase, reductase, isomerase and
chaperone [10]. Strikingly, tail-less ERp44 formed
mixed disulfides with endogenous proteins in several
cell types [10], suggesting that the C-terminal cap of
the substrate binding domains contributes to the speci-
ficity of ERp44. How the action of the C-terminal tail
of ERp44 is regulated in cells is an intriguing question.
Structural studies of two other major PDIs, ERp57
and ERp72, showed that they do not contain hydro-
phobic pockets [16,18]. Structurally, ERp57 lacks a
protein substrate binding site in its b¢ domain. Instead,
the a1–
a3 surface is mostly negatively charged. The
corresponding surface of ERp72 is likewise polar and
is unable to bind hydrophobic peptides. The residues
Arg398 and Glu459 of ERp72 form a salt bridge to
occlude a potential substrate binding cavity (Fig. 3C).
This interaction is stabilized by a hydrogen bond
between Tyr416 and Glu459. These two positions are
characteristic of b¢ domains of protein disulfide isome-
rases that do not bind directly to hydrophobic
stretches. In ERp57, Gln256 forms a salt bridge with
the corresponding Glu310 that is stabilized by a hydro-
gen bond with Tyr264 (Fig. 3D). The charged nature
of the a1–a3 surface explains the inability of ERp57
and ERp72 to bind model peptides in vitro [40,41].
In contrast, several other PDIs are predicted to bind
to substrates via hydrophobic pockets in their b¢
domains. PDIp (pancreas-specific protein disulfide iso-
merase, also known as PDIA2), PDILT and ERp27

have mostly identical or similar hydrophobic residues
A
B
Fig. 2. Role of arginines in the ERp72 catalytic thioredoxin-like
domains in modulating disulfide isomerization activity. (A) Phe97
and Pro138 of the a
0
domain restrict access of Arg155 to the
Cys-x-x-Cys active site. (B) A conformational change in the a
domain a2 helix allows Arg270 to enter the hydrophobic core and
form a salt bridge with the conserved buried residue Glu200.
G. Kozlov et al. PDI family of proteins
FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS 3927
as PDI in their hydrophobic pockets (Fig. 3E). PDIp
can bind the amphipathic peptide D-somatostatin [42],
as can ERp27 [24]. The redox-inactive bb¢ domains of
PDIp exhibit chaperone activity in vitro and in vivo
[43]. Binding of liver- and testis-specific PDILT to
D-somatostatin and unfolded bovine pancreatic trypsin
inhibitor has been demonstrated [26], and PDILT has
been shown to form mixed disulfides with substrates in
HeLa cells via its unusual Ser-x-x-Cys motif [25]. The
lack of a hydrophobic substrate binding site in the b
and b¢ domains of ERp57 and ERp72 indicates that
these PDIs either require protein partners that assist in
substrate recognition, or that they directly interact
with substrates through the active sites of their cata-
lytic domains.
ERp29 utilizes an alternative site for substrate recog-
nition. The putative peptide binding site of its single

thioredoxin-like domain is located in the b2–a2 and
a3–b4 loop area of its N-terminal thioredoxin-like
domain [14]. This is opposite to the a1–a3 surface used
by PDI. ERp29 forms a tight dimer, and its b domain
is sufficient for peptide and substrate binding. It binds
peptides with two or more aromatic residues, and
favors peptides with basic character [14].
The structure of ERp18 revealed that this protein
also adopts a thioredoxin-like fold and has a conserved
Pro113 that results in an unusually bent a2 helix when
ERp18 is in its oxidized form [44]. This conserved pro-
line might be important for ERp18 function, although
a specific requirement for ERp18 function has yet to
be determined. ERp18 shows specificity for a compo-
nent of the complement cascade, the pentraxin-related
protein PTX3 [45], and has been implicated in the
reduction of gonadotropin-releasing hormone [46].
The recently determined ERp57–tapasin structure
provided the first structural insights into protein bind-
ing by the catalytic domains of a mammalian PDI [13].
In the structure, the a domain of full-length ERp57 is
linked to tapasin by a disulfide bond. Tapasin is a
chaperone associated with editing the peptide cargo of
the major histocompatibility complex class I. ERp57
specificity for tapasin appears to be determined pri-
marily by the catalytic domains [47]. It has been sug-
gested that the interdomain distance between the a and
AB
C
E

D
Fig. 3. A cavity on the a1–a3 surface of the
b¢ domain defines the ability of the PDI to
bind hydrophobic stretches of protein sub-
strates. The stretches after the b¢ domain of
PDI (A) and ERp44 (B) interact with the
hydrophobic surface in the corresponding
crystal structures. The residues that bind to
the hydrophobic groove are labeled. The cor-
responding surfaces in ERp72 (C) and
ERp57 (D) are occluded by polar interactions
involving conserved glutamates that also
form hydrogen bonds with tyrosine side
chains. Hydrogen bonds are shown as black
dashed lines, and the residues involved are
labeled. (E) Structure-based sequence align-
ment of the b¢ domains from human PDIs
and rat ERp72. The glutamates that contrib-
ute to the inability of the ERp57 and ERp72
b¢ domains to bind hydrophobic stretches
are highlighted in pink, and their polar equiv-
alents are shown in turquoise. The residues
potentially involved in substrate binding are
highlighted in gray. The alignment includes
the N-terminal part of the x-linker that
appears to be an integral part of the b¢
domain. The consensus secondary structure
is shown above the alignment.
PDI family of proteins G. Kozlov et al.
3928 FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS

a¢ domains makes ERp57 particularly suited for bind-
ing to tapasin; however, the mobility of the a and a¢
domains relative to the bb¢ base for each of these pro-
teins (discussed further below) suggests that the a to a¢
interdomain distance is not strictly maintained
(Fig. 4A). The suggestion that the interaction is stabi-
lized by cumulative complementary protein–protein
interactions seems more likely, with the a and a¢
domains together providing an avidity effect that
secures binding to tapasin. Although most ERp57 resi-
dues within 4.5 A
˚
of tapasin in the complex are con-
served in ERp57, ERp72 and PDI, three (K366, C92
and R448) are unique to ERp57. Further studies are
required to understand ERp57 specificity for tapasin,
but these results illustrate the importance of PDI–sub-
strate complementarity within catalytic domains.
Protein partner binding sites
PDI is highly abundant in the ER, and is a member of
distinct protein complexes with specific functions. PDI
is the b-subunit of the prolyl-4-hydroxylase complex
that is important for hydroxylating proline residues of
collagen [48], and also is a subunit of the microsomal
triglyceride transfer protein [49]. Binding of PDI to the
prolyl-4-hydroxylase complex minimally requires intact
b¢ and a¢ domains of PDI, but the assembly and activ-
ity of the complex is further enhanced by the addition
of a and b domains [50]. Mutagenesis of individual
residues in PDI confirmed the importance of the a and

a¢ domains in the assembly of an active complex but,
surprisingly, mutations in the hydrophobic substrate-
binding site in the b¢ domain had no effect [51], for
assembly of the prolyl-4-hydroxylase complex.
Rather than directly binding to substrates, ERp57
requires a protein partner to assist in substrate protein
folding [41]. The partner protein, either calnexin
(CNX) or calreticulin (CRT), recruits glycoprotein
substrates through a lectin domain. NMR titrations
and mutagenesis studies mapped CNX ⁄ CRT binding
to a site centered on the N-terminal half of helix a2of
the b¢ domain of ERp57 [16]. This area is abundant in
positively charged residues and displays charge com-
plementarity to the negatively charged tip of the
CNX ⁄ CRT P-domain, which is responsible for ERp57
binding [52]. In particular, mutations R282A and
K214A in ERp57 abrogate or greatly decrease CNX
P-domain binding in vitro [16] and inhibit substrate
interactions in vivo [31]. This region of ERp57 has also
been shown to mediate binding to the PDI ERp27
[24]. ERp27 has a hydrophobic peptide binding site on
its second thioredoxin-like domain and may recruit
substrates to ERp57. ERp27 has no catalytic cysteines
of its own.
A yeast homolog of CNX, Cne1p, interacts with the
oxidoreductase Mpd1p. Mpd1p and ERp57 present
very different overall architectures. Mpd1p contains
only two domains: an N-terminal catalytic domain and
a C-terminal non-catalytic domain. The recently deter-
mined crystal structure of Mpd1p has a positively

charged surface at the beginning of the second thiore-
doxin-like domain that has been suggested to be a
potential Cne1p binding site [15].
ERp72 also does not possess a hydrophobic sub-
strate binding site in its b and b¢ domains. Compared
to PDI and ERp57, ERp72 contains an additional cat-
alytic a
0
domain at its N-terminus. The recently deter-
mined high-resolution crystal structure of the bb¢
fragment of ERp72 reveals strong structural similarity
to ERp57 in terms of both the individual domains and
relative domain orientation [18]. The ERp72 surface
corresponding to the CNX binding site of ERp57 is
AB
a′
a′
b′
b′
Fig. 4. Involvement of the catalytic domains in protein binding. (A) Representation of the ERp57–tapasin structure showing that only the cat-
alytic a and a¢ domains of ERp57 (green) interact with tapasin (turquoise). The ERp57 residues that interact with CNX are shown in blue
[16]. (B) Structural model of ERp72 based on structures of the a
0
a (pink), bb¢ (turquoise) and a¢ (yellow) domain fragments overlaid upon full-
length ERp57. The catalytic cysteines and adjacent hydrophobic residues in ERp57 and ERp72 are shown in orange and gray, respectively.
G. Kozlov et al. PDI family of proteins
FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS 3929
negatively charged, and consequently does not bind
the CNX P-domain, but shows high sequence conser-
vation among ERp72 proteins from various species,

suggesting functional importance.
Given that ERp72 does not interact with CNX, and
lacks a hydrophobic substrate binding site, how might
it interact with substrates? Perhaps ERp72 relies on its
three catalytic domains for specificity toward sub-
strates or partners. The relative contributions of each
of the catalytic Cys-x-x-Cys motifs of ERp72 to reduc-
tase activity on insulin in vitro suggest unequal contri-
butions to binding and catalysis [53]. Cysteine-to-serine
mutations in the N-terminal a
0
domain affect the k
cat
for insulin reduction, but the K
m
is unaffected. The
same mutations in the a domain have intermediate
effects on both k
cat
and K
m
, while loss of the catalytic
motif of the a¢ domain primarily affects K
m
[53]. These
results were interpreted as indicating that the a
0
domain is primarily involved in catalysis, the a domain
has intermediate roles in catalysis and binding, and the
a¢ domain functions primarily to bind substrates. How-

ever, the unequal contributions to binding and cataly-
sis of the catalytic domains could also indicate that
other structural features are important for the activi-
ties. These kinetic studies on ERp72 must be inter-
preted carefully, because PDI-catalyzed oxidoreductase
reactions do not necessarily follow simple Michaelis–
Menten rules. Compared to the other abundant PDIs,
relatively few endogenous substrates were identified for
ERp72 in HT1080 human fibroblasts [45]. However,
due to the use of Cys-x-x-Ala substrate-trapping
mutants that form mixed disulfides during reduction or
isomerization reactions, substrates oxidized by ERp72
may have been missed. ERp72 may play a more spe-
cialized role in protein oxidation, or act on specific
substrates not readily detected by the methods used.
Further work is required to understand ERp72–sub-
strate and ERp72–partner interactions.
Recently, the transmembrane PDI TMX4 was char-
acterized and found to interact with ERp57 and caln-
exin [54]. Unlike ERdj5, over-expression of TMX4
does not accelerate ER-associated decay of the NHK
(null-Hong Kong) variant of a1-antitrypsin. Interac-
tion of TMX4 with ERp57 was dependent on its cata-
lytic active site, suggesting that it may reduce ERp57
in the ER. On the other hand, the interaction of
TMX4 with CNX did not require an intact Cys-x-x-
Cys motif. Further work is necessary to determine how
TMX4 might interact with substrates, although sub-
strate recruitment by CNX in a fashion analogous to
the ERp57-CNX complex is a possibility. While

TMX4 lacks a b¢-like domain that can interact with
the P-domain of CNX, other mechanisms are possible.
TMX4 does not isomerize scrambled RNase A in vitro,
suggesting that it requires a co-factor ⁄ partner for sub-
strate recruitment.
Interdomain mobility
The question of interdomain flexibility is relevant for
PDIs that comprise multiple thioredoxin-like domains.
Recent crystallographic and small-angle X-ray scatter-
ing (SAXS) studies provide support for interdomain
mobility. This may be the main reason why PDI was
resistant to crystallization efforts for a long time. Only
two-four-domain PDIs (ERp57 and yeast PDI) have
been crystallized, and both represent partner- or
pseudo-substrate-bound structures. ERp57 was crystal-
lized as a heterodimer with tapasin, while yeast PDI
was crystallized with another yeast PDI molecule mim-
icking a bound substrate. Crystallization of the three-
domain ERp44 structure was potentially favored by
reduced mobility due to binding of the protein C-ter-
minus to the thioredoxin-like domains. Apart from
these examples, only the two-domain proteins ERp29
and Mpd1p, and protein fragments of ERp57 and
ERp72 (bb¢ for ERp57 and a
0
a and bb¢ for ERp72)
have been crystallized. The best strategy for crystalliz-
ing PDIs with domains connected by flexible linkers
appears to be immobilization of their domains via sub-
strate or partner binding, or focusing on smaller frag-

ments instead of the whole protein.
The clearest evidence of interdomain flexibility
comes from the two crystal structures of yeast PDI
(Fig. 5A) [12,55]. When the structures are superposed,
the catalytic domains clearly adopt different positions.
The ability of human PDI to adopt open and closed
conformations was demonstrated by sedimentation
equilibrium and SAXS experiments [56,57]. Although
only one crystal form of ERp57 is available, compari-
son of a SAXS reconstruction of the protein free in
solution with the ERp57 ⁄ tapasin crystal structure sug-
gests mobility of the catalytic a and a¢ domains [13,16].
Among PDIs with x-linkers, mobility between the b¢
and a¢ domains is likely to be much more pronounced
than between the a and b domains, which have a short
interdomain linker. A recent study of domain mobility
in human PDI showed that the sites of greatest prote-
ase sensitivity are located between the b¢ and a¢
domains [58].
Another striking example of domain flexibility was
provided by recent SAXS, crystallographic and NMR
studies of ERp72 [18,38]. The crystal structures of the
a
0
a and bb¢ fragments allow modeling of the full-length
protein by overlaying the a and bb¢ domains onto the
ERp57 structure (Fig. 4B). Interestingly, this generates
PDI family of proteins G. Kozlov et al.
3930 FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS
a potential substrate binding site comprising the cata-

lytic a
0
, a and a¢ domains. The ERp72 model presents
one possible orientation of the domains, but does not
illustrate the full range of possible interdomain confor-
mations. SAXS measurements of full-length ERp72
revealed multiple relative orientations of the domains,
with the greatest mobility between the a
0
and a
domains and the b¢ and a¢ domains [18]. Limited prote-
olysis revealed that the a¢ domain is cleaved most
rapidly from ERp72, probably due to flexibility of the
x-linker (P.M., unpublished data). The recently deter-
mined a
0
a structure illustrates the difficulty in drawing
conclusions about interdomain mobility based on crys-
tal structures alone [38]. Although the a
0
a crystal
structure suggests a single conformation, NMR chemi-
cal shifts in the a
0
a fragment and a
0
domains suggest
that the two domains do not form a rigid pair in solu-
tion (G.K., unpublished data).
The bb

¢
domains form a rigid spacer
between catalytic domains
Although future studies will better define the structural
diversity of PDIs, an obvious feature is the limited
mobility of the bb¢ fragment in the four- and five-
domain PDIs. A structural overlay of this region from
human PDI, ERp57, ERp72 and yeast PDI showed
striking similarity in the domain orientations (Fig. 5).
As there are two crystal structures available for yeast
PDI (at 4 °C and room temperature) and for ERp57
(full-length and the bb¢ fragment), more conclusive
comparisons can be made concerning the rigidity of
these bb¢ pairs. The bb¢ domains of the two yeast PDI
structures superpose with an rmsd of 1.7 A
˚
over 210
Ca atoms. Much greater variability is observed in the
positions of the catalytic a and a¢ domains (Fig. 5A).
The bb¢ domain orientation is also similar in human
and yeast PDI (Fig. 5B). Likewise, overlay of the bb¢
structures from ERp57 and ERp72 results in an rmsd
of 1.7 A
˚
for backbone atoms (Fig. 5C). The difference
results from a small (10°) rotation at the interdomain
interface. A similar comparison between the two full-
length ERp57 molecules in the crystal structure with
tapasin shows the bb¢ domains overlay with an rmsd
of 1.1 A

˚
[13]. These examples indicate that the bb¢ tan-
dem forms a relatively rigid base, providing a spacer
for the attachment of more mobile active site domains
that can access substrates from opposite sides simulta-
neously. The bb¢ domains may also jointly contribute
to functional substrate or protein binding sites. As one
example, the CNX binding site of ERp57 includes a
contribution from Lys214 in the b domain in addition
to the residues in the b¢ domain, which form the
majority of the binding site [16]. In contrast to the bb¢
base, the catalytic domains in the available structures
show a much larger degree of mobility, which may be
important for recognition of protein substrates of vari-
able sizes as well as adjustment to conformational
changes in substrate during folding and disulfide rear-
rangement. In the proteins most closely related to
PDI, the a–b linker is generally very short, while the
b¢–a¢ linker (x-linker) is significantly longer. It is very
likely that other PDI-like proteins such as PDIp and
PDILT will display a similar structural arrangement of
their domains.
The recently determined structure of yeast Mpd1p
shows a very different orientation of its two thioredox-
A
BC
DE
Fig. 5. The non-catalytic bb¢ fragment provides a relatively rigid
base in PDIs containing four or five thioredoxin-like domains, while
allowing greater mobility of the catalytic domains. (A) An overlay of

the yeast PDI structures crystallized at 4 °C (pink) and room tem-
perature (yellow) shows high similarity in the orientation of the bb¢
domains and significant differences in orientation of their a and a¢
domains. The catalytic cysteines (orange) of the 4 °C yeast PDI
structure face each other. (B) The b and b¢ domains (gray) of
human PDI are oriented similarly to those from yeast PDI crystal-
lized at room temperature (pink). (C) An overlay of the bb¢ struc-
tures of ERp57 (green) and ERp72 (turquoise) shows a very similar
domain orientation. (D) An overlay of the bb¢ structures of ERp57
(green) and ERp44 (brown) shows roughly similar domain orienta-
tion. (E) Representation of the Mpd1p structure with the N-terminal
a domain shown in similar orientation to the b domains in (A)–(D).
The structure, colored blue to red from the N- to C-terminus,
reveals the C-terminal helix contacts the a domain.
G. Kozlov et al. PDI family of proteins
FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS 3931
in-like domains, which are locked in a rigid orientation
by numerous interdomain contacts (Fig. 5E) [15]. This
led to suggestions that other PDIs cannot be reliably
modeled using yeast PDI and ERp57 structures. Nev-
ertheless, there are several reasons why this argument
may not apply to most mammalian PDIs. Mpd1p does
not have a close structural homolog in the mammalian
ER. Only its N-terminal catalytic domain has signifi-
cant sequence homology to human PDIs P5 (39%
identity) and ERdj5 (34% identity), while the C-termi-
nal non-catalytic domain has no detectable sequence
similarity to human proteins. In contrast, mammalian
PDIs have significant (30–40%) sequence identity
(Table 1) that translates into structural similarity.

Although the non-catalytic domains have low sequence
similarity, there is strong sequence similarity between
the human PDI bb¢ domains (residues 136–354) and
PDIp (39% identity), ERp27 (32%), PDILT (27%)
and ERp72 (24%). The sequence identity of 28%
between the bb¢ domains of ERp57 and ERp72 results
in a strikingly similar domain orientation, and the bb¢
domains of PDI, ERp57 and ERp72 adopt similar rel-
atively rigid conformations. Based on this, the non-cat-
alytic domains of ERp27 are expected to adopt a very
similar structure. Although the bb¢ domains of ERp44
show a somewhat different interdomain angle, the ori-
entation is still largely similar to other known struc-
tures of mammalian PDIs (Fig. 5D), and could reflect
the effect of the protein C-terminus.
In contrast, the a–b interfaces of PDIs are more dif-
ficult to compare to one another. Indeed, even within
the same protein, the a–b or b¢–a¢ interfaces assume
different orientations (Fig. 5A). As Mpd1p has only
two domains, it may adopt a unique orientation to
provide the catalytic, substrate binding and partner
binding sites afforded by the four domains in ERp57.
These observations suggest that Mpd1p is a structural
outlier when compared to mammalian PDIs.
Table 1. Sequence identity (%) between human protein disulfide
isomerases.
Domains PDI PDIp PDILT ERp57 ERp72
PDI (residues
26–471)
abb¢a –49 32 34 35

PDIp (residues
44–492)
abb¢a¢ 49 – 33 30 31
PDILT (residues
45–490)
abb¢a¢ 32 33 – 23 25
ERp57 (residues
27–478)
abb¢a¢ 34 30 23 – 42
ERp72 (residues
179–632)
abb¢a¢ 35 31 25 42 –
A
BC
DE
Fig. 6. Structural organization of the x-linker. (A) Representation of
the full-length ERp57 structure with the region recognized as the
x-linker colored in red. (B) The N-terminal part of the x-linker folds
against the b¢ domain in the structures of the ERp57 bb¢ fragment
(blue), full-length ERp57 with tapasin (green) and the ERp72 bb¢
fragment (turquoise). The side chain of Leu361 in the ERp57 x-lin-
ker inserts into a cavity formed by three aromatic side chains. This
interaction is also observed in ERp72, involving residues Leu508 of
the x-linker and residues Phe484, Phe499 and Phe503 of the b¢
domain. Towards the middle of the x-linker, Tyr364 of ERp57 (and
Val511 of ERp72) fit into a small hydrophobic pocket below the
C-terminus of helix a3 of the b¢ domain. For clarity, ERp72 residues
are not labeled and side chains of the ERp57–tapasin structure are
not shown. (C) The structures of the b¢ domains of human PDI
(gray) and the two crystal forms of yeast PDI (yellow and pink)

show similar interactions. Ile334 of the x-linker is inserted into the
pocket formed by the three aromatic side chains Phe325, Phe329
and Tyr310 of human PDI (gray). In yeast PDI, the corresponding
residues are Ala361 from the x-linker and Tyr325, Leu352 and
Phe356 of the b¢ domain. (D) An overlay of the a¢ domain from full-
length ERp57 (green) and the isolated domain (brown) of ERp57
shows a similar structure for the C-terminal part of the x-linker
between residues L365 and P377. The N-terminal part of the x-lin-
ker is disordered in the NMR structure of the isolated a¢ domain.
Only one of many possible conformations is shown. (E) An overlay
of the a¢ domains of full-length yeast PDI crystallized in various
crystal forms at 4 °C (yellow) and room temperature (pink) shows
very similar conformations of the C-terminal region of the x-linker
between Ser367 and Ser377.
PDI family of proteins G. Kozlov et al.
3932 FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS
The x-linker consists of distinct
structural regions
The x-linker is a conserved stretch of approximately 20
residues connecting the b¢ and a¢ domains [5]. Recent
progress in the structural characterization of a number
of PDIs has provided new insights into the structural
role of this region. The structures of the full-length
proteins ERp57 and yeast PDI reveal that the linker
consists of two distinct regions (Fig. 6A) [12,13]. The
N-terminal part (approximately seven residues) folds
onto the b¢ domain and runs perpendicular to the
strand b5. The interactions are mostly mediated by the
hydrophobic residues (Leu361 and Tyr364 in ERp57)
that contact the hydrophobic surface at the edge of the

b sheet (Fig. 6B). In ERp57, the side chain of Leu361
occupies a hydrophobic pocket formed by the aromatic
rings of Phe336, Phe352 and Tyr356 (Fig. 6B). These
interactions are conserved in the structures of ERp72
and PDI, suggesting that the N-terminal region of the
linker is an integral part of the b¢ domain (Fig. 6B,C).
In agreement with this conclusion, removal of the
x-linker decreases the midpoint for denaturation of the
PDI b¢ domain from 2.32 m guanidinium chloride to
1.65 m [7].
In contrast, the C-terminal portion of the x-linker
takes on strikingly different conformations in the
structures of the isolated b¢ domain and full-length
proteins. In the crystal structure of the b¢x fragment of
human PDI, the C-terminal part of the x-linker turns
back to contact the b¢ hydrophobic surface between
helices a1 and a3 [17]. In the full-length proteins
ERp57 (Fig. 6D) and yeast PDI (Fig. 6E), the C-termi-
nal half of the x-linker is structurally associated with
the a¢ domain, and displays an irregular conformation
without conserved salt bridges or hydrophobic interac-
tions with the a¢ domain. Overlay of structures from
full-length ERp57 and the isolated ERp57 a¢ domain
(PDB accession numbers 3f8u and 2dmm) shows that
the linker structure is preserved even in the absence of
the preceding domain (Fig. 6D). At least three NMR
solution structures of a¢ domains from human PDI
(PDB accession number 1x5c), human ERp72 (PDB
accession number 2dj3) and H. insolens PDI (PDB
accession number 2djj) have been obtained without

residues comprising the x-linker. The a¢ domain from
rat ERp72 is also well-folded on its own [38]. This sup-
ports the idea that the C-terminal part of the x-linker
loosely interacts with the a¢ domain and does not con-
tribute to its structural integrity. As discussed previ-
ously, studies with both PDI and ERp72 have shown
significant interdomain mobility at the b¢a¢ domain
interface.
N- and C-terminal extensions
A number of PDIs contain N- or C-terminal tails out-
side the thioredoxin-like domains (Fig. 1). The C-ter-
minal tail of yeast PDI forms a protruding a helix [12].
The C-terminus of ERp44 forms short helical turns
while folding back and interacting with the b¢ and a
domains [10]. Likewise, the C-terminus of Mpd1p
binds to the N-terminal domain as an a helix [15].
Despite these examples, the tails of many mammalian
PDIs are unlikely to be structured due to their low
sequence complexity and highly charged nature. In
particular, the C-terminal tail of PDI and N-terminal
tail of ERp72 are very acidic, while the C-terminus of
ERp57 is positively charged. NMR spectra suggest
that the above-mentioned acidic stretches are unstruc-
tured in solution (unpublished data). They could
become more structured during interaction with
ligands or protein partners. These acidic extensions
have been previously implicated in calcium binding,
and recent circular dichroism measurements showed
that a similar extension of the ER luminal chaperone
CRT becomes structured upon binding calcium [59].

Although not generally critical for the disulfide isomer-
ase activity, the N- and C-termini of PDIs may also be
important for mediating protein–protein interactions
with other ER chaperones [60].
Concluding remarks
The mammalian PDI family currently consists of 20
proteins with diverse functions in oxidative folding of
protein substrates in the ER. As the availability
of structures for the PDI family grows, the functions
of its members are becoming clearer. Most PDIs con-
sist of multiple thioredoxin-like domains with a similar
organization: central non-catalytic domains that often
form a rigid scaffold for binding substrate or partner
chaperones, surrounded by more mobile catalytic
domains with active site cysteines. The mobility of cat-
alytic domains may be beneficial when acting upon
incorrectly disulfide-bonded proteins and substrates of
various sizes. This model of PDI function is consistent
with sequence analysis showing that the non-catalytic
domains have the greatest sequence diversity while the
catalytic domains are more highly conserved, especially
in regions flanking the Cys-x-x-Cys catalytic sites.
It is currently unclear why most PDIs have multiple
catalytic domains. One possibility is that these
domains are involved in oxidative folding of complex
substrates with many disulfide bonds. Another possi-
bility is that multiple catalytic domains enhance
the avidity for substrates, or provide modules that
G. Kozlov et al. PDI family of proteins
FEBS Journal 277 (2010) 3924–3936 ª 2010 The Authors Journal compilation ª 2010 FEBS 3933

determine substrate or partner specificity. Structural
studies of PDI–substrate complexes using trapping
mutants may provide further insight into the roles of
the multiple catalytic domains.
PDIs are the workhorses of oxidative protein folding
in cells. Future research should better define the pool
of substrates for each particular PDI. Structures pro-
vide valuable clues as to how interactions with sub-
strates are mediated, whether directly or through
partner proteins. Although many PDI structures
remain unsolved, the recent surge of structural studies
of PDIs has led to clear advances in understanding
how PDIs function in cells.
Acknowledgements
K.G. and D.Y.T. acknowledge the financial support
received from the Canadian Institutes of Health
Research. K.G. is a Chercheur National of the Fonds
de la Recherche en Sante
´
de Que
´
bec. P.M. is sup-
ported by a Bourse de Doctorat from the Fonds de la
Recherche en Sante
´
de Que
´
bec.
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