Báo cáo khoa học: The periplasmic peptidyl prolylcis–transisomerases PpiD and SurA have partially overlapping substrate specificities doc

The periplasmic peptidyl prolyl cis–trans isomerases
PpiD and SurA have partially overlapping substrate
specificities
Krista H. Stymest and Peter Klappa
Department of Biosciences, University of Kent, Canterbury, UK
During the process of protein folding, several poten-
tially rate-limiting steps can occur, one of which is the
cis–trans isomerization of proline peptide bonds, catal-
ysed by a ubiquitously expressed group of peptidyl
prolyl cis–trans isomerases (PPIases) [1,2]. Three major
classes of PPIases have been identified in both prok-
aryotes and eukaryotes: the cyclophilins, i.e. cyclospo-
rin A-binding proteins, the FK506-binding proteins,
and PPIases that have sequence similarity with the
catalytic domain of the small PPIase parvulin [3,4].
The periplasmic space of Escherichia coli contains all
three classes of PPIases, namely PpiA, a cyclophilin-
type PPIase, FkpA, a FK506-binding PPIase, and two
members of the parvulin-family, SurA and PpiD.
SurA was originally isolated as a protein product of
stationary-phase survival genes of E. coli [5–7]. Null
mutants or mutants with reduced levels of SurA in the
periplasm lead to a significant decrease in folding of
the maltose-inducible porin LamB and the trimeric
porins OmpC and OmpF [5,6]. It has been shown that
SurA is important in maintaining outer membrane
integrity by facilitating maturation of integral outer
membrane proteins [8–10]. SurA contains two domains
with high sequence similarity to parvulin, in addition
Keywords
peptidyl prolyl cis–trans isomerase;

periplasmic space; protein folding; protein–
protein interaction; substrate specificities
Correspondence
P. Klappa, Department of Biosciences,
University of Kent, Canterbury CT2 7NJ, UK
Fax: +44 1227 763912
Tel: +44 1227 823515
E-mail:
(Received 19 February 2008, revised 14
April 2008, accepted 6 May 2008)
doi:10.1111/j.1742-4658.2008.06493.x
One of the rate-limiting steps in protein folding has been shown to be the
cis–trans isomerization of proline residues, catalysed by a range of peptidyl
prolyl cis–trans isomerases (PPIases). In the periplasmic space of Escherichia
coli and other Gram-negative bacteria, two PPIases, SurA and PpiD, have
been identified, which show high sequence similarity to the catalytic domain
of the small PPIase parvulin. This observation raises a question regarding
the biological significance of two apparently similar enzymes present in the
same cellular compartment: do they interact with different substrates or do
they catalyse different reactions? The substrate-binding motif of PpiD has
not been characterized so far, and no biochemical data were available on
how this folding catalyst recognizes and interacts with substrates. To char-
acterize the interaction between model peptides and the periplasmic PPIase
PpiD from E. coli , we employed a chemical crosslinking strategy that has
been used previously to elucidate the interaction of substrates with SurA.
We found that PpiD interacted with a range of model peptides indepen-
dently of whether they contained proline residues or not. We further dem-
onstrate here that PpiD and SurA interact with similar model peptides, and
therefore must have partially overlapping substrate specificities. However,
the binding motif of PpiD appears to be less specific than that of SurA,

indicating that the two PPIases might interact with different substrates. We
therefore propose that, although PpiD and SurA have partially overlapping
substrate specificities, they fulfil different functions in the cell.
Abbreviations
DSG, disuccinimidyl glutarate; PDI, protein disulfide isomerase; PPIase, peptidyl prolyl cis–trans isomerase; scRNase, ‘scrambled’
ribonuclease A.
3470 FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS
to an N-terminal peptide-binding domain (Fig. 1).
In vitro, this N-terminal domain has the properties of
a molecular chaperone, i.e. it prevents heat-denatured
citrate synthase from aggregation [8], and is essential
and sufficient for interaction with model peptides [11].
Recently, it was reported that SurA has a bi-partite
substrate-binding area: while the N-terminal peptide-
binding site appears to interact with unfolded proteins,
the first parvulin domain confers substrate specificity
by interacting specifically with aromatic residues in the
tested peptides [12]. Studies with a recombinant frag-
ment of SurA revealed that only the second, C-termi-
nal, parvulin domain has catalytic activity [8].
PpiD was discovered in a genetic screen as a multi-
copy suppressor of a surA deletion strain [13]. PpiD is
anchored to the inner membrane via a single N-termi-
nal transmembrane segment, with the catalytic parvu-
lin-like domain (Fig. 1) facing the periplasmic space
[13]. Deletion of the ppiD gene leads to an overall
reduction in the level and folding of outer membrane
proteins. The simultaneous deletion of both ppiD and
surA genes was found to confer synthetic lethality, and
therefore it was suggested that these PPIases have an

overlapping substrate specificity [14]. However, this
observation was recently disputed by Justice et al. [15]:
in their experiments, a ppiD ⁄ surA double mutant did
not show any loss in viability and only a quadruple
mutant lacking all four periplasmic PPIases (SurA,
PpiD, PpiA and FkpA) showed a significant decrease
in the growth rate.
The presence of two enzymes with apparently similar
catalytic properties in the same compartment raises a
question regarding their biological function. Do they
catalyse different reactions or do they interact with dif-
ferent substrates? The substrate-binding motif of SurA
was recently identified, and the enzyme was shown to
bind to proteins with the motif Ar-X-Ar, or modifica-
tions of it, where Ar is an aromatic residue [16,17].
However, the substrate-binding motif of PpiD has not
been characterized so far, and there are no biochemical
data available on how this folding catalyst recognizes
and interacts with its substrates.
To address the question of how PpiD interacts with
its substrates, we used chemical crosslinkers, which are
powerful tools for the study of interactions between
proteins, and can be applied to proteins that are avail-
able in small amounts even in crude cell extracts
[18–20].
Results
To investigate how PpiD interacts with its substrates,
and to compare its substrate specificity with that of
SurA, we overexpressed PpiD as an N-terminally
hexahistidine-tagged protein without leader sequence

or transmembrane domain (Fig. 1). SurA was
expressed as full-length mature protein in which a
hexahistidine tag replaced the leader sequence, as pre-
viously described [11]. Expression of both PPIases
resulted in high yields of the respective proteins. The
molecular masses for SurA and PpiD were approxi-
mately 45 and 62 kDa, respectively. Sedimentation
analysis showed that both proteins were found pre-
dominantly in the soluble fraction, with < 5% in the
pellet (data not shown).
Using purified recombinant proteins for crosslinking
experiments has occasionally been observed to produce
false-positive results, as model substrates with very
weak binding affinities can also be crosslinked to the
purified protein (P. Klappa, unpublished results). To
avoid such crosslinking artefacts, all crosslinking
experiments were routinely carried out with whole
E. coli cell lysates expressing the respective PPIases
unless otherwise stated.
We observed that addition of the chemical cross-
linker disuccinimidyl glutarate (DSG) to a lysate exp-
ressing recombinant PpiD resulted in crosslinking
products [collectively indicated as (b)] with an appar-
ent molecular mass of approximately 80 kDa (Fig. 2,
lane 4), in addition to unmodified PpiD (a). Identical
crosslinking products were also detected with purified
PpiD (Fig. 2, lane 2), indicating that these additional
crosslinking products were not proteins from the
E. coli lysate interacting with PpiD. Both (a) and (b)
were recognized by an antibody directed against PpiD

(data not shown), whereas anti-hexahistidine serum
only recognized band (a) (compare with Fig. 3, lanes 6
and 7). Electrospray mass spectroscopy analysis indi-
Fig. 1. Domain constructs of recombinant SurA and PpiD. The
numbering is based on the full-length sequences of SurA and PpiD.
S, signal sequence; Pep, peptide-binding domain; TM, transmem-
brane portion; Parv, parvulin-like domain; ?, segments of unknown
function. The substrate-binding domain of SurA has been reported
to be located in the peptide-binding domain and the first parvulin
domain [12].
K. H. Stymest and P. Klappa Substrate interactions of periplasmic PPIases
FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS 3471
cated that addition of DSG generates a heterogeneous
mixture of DSG–PpiD adducts. However, as DSG
modifies lysine residues and thus changes the m ⁄ z
ratio, a detailed analysis of the crosslink products (b)
was not possible. From the observation that all bands
were recognized by anti-PpiD serum, but only band (a)
was detected using anti-hexahistidine serum, we pro-
pose that, in conformation (b), the N-terminal hexahis-
tidine tag is no longer exposed and is therefore not
recognized by the hexahistidine antibody. No such
effect was observed for chemical crosslinking of SurA
(data not shown), indicating that the reduced mobility
of conformation (b) did not merely result from the
attachment of the chemical crosslinker to a hexahisti-
dine tag.
We believe that the bands (b) are most likely cross-
linking artefacts that change the mobility of PpiD in
SDS–PAGE, but do not affect substrate binding.

PpiD, like SurA, interacts with a misfolded
protein
To investigate whether PpiD interacts with a misfolded
protein, we used ‘scrambled’ ribonuclease A (scRNase)
as a model substrate. We previously demonstrated that
biotinylated scRNase could be chemically crosslinked
to purified SurA [11]. Here we extended our studies to
determine whether this technique could also be
employed to study the binding properties of PpiD. To
avoid potential artefacts through interaction with the
biotin moiety, we used unmodified scRNase, which
was incubated with crude E. coli lysates expressing
recombinant SurA or PpiD. Samples without scRNase
and DSG served as controls. Analysis was carried out
by Western blotting of the gels, with subsequent detec-
tion using anti-hexahistidine serum (Fig. 3). In the
absence of DSG or scRNase, no crosslinking products
could be detected (lanes 1–3 for SurA and 6–8 for
PpiD, respectively). In the presence of DSG and scRN-
ase, a single specific crosslinking product was observed
for PpiD, with an approximate molecular mass of
75 kDa (lane 9). Crosslinking of scRNase to an E. coli
lysate expressing SurA resulted in a crosslinking prod-
uct with an approximate molecular mass of 60 kDa
(lane 4). This result clearly showed that PpiD, like
SurA, interacts with misfolded proteins. That this
interaction was dependent on the native conformation
of the PPIases was demonstrated by heat inactivation
of the lysates. After heat treatment of the lysates
(5 min at 95 °C) prior to the addition of scRNase and

chemical crosslinking, the interaction between scRNase
and the PPIases was strongly reduced (lanes 5 and 10,
respectively).
PpiD, like SurA, interacts with a radiolabelled
model peptide without proline residues
To facilitate analysis of the recognition motif of PpiD,
we used a radiolabelled peptide, D-somatostatin, with
the amino acid sequence AGSKNFFWKTFTSS,
as a model substrate. [
125
I]-Bolton–Hunter-labelled
D-somatostatin was chemically crosslinked to recombi-
Fig. 2. Chemical crosslinking of PpiD. Purified PpiD (5 lM, Pur) or
E. coli lysate (lys) expressing recombinant PpiD in a total volume of
10 lL were incubated with DSG (final concentration 0.5 m
M) in buf-
fer B for 60 min at 0 °C or were left untreated. The samples were
then analysed on 10% polyacrylamide gels with subsequent
Coomassie Brilliant Blue staining. M, molecular mass marker.
Fig. 3. Interaction of PPIases with scRNase. E. coli lysates
expressing recombinant SurA or PpiD, respectively, were heat-inac-
tivated at 95 °C for 5 min (lanes 5 and 10) or left untreated. The
samples were then incubated with 100 l
M scRNase or buffer B
prior to crosslinking with DSG. After crosslinking, the samples were
analysed on 10% polyacrylamide gels with subsequent electro-
transfer onto poly(vinylidene) fluoride (PVDF) membranes. The sam-
ples were probed with primary anti-hexahistidine serum, raised in
mouse, and secondary anti-mouse serum conjugated to horseradish
peroxidise, and visualized by enhance chemiluminescence. Cross-

linking products are indicated with an asterisk. The positions of the
molecular mass markers are indicated.
Substrate interactions of periplasmic PPIases K. H. Stymest and P. Klappa
3472 FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS
nant PpiD overexpressed in E. coli cell lysate
(Fig. 4A). Crosslinking of the radiolabelled peptide to
a cell lysate expressing SurA served as a positive con-
trol, while a cell lysate without recombinant SurA or
PpiD, served as a negative control (lanes 1 and 2). Cell
lysates that contained recombinant PPIases showed
crosslinking products, indicated by arrows (lanes 4 and
7, respectively). The lysate expressing SurA showed
only one specific crosslinking product, with an approx-
imate molecular mass of 47 kDa, but the lysate
expressing PpiD showed two specific crosslinking prod-
ucts with molecular masses of 64 and 82 kDa. As the
addition of chemical crosslinker to PpiD resulted in
two bands [(a) and (b), see Fig. 2], we believe that
these crosslinking products are most likely due to
interaction of the two PpiD bands with the radio-
labelled peptide. In the absence of DSG, no crosslink-
ing products were detected (Fig. 4A, lanes 3 and 6).
Heat inactivation of the lysates (5 min at 95 °C) prior
to addition of the peptide and chemical crosslinking
inhibited the interaction between radiolabelled peptide
and the PPIases (lanes 5 and 8), indicating that the
interaction was specific for native proteins. We
observed that all cell lysates, irrespective of whether
they expressed recombinant PPIases or not, showed an
unidentified crosslinking product of approximately

25 kDa, marked x, which has been observed previously
[20]. We also noted that the interaction between PpiD
and radiolabelled D-somatostatin was much weaker
A
B
C
Fig. 4. Interaction of PPIases with radiolabelled D-somatostatin.
(A) [
125
I]-Bolton–Hunter-labelled D-somatostatin (33 lM) was incu-
bated with E. coli lysates expressing recombinant SurA, PpiD or nei-
ther of the PPIases (Lys) in buffer B for 10 min at 0 °C in a total
volume of 10 lL. As controls, E. coli lysates expressing recombinant
SurA or PpiD, respectively, were heat-inactivated at 95 °C for 5 min
(lanes 5 and 8) prior to addition of the radiolabelled peptide. Samples
were subsequently incubated with DSG (final concentration 0.5 m
M)
for 60 min at 0 °C or were kept untreated. After crosslinking, the
samples were analysed on 10% polyacrylamide gels with subse-
quent autoradiography. The positions of the molecular mass markers
are indicated. (B) [
125
I]-Bolton–Hunter-labelled D-somatostatin
(33 l
M) was incubated with E. coli lysates expressing recombinant
SurA or PpiD in the presence of 0.2% w ⁄ v Triton X-100 prior to
crosslinking with DSG. After cross-linking, the samples were analy-
sed on 10% polyacrylamide gels with subsequent autoradiography.
The positions of the molecular mass markers are indicated. (C) [
125

I]-
Bolton–Hunter-labelled D-somatostatin (D-som, 10 l
M) was incu-
bated with an E. coli lysate expressing recombinant PpiD in the
absence or presence of 50 l
M (+) or 100 lM (++) scRNase. After
crosslinking with DSG, the samples were analysed on 10% poly-
acrylamide gels with subsequent electrotransfer onto poly(viny-
lidene) fluoride (PVDF) membranes. The membranes were
subjected to autoradiography (upper panel) and subsequently probed
with primary anti-hexahistidine serum, raised in mouse, and second-
ary anti-mouse serum conjugated to horseradish peroxidise, and
visualized by enhance chemiluminescence (Western blot, lower
panel). Crosslinking products are indicated by arrows. Exposure
times for samples containing PpiD were on average 20 times longer
than for samples containing SurA. ‘a’, crosslinking to PpiD band (a);
‘b’, crosslinking to PpiD band (b); x, unspecified crosslinking product.
K. H. Stymest and P. Klappa Substrate interactions of periplasmic PPIases
FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS 3473
than that between SurA and the peptide, as exposure
of the PpiD crosslinking autoradiographs for 20 times
longer was required to obtain similar intensities of the
crosslinking products. As the intensities of the scRN-
ase crosslinking products were comparable between
SurA and PpiD (compare with Fig. 3), we speculate
that PpiD might bind preferentially to misfolded pro-
teins rather than short peptides (see below).
Qualitatively and quantitatively identical results were
obtained when we substituted the E. coli lysates by
purified SurA or PpiD, with the exception that the

unspecific crosslinking product, denoted x, was no
longer detectable (data not shown). Using an alterna-
tive chemical crosslinker, bis(sulfosuccinimidyl) suber-
ate, gave quantitatively and qualitatively identical
results (data not shown).
It is important to point out that D-somatostatin
does not contain any proline residues, and therefore it
is clear that the presence of a proline residue in the
model peptide is not essential for interaction with
PpiD.
We recently demonstrated that the binding of pep-
tides to purified SurA required hydrophobic interac-
tions [11], and we therefore wished to determine
whether this is also the case for the interaction of pep-
tides with PpiD. Radiolabelled D-somatostatin was
chemically crosslinked to a cell lysate overexpressing
recombinant PpiD in the absence or presence of Tri-
ton X-100. A cell lysate expressing SurA served as a
positive control. As with SurA, binding of radiola-
belled D-somatostatin to PpiD was strongly inhibited
in the presence of Triton X-100 (Fig. 4B). This experi-
ment demonstrated that the interaction of PpiD with
peptides, like that of SurA, is detergent-sensitive and
presumably requires hydrophobic interactions. A simi-
lar observation was made for interaction between the
radiolabelled peptide and the unidentified protein
giving rise to the crosslinking product indicated x.
To demonstrate that the peptide-binding site in
PpiD is identical to the site that interacts with a mis-
folded protein, we carried out a competition experi-

ment (Fig. 4C). Chemical crosslinking of 10 lm
radiolabelled D-somatostatin to a cell lysate expressing
PpiD was reduced in the presence of excess scRNase
(100 or 50 lm) (Fig. 4C, upper panel, lane 1 versus
lanes 2 and 3). To show that the competition is not
due to a crosslinking artefact, e.g. quenching of the
crosslinker by the excess of scRNase, we also per-
formed a Western blot of the same gel, and detected a
crosslinking product between scRNase and PpiD using
anti-hexahistidine serum (lower panel, lanes 2 and 3).
From this experiment, we concluded that excess of
scRNase could replace the radiolabelled peptide. We
therefore suggest that peptides and misfolded proteins
interact with the same substrate binding site in PpiD.
Interestingly, when we carried out a competition
experiment using an excess of unlabelled D-somato-
statin (100 lm) to inhibit the binding of scRNase
(20 lm), we could not detect any competitive effects
(data not shown). The most likely interpretation of this
result is that PpiD has a higher affinity for the mis-
folded protein than for a small peptide (see below).
Taken together, our results indicated that there is no
principal difference between the two PPIases SurA and
PpiD with respect to interaction with a misfolded pro-
tein or a radiolabelled model peptide. PpiD interacted
with a misfolded protein and a radiolabelled model
peptide that did not contain any proline residues. The
interactions were detergent-sensitive and required
native PpiD.
PpiD and SurA interact with different peptides

To address the question of whether PpiD showed simi-
lar substrate specificity to that of SurA, we chemically
crosslinked various radiolabelled peptides to SurA or
PpiD (Fig. 5A). A lysate expressing human archetypal
protein disulfide isomerase (PDI) served as a control
to demonstrate that absence of a crosslinking product
is not due to low efficiency of radiolabelling of the
peptide. PDI interacted with all the peptides tested to
a similar extent, indicating that each peptide could be
crosslinked with similar efficacy. We found that SurA
showed strong interactions with D-somatostatin and
somatostatin, while other peptides gave only weak
crosslinking signals. In contrast, PpiD showed a strong
interaction with most peptides tested. However, we
noted that the interaction with radiolabelled D-somato-
statin was rather weak compared to that with other
peptides. For all the peptides tested, we observed
crosslinking products related to the PpiD bands (a)
and (b).
For efficient crosslinking of a peptide to a protein,
correct spatial orientation of the crosslinked residues
in the target molecules is essential. It is therefore con-
ceivable that failure to detect crosslinking products in
the case of SurA and peptides other than D-somato-
statin and somatostatin was due to unfavourable ori-
entation of crosslinkable residues. We therefore carried
out competition experiments, using cell lysates express-
ing recombinant SurA or PpiD, and radiolabelled
D-somatostatin (10 lm) in the presence of an excess of
unlabelled peptides (100 lm). Although D-somatostatin

did not show very strong binding to PpiD, we used it
in our experiments to enable direct comparison with
SurA. Competition between radiolabelled D-somato-
Substrate interactions of periplasmic PPIases K. H. Stymest and P. Klappa
3474 FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS
statin (10 lm) and unlabelled D-somatostatin (100 lm)
served as a positive control. Figure 5B shows a typical
example of these experiments.
Unlabelled peptide M did not compete with radiola-
belled D-somatostatin for binding to SurA, but showed
competition for binding to PpiD. This result is in
excellent agreement with the results in Fig. 5A, in
which radiolabelled peptide M interacts with PpiD,
but not SurA. Unlabelled peptide A, however, inhib-
ited the interaction between radiolabelled D-somato-
statin and SurA, but was far less efficient with PpiD.
This result is in line with experiments using radio-
labelled peptide A: while radiolabelled peptide A
bound to SurA, no interaction with PpiD was detected
(data not shown).
Table 1 summarizes the competition experiments.
We used the competition between radiolabelled
D-somatostatin and unlabelled D-somatostatin as the
reference point: strong competition between radiola-
belled D-somatostatin and unlabelled peptide is indi-
cated by +++, no competition by ).
A
B
Fig. 5. Interaction of PPIases with model peptides. (A) The indi-
cated [

125
I]-Bolton–Hunter-labelled peptides (30 lM) were incubated
with E. coli lysates expressing recombinant SurA, PpiD or human
PDI in buffer B for 10 min at 0 °C in a total volume of 10 lL. After
crosslinking with DSG, the samples were analysed on 10% poly-
acrylamide gels with subsequent autoradiography. The positions of
the molecular mass markers are indicated. (B) [
125
I]-Bolton–Hunter-
labelled D-somatostatin (D-som, 10 l
M) was incubated with E. coli
lysates expressing recombinant SurA or PpiD in the presence of
100 l
M of the indicated unlabelled peptides. A sample without unla-
belled peptides served as controls. After crosslinking with DSG, the
samples were analysed on 10% polyacrylamide gels with sub-
sequent autoradiography. ‘a’, crosslinking to PpiD band (a); ‘b’,
crosslinking to PpiD band (b); x, unspecified crosslinking product.
Table 1. Competition between peptides and radiolabelled
D-somatostatin for binding to SurA and PpiD. E. coli cell lysates
expressing recombinant SurA or PpiD were incubated with 10 l
M
radiolabelled D-somatostatin in the presence of a 10-fold excess of
the indicated unlabelled peptides prior to crosslinking. Samples
were subsequently incubated with DSG (final concentration
0.5 m
M) for 60 min at 0 °C. After crosslinking, the samples were
analysed on 10% polyacrylamide gels with subsequent autoradiog-
raphy. Quantification was performed with a Bio-Rad (Hemel Hemp-
stead, UK) phosphoimager. A sample without competing peptides

served as a control. Competition was expressed as reduction in the
intensity of the respective crosslinking product relative to the con-
trol without unlabelled peptides. +++, strong competition (intensity
of crosslinking product < 10% of control); ++, moderate competi-
tion (intensity of crosslinking product between 10% and 30% of
control); +, weak competition (intensity of crosslinking product
between 50% and 80% of control); ), no competition (intensity of
crosslinking product > 80% of control).
Peptide SurA PpiD
AGSKNFFWKTFTSS (D-som) +++ +++
AGCKNFFWKTFTSC (somatostatin) +++ +++
AASKNFFWKTFTSS +++ +++
AGAKNFFWKTFTSS +++ ++
AGSKAFFWKTFTSS ++ ++
AGSKNAFWKTFTSS (F6A) + +++
AGSKNFAWKTFTSS (F7A) + +
AGSKNFFAKTFTSS (W8A) ) +
AGSKNFFWKAFTSS +++ +++
AGSKNYFWKTFTSS (F6Y) + +++
AGSKNYFAKTFTSS (F7Y) ) +
AGSKNWFWKTFTSS +++ +++
AGSKNFFWKTFT +++ )
AGSKNFFWKT +++ )
AGSKNYFWKSAS (peptide S) ))
AGSKNFFWKS (peptide A) +++ )
AASKAFFWKS +++ )
AGSKNFFWAT +++ )
TKWFFNKSGA +++ )
- -SKNFFWKTFT +++ )
- -SKNFFWKT ))

- - - -NFFWKT ))
INLKALAALAKKIL (peptide M) ) +++
WEYIPNV + )
WEYIP ))
PTIKFFNGDTASPK (peptide P) ) +++
K. H. Stymest and P. Klappa Substrate interactions of periplasmic PPIases
FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS 3475
Our results demonstrate that changing the phenylal-
anine at position 6 to alanine (F6A) in the D-somato-
statin sequence prevented this peptide from competing
with the binding of radiolabelled D-somatostatin to
SurA. Interestingly, the same modified peptide showed
strong competition for the interaction of radiolabelled
D-somatostatin with PpiD. Changing the phenylalanine
at position 7 to alanine (F7A) reduced the competitive
effect of the peptide for the interaction with both SurA
and PpiD. A similar result was observed for the tryp-
tophan to alanine (W8A) modification. Changing the
phenylalanine at position 6 to a tyrosine (F6Y) pre-
vented the peptide from competing with the binding of
radiolabelled D-somatostatin to SurA, but not to
PpiD.
We noticed that peptides shorter than 14 amino
acids did not efficiently compete for the binding of
radiolabelled D-somatostatin to PpiD. Interestingly,
peptide S did not compete for the binding to PpiD,
although radiolabelled peptide S interacted with PpiD
(Fig. 5A, lane 13). This result is most likely due to the
increase in the size of peptide S during direct radio-
labelling: treatment of peptides with [

125
I] Bolton–
Hunter labelling reagent results in an increase in the
length of the peptide chain by one residue. We there-
fore suggest that the interaction of PpiD with model
peptides is dependent on their size.
Taken together, our results show that the interaction
between peptides and SurA is strongly dependant on
the motif FFW in the peptide, whereas the substrate
specificity for PpiD appears to be less specific.
Discussion
In native polypeptides, about 5–7% of the peptidyl
prolyl bonds are in the cis configuration, and almost
half of the 1453 non-redundant protein structures in
the protein database contain at least one cis peptidyl
prolyl bond [21]. Conversion of the trans peptidyl
prolyl bond into the cis conformation, which is cataly-
sed by peptidyl prolyl cis–trans isomerases, has been
reported to be essential for the correct folding of many
proteins, e.g. the folding of outer membrane proteins
[5,13] and periplasmic proteins as well as certain toxins
[22] in prokaryotes.
In the periplasmic space of E. coli, two PPIases,
SurA and PpiD, with sequence similarity to the cata-
lytic domain of parvulin have been identified. This
observation raises a question regarding the biological
significance of these two PPIases in the same cellular
compartment: do they interact with different sub-
strates or do they catalyse different reactions? The
simultaneous deletion of both ppiD and surA genes

was reported to confer synthetic lethality, and hence
it was suggested that the PPIases have overlapping
substrate specificity [14]. However, Justice et al. [15]
challenged this observation, showing that a double
deletion of the ppiD and surA genes did not result in
loss of viability.
To investigate how these PPIases interact with their
substrates and whether they have potentially overlap-
ping substrate specificities, we employed a crosslinking
approach, which we had used previously to determine
the interaction between peptides and other folding
catalysts [18,20,23].
PpiD interacts with model peptides and
a misfolded protein
We recently showed that model peptides and a mis-
folded protein, ‘scrambled’ RNase A, interacted specif-
ically with purified recombinant SurA from E. coli
[11]. Using a similar crosslinking approach, we demon-
strate here that a recombinant fragment of PpiD, lack-
ing the leader sequence and the transmembrane
segment, also interacted specifically with radiolabelled
model peptides and scRNase.
As with SurA, we found that the interaction
between PpiD and model peptides was independent of
the presence of a proline residue within the peptide.
This result is in line with the results for other protein
isomerases, such as PDI [18] and the PPIases trigger
factor [24] and FkpA [25]. As with PDI [18] and SurA
[11], the interaction of PpiD with model peptides is
sensitive to the presence of Triton X-100, which indi-

cates that hydrophobic interactions play a role in the
initial binding of peptides to PpiD. Triton X-100 is
widely used for the functional recovery of intracellular
soluble and membrane-bound proteins, and therefore
it is unlikely that the detergent interferes with the
structure of the binding site. This was confirmed by
our observation that addition of Triton X-100 did not
interfere with the interaction between scRNase and
PpiD or SurA (data not shown). We speculate that
the interaction between PPIases and a misfolded pro-
tein is stronger than that with a short peptide, proba-
bly due to a more extensive array of interactions,
which include interactions other than hydrophobic
interactions, e.g. electrostatic interactions and hyd-
rogen bonds.
Interaction of PPIases with model peptides
The substrate-binding motif of SurA was recently
identified, and the enzyme was shown to bind to
peptides with the motif Ar-X-Ar, or modifications of
Substrate interactions of periplasmic PPIases K. H. Stymest and P. Klappa
3476 FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS
it, where Ar is an aromatic residue [10,16,17]. Recent
reports also showed that SurA has affinity for pep-
tides enriched in aromatic residues with positively
charged residues in the vicinity [26]. Our results con-
firmed these observations; however, based on our
crosslinking competition data, we speculate that the
substrate specificity for SurA is more extensive and
comprises more amino acids. Furthermore, our data
support previous reports indicating that the conforma-

tion of the peptide plays an important role in binding
to SurA [8–10]. For example, peptide S contained the
required binding motif Ar-X-Ar, but was not found
to interact with SurA. It is unlikely that the lack of
the phenylalanine residue in position 11 (F11) was the
sole reason for this result, as peptide A, which also
lacked F11, showed efficient binding to SurA. We
therefore propose that the alanine and serine residues
at the C-terminus of peptide S induce a structural
change such that the Ar-X-Ar motif is no longer
accessible. Replacement of phenylalanine 6 (F6) with
alanine in D-somatostatin strongly reduced the inter-
action with SurA, but did not affect the interaction
with PpiD.
Interestingly, scRNase does not contain an Ar-X-Ar
motif; however, the efficient interaction between this
misfolded protein and SurA indicates that SurA
might recognize hydrophobic patches in a tertiary
structure, which places two or more aromatic amino
acids in close proximity. Alternatively, scRNase might
bind predominantly to the general substrate-binding
site as identified by Xu et al. [12]. Based on their
extensive study of interactions between model pep-
tides and SurA by X-ray crystallography, it was sug-
gested that the interaction between peptides and SurA
is facilitated by the N-terminal binding domain, as
well as the first parvulin domain. The N-terminal
binding domain appears to act as a general interac-
tion site for misfolded proteins, whereas the first
parvulin domain confers substrate specificity through

very specific interactions between the aromatic resi-
dues in substrate molecules and the binding site of
SurA [12].
PpiD, like SurA, can interact with aromatic residues
in the model peptide D-somatostatin. However, the
substrate specificity for the interaction with PpiD does
not appear to be restricted to aromatic residues, as
demonstrated by the binding of peptide M, which does
not contain aromatic residues. We therefore propose
that the substrate specificity of PpiD is less specific
than that for SurA. We speculate that the substrate
specificity of PpiD is determined more by the hydro-
phobicity of residues in the model peptides than the
presence of aromatic residues.
Biological significance
In our experiments, we confirmed the finding by Bitto
& McKay that the Ar-X-Ar binding motif is essential
for the interaction between SurA and model peptides
[16,17]. It was demonstrated that this is the signature
motif for specific outer membrane proteins, which have
been proposed to be the predominant substrates of
SurA [9,10]. Our results show that even short peptides
(< 11 amino acids) interacted with SurA, as long as
they contained this signature motif and probably
exhibited some other structural requirements [9]. The
interaction with PpiD, however, required a longer pep-
tide chain, with at least 13 amino acids. This difference
might reflect the different biological functions of the
two PPIases. We propose that the main biological
function of SurA is to facilitate the folding of predom-

inantly outer membrane proteins, which might contain
a simple signature motif for the interaction with SurA.
Although the precise mechanisms of targeting of outer
membrane proteins to the outer membrane has not yet
been established [27], it is likely that translocation of
most outer membrane proteins from the inner to the
outer membrane occurs via the periplasmic space, thus
allowing soluble SurA to efficiently interact with incor-
rectly folded substrates. PpiD, however, is anchored to
the inner membrane, with the catalytically active
site facing the lumen of the periplasmic space. This
particular localization makes it likely that PpiD is pre-
dominantly involved in the folding of inner membrane-
associated proteins rather than soluble proteins. PpiD
might therefore interact with a variety of slowly fold-
ing proteins, for which a simple and specific recogni-
tion motif does not exist. We therefore propose that
PpiD has a broader substrate specificity than that of
SurA: while PpiD acts as a general folding catalyst rec-
ognizing various binding motifs, SurA requires only a
short and characteristic recognition motif specific for
outer membrane proteins.
Experimental procedures
‘Scrambled’ RNase A, the homobifunctional crosslinking
reagent disuccinimidyl glutarate (DSG), and all other chem-
icals were obtained from Sigma (Poole, UK). The [
125
I] Bol-
ton–Hunter labelling reagent, anti-hexahistidine serum,
enhanced chemiluminescence reagent and X-ray films were

purchased from Amersham GE Healthcare (Little Chalfont,
UK). Peptides were synthesized as described previously for
other peptides [18], and peptide sequences are given in
Table 1. Antibodies against recombinant SurA and PpiD
were raised by injection of the purified proteins (see below)
into New Zealand White rabbits as described previously
K. H. Stymest and P. Klappa Substrate interactions of periplasmic PPIases
FEBS Journal 275 (2008) 3470–3479 ª 2008 The Authors Journal compilation ª 2008 FEBS 3477
[23]. Secondary antibodies coupled to horseradish peroxi-
dase were purchased from Dako (Glostrup, Denmark).
Cloning of PpiD and purification of proteins
The cloning of the surA gene from E. coli has been
described previously [11]. The gene encoding mature PpiD
without the transmembrane portion (positions 37–623) was
amplified from the E. coli genome by PCR using the fol-
lowing primers: forward primer, 5’-TTTTTTTTCATAT
GGGAGGCAATAACTACGCCGCAAAAG-3’; reverse
primer, 5’-TTTTTTTTCTCGAGCTATTATTGCTGTTC
CAGCGCATCGC-3’. These primers allowed the insertion
of an NdeI site at the N-terminus and an Xho I site at the
C-terminus. The primers complementary to the 3’ end
included a stop codon. The inserts were cloned between the
NdeI and XhoI sites of pLWRP51, a modified pET23d vec-
tor, which contained an insert coding for an initiating
methionine residue followed by a hexahistidine tag, as
described previously [11]. The construct was verified by
DNA sequencing.
Protein expression and purification of PpiD were per-
formed as described for SurA [11]. Cloning and protein
expression of human PDI were performed as described previ-

ously [11]. [
125
I]-Bolton–Hunter labelling of peptides was per-
formed as recommended by the manufacturer of the reagent.
Binding of peptides and scRNase
After precipitation with trichloroacetic acid, the radio-
labelled peptides were dissolved in buffer B (100 mm NaCl,
25 mm KCl, 25 mm sodium phosphate buffer pH 7.5).
Labelled peptides or scRNase were added to E. coli lysates
expressing recombinant SurA, PpiD or human PDI in buf-
fer B. The samples (10 lL) were incubated for 10 min on
ice prior to crosslinking [11].
Chemical crosslinking
Crosslinking was performed using the homobifunctional
crosslinking reagent disuccinimidyl glutarate (DSG)
[11]. Crosslinking solution (2.5 mm DSG in buffer B) was
added to the samples at one-fifth of the sample volume.
The reaction was carried out for 60 min at 0 °C. Crosslink-
ing was stopped by the addition of SDS–PAGE sample
buffer [11]. The samples were subjected to electrophoresis
in 10% SDS polyacrylamide gels.
Acknowledgements
We wish to thank Kevin Howland and Judy Hardy
(Department of Biosciences, University of Kent, Can-
terbury, UK) for synthesis of the peptides, and the
Wellcome Trust for establishment of a Protein Science
Facility. This work was supported by a Biotechnology
and Biological Sciences Research Council PhD
studentship to KHS.
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