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

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.. PpiD, like SurA, interacts wit

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 eukprok-aryotes: 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: p.klappa@kent.ac.uk

(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 coliand 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.

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

surAgenes 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].

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 95C) 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 [125I]-Bolton–Hunter-labelled D-somatostatin was chemically crosslinked to

recombi-Fig 2 Chemical crosslinking of PpiD Purified PpiD (5 l M , 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.

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 95C) 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 l M ) 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 l M (++) 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.

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 somatoD-somato-statin 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-statin (10 lm) and unlabelled D-somatoD-somato-statin (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 unlaradiola-belled 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 l M ) 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).

Our results demonstrate that changing the

phenylal-anine at position 6 to alphenylal-anine (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 [125I] 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

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 interinter-action

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 [125I] 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

[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 XhoI 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] [125I]-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 0C

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

References

1 Fischer G, Tradler T & Zarnt T (1998) The mode of action of peptidyl prolyl cis⁄ trans isomerases in vivo: binding vs catalysis FEBS Lett 426, 17–20

2 Bang H, Pecht A, Raddatz G, Scior T, Solbach W, Brune

K & Pahl A (2000) Prolyl isomerases in a minimal cell Catalysis of protein folding by trigger factor from Myco-plasma genitalium Eur J Biochem 267, 3270–3280

3 Schmid FX, Mayr LM, Mucke M & Schonbrunner ER (1993) Prolyl isomerases: role in protein folding Adv Protein Chem 44, 25–66

4 Gothel SF & Marahiel MA (1999) Peptidyl-prolyl cis– trans isomerases, a superfamily of ubiquitous folding catalysts Cell Mol Life Sci 55, 423–436

5 Lazar SW & Kolter R (1996) SurA assists the folding

of Escherichia coli outer membrane proteins J Bacteriol

178, 1770–1773

6 Lazar SW, Almiron M, Tormo A & Kolter R (1998) Role of the Escherichia coli SurA protein in stationary-phase survival J Bacteriol 180, 5704–5711

7 Tormo A, Almiron M & Kolter R (1990) surA, an Escherichia coligene essential for survival in stationary phase J Bacteriol 172, 4339–4347

8 Behrens S, Maier R, de Cock H, Schmid FX & Gross

CA (2001) The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity EMBO J 20, 285–294

9 Behrens S (2002) Periplasmic chaperones – new structural and functional insights Structure 10, 1469– 1471

10 Hennecke G, Nolte J, Volkmer-Engert R, Schneider-Mergener J & Behrens S (2005) The periplasmic chaper-one SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recogni-tion J Biol Chem 280, 23540–23548

11 Webb HM, Ruddock LW, Marchant RJ, Jonas K & Klappa P (2001) Interaction of the periplasmic pepti-dylprolyl cis–trans isomerase SurA with model peptides The N-terminal region of SurA is essential and suffi-cient for peptide binding J Biol Chem 276, 45622– 45627

12 Xu X, Wang S, Hu YX & McKay DB (2007) The peri-plasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues

J Mol Biol 373, 367–381

13 Dartigalongue C & Raina S (1998) A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli EMBO J 17, 3968–3980

14 Rizzitello AE, Harper JR & Silhavy TJ (2001) Genetic

evidence for parallel pathways of chaperone activity in

the periplasm of Escherichia coli J Bacteriol 183, 6794–

6800

15 Justice SS, Hunstad DA, Harper JR, Duguay AR,

Pinkner JS, Bann J, Frieden C, Silhavy TJ & Hultgren

SJ (2005) Periplasmic peptidyl prolyl cis–trans isomerases

are not essential for viability, but SurA is required for

pilus biogenesis in Escherichia coli J Bacteriol 187, 7680–

7686

16 Bitto E & McKay DB (2003) The periplasmic molecular

chaperone protein SurA binds a peptide motif that is

characteristic of integral outer membrane proteins

J Biol Chem 278, 49316–49322

17 Bitto E & McKay DB (2004) Binding of

phage-display-selected peptides to the periplasmic chaperone protein

SurA mimics binding of unfolded outer membrane

proteins FEBS Lett 568, 94–98

18 Klappa P, Hawkins HC & Freedman RB (1997)

Inter-actions between protein disulphide isomerase and

pep-tides Eur J Biochem 248, 37–42

19 Freedman RB & Klappa P (1998) Protein

disulphide-isomerase: a catalyst of thiol:disulphide interchange and

associated protein folding In Molecular Biology of

Chaperones and Folding Catalysts(Bukau B, ed), pp

437–459 Harwood Academic Press, London

20 Klappa P, Ruddock LW, Darby NJ & Freedman RB

(1998) The b’ domain provides the principal

peptide-binding site of protein disulfide isomerase but all

domains contribute to binding of misfolded proteins

EMBO J 17, 927–935

21 Reimer U, Scherer G, Drewello M, Kruber S, Schut-kowski M & Fischer G (1998) Side-chain effects on peptidyl-prolyl cis⁄ trans isomerisation J Mol Biol 279, 449–460

22 Ruddock LW, Coen JJ, Cheesman C, Freedman RB & Hirst TR (1996) Assembly of the B subunit pentamer of Escherichia coliheat-labile enterotoxin Kinetics and molecular basis of rate-limiting steps in vitro J Biol Chem 271, 19118–19123

23 Klappa P, Stromer T, Zimmermann R, Ruddock LW & Freedman RB (1998) A pancreas-specific glycosylated protein disulphide-isomerase binds to misfolded proteins and peptides with an interaction inhibited by oestro-gens Eur J Biochem 254, 63–69

24 Scholz C, Mucke M, Rape M, Pecht A, Pahl A, Bang

H & Schmid FX (1998) Recognition of protein sub-strates by the prolyl isomerase trigger factor is indepen-dent of proline residues J Mol Biol 277, 723–732

25 Bothmann H & Pluckthun A (2000) The periplasmic Escherichia colipeptidylprolyl cis,trans-isomerase FkpA

I Increased functional expression of antibody fragments with and without cis-prolines J Biol Chem 275, 17100– 17105

26 Alcock FH, Grossmann JG, Gentle IE, Likic VA, Lith-gow T & Tokatlidis K (2008) Conserved substrate bind-ing by chaperones in the bacterial periplasm and the mitochondrial intermembrane space Biochem J 409, 377–387

27 Danese PN & Silhavy TJ (1998) Targeting and assembly

of periplasmic and outer-membrane proteins in Escheri-chia coli Annu Rev Genet 32, 59–94