doi:10.1016/j.jmb.2007.10.088 J. Mol. Biol. (2008) 376, 414–426

Available online at www.sciencedirect.com

The Peptidyl–Prolyl Isomerase and Chaperone Par27of Bordetella pertussis as the Prototype for a NewGroup of Parvulins

Hélène Hodak1,2,3, Alexandre Wohlkönig3,4, Caroline Smet-Nocca5,Hervé Drobecq3,4, Jean-Michel Wieruszeski6, Magalie Sénéchal3,4,Isabelle Landrieu6, Camille Locht1,2,3, Marc Jamin7⁎and Françoise Jacob-Dubuisson1,2,3⁎

1INSERM U629, Lille, France2Institut Pasteur de Lille,1 rue Professeur Calmette,F-59019 Lille Cedex, France3IFR142, Lille, France4UMR 8161, IBL, Lille, France5Institut de RechercheInterdisciplinaire, CNRSFRE2963, 1 rue ProfesseurCalmette, F-59019 Lille Cedex,France6Unité de GlycobiologieStructurale et Fonctionnelle,UMR CNRS 8576-Universitéde Lille1, Villeneuve d'Ascq,France7Unit of Virus Host CellInteractions, UVHCI UMR5233 UJF-EMBL-CNRS, 6,rue Jules Horowitz, B.P. 181,F-38042 Grenoble Cedex 9, France

Received 21 August 2007;received in revised form26 October 2007;accepted 31 October 2007Available online31 December 2007

*Corresponding authors. Institut PaE-mail addresses: jamin@embl-grenAbbreviations used: PPIase, peptid

TPS, two-partner secretion; GB1, B1 dchromatography; MALLS, multianglGengou; SSmedium, Stainer–Scholtemixing time; PBST, phosphate-buffer

0022-2836/$ - see front matter © 2007 E

Proteins that pass through the periplasm in an unfolded state are highlysensitive to proteolysis and aggregation and, therefore, often requireprotection by chaperone-like proteins. The periplasm of Gram-negativebacteria is well equipped with ATP-independent chaperones and foldingcatalysts, including peptidyl–prolyl isomerases (PPIases). The filamentoushemagglutinin of Bordetella pertussis, which is secreted by the two-partnersecretion pathway, crosses the periplasm in an unfolded conformation. Byaffinity chromatography, we identified a new periplasmic PPIase of theparvulin family, Par27, which binds to an unfolded filamentous hemagglu-tinin fragment. Par27 differs from previously characterized bacterial andeukaryotic parvulins. Its central parvulin-like domain is flanked by atypicalN- and C-terminal extensions that are found in a number of putativePPIases present mostly in β proteobacteria. Par27 displays both PPIase andchaperone activities in vitro. In vivo, Par27 might function as a generalperiplasmic chaperone in B. pertussis.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: peptidyl–prolyl isomerase; parvulin; periplasmic chaperone;filamentous hemagglutinin; Bordetella pertussis

Edited by M. Gottesman

steur de Lille, 1 rue Professeur Calmette, F-59019 Lille Cedex, France.oble.fr; francoise.jacob@ibl.fr.yl–prolyl isomerase; OMP, outermembrane protein; FHA, filamentous hemagglutinin;omain of the streptococcal G protein; IgG, immunoglobulin G; SEC, size-exclusione laser light scattering; RI, refractometry; GST, glutathione S-transferase; BG, Bordet–medium;OD600, optical density at 600 nm; EDTA, ethylenediaminetetraacetic acid;MT,ed saline containing 0.1% Tween 20; RCM-T1, reduced carboxymethylated RNase T1.

lsevier Ltd. All rights reserved.

Fig. 1. Schematic representation of the FHA deriva-tives used in this work and identification of a periplasmicprotein with affinity for an FHA fragment. (a) The Fha30model protein is the N-terminal 304-residue portion ofFHA. It represents the smallest FHA fragment that can beefficiently secreted by B. pertussis and correspondsessentially to the TPS domain. The TPS domain (inblack) comprises residues 1 to 245, while the gray boxrepresents the first portion of the large central region ofFHA. (b) The chimeric FhaB derivative GB1-Fha15–133-His6was used as an affinity bait. The portion of the TPSdomain included in the recombinant protein is shownin black, while the gray boxes at the N- and C-terminirepresent the GB1 motif and a 6-His tag, respectively. (c)IgG beads coated or not with GB1-Fha15–133-His6 wereincubated with the B. pertussis periplasmic extract or withbuffer, as indicated. The proteins retained on the beadswere eluted, analyzed by SDS-PAGE, and Coomassieblue stained. Par27 (indicated with an asterisk) was iden-tified by mass fingerprinting analysis as a putative PPIase.The symbol indicates the bait chimera and the fuzzyband in lane 1 is the light chain of IgG that was partiallyreleased from the beads by the acid pulse. The molecularweights of the marker proteins are indicated on the left.

415Par27 as Prototype for a New Group of Parvulins

Introduction

Many proteins of Gram-negative bacteria des-tined to the periplasm, the outer membrane, orbeyond enter the periplasm in an unfolded stateand face potential aggregation and/or proteolysisprior to adopting their native conformation. Specificor broad-spectrum ATP-independent chaperonesand folding catalysts, such as peptidyl–prolylisomerases (PPIases) and protein-disulfide oxidasesand isomerases, mediate their protection or facil-itate their folding.1,2 Among these, PPIases areubiquitously found in eukaryotes, eubacteria, andarchaea and have been classified into three distinctfamilies: cyclophilins, FK506-binding proteins, andparvulins.3–6The parvulin family of PPIases was discovered

most recently. Its prototypic member is a 10-kDaprotein found in the cytoplasm of Escherichia coli thatwas termed parvulin from the Latin word parvulus(small), or Par10.7 Par10 corresponds to the smallestPPIase core known. Par10 homologues describedthus far are all larger proteins characterized by aconserved region that corresponds to the Par10PPIase domain, called the “parvulin box.”6,8 TheE. coli periplasm contains two such large parvulins,called PpiD and SurA. PpiD is a 68-kDa membrane-anchored protein involved in outer membraneprotein (OMP) biogenesis and the extracytoplasmicstress response,9 whereas SurA is a 47.4-kDa proteininvolved in OMP biogenesis and cell survival.10–12

SurA in particular and various PPIases from otherfamilies display a chaperone activity in vitro andwere thus proposed to contribute to protein foldingin vivo.13–16Filamentous hemagglutinin (FHA), a major adhe-

sin of the whooping cough agent Bordetella pertussis,is a suitable model protein in studying the role ofperiplasmic chaperones and folding catalysts. Theprecursor of FHA, FhaB, is a very large protein(∼360 kDa) that crosses the periplasm in an un-folded state and has a strong tendency to aggregateand to be degraded if secretion is blocked (Ref. 17and our unpublished data). FHA is secreted by thetwo-partner protein secretion (TPS) pathway, whichis widespread in Gram-negative pathogens.18 TheFHA protein belongs to the TpsA family, whichis characterized by the presence of a conservedN-terminal TPS domain containing secretion de-terminants required for transport across the outermembrane.19 The specific transporter of FHA isFhaC of the Omp85/TpsB family.20,21 FHA mostlikely acquires its native structure at the bacterialsurface where it folds into a 50-nm-long β helix.22,23

Because its precursor is highly aggregative andprone to proteolytic degradation in the periplasm,17

we hypothesized that periplasmic chaperones maybe involved in its secretion.In this work, we identified, by way of affinity

chromatography, a 27-kDa periplasmic PPIase of theparvulin family that we have called Par27, whichbinds to a nonnative FHA fragment. Unlike pre-viously characterized parvulins, Par27 is a dimeric

protein with atypical N-terminal and C-terminalextensions. It exhibits both PPIase and chaperoneactivities in vitro. Comparative genomic analysesshowed that putative Par27-like proteins have anarrow distribution among bacteria and are presentexclusively in Proteobacteria, predominantly withinthe β class.

Results

Identification of Par27 in the periplasm ofB. pertussis

In an attempt to identify putative periplasmicchaperones and/or folding catalysts of FHA, weperformed an affinity chromatography with a peri-plasmic extract of B. pertussis using an FHAderivative as bait. We first developed a protocolfor the extraction of B. pertussis periplasmic proteins.This protocol allowed us to obtain a fraction inwhich most proteins identified by mass fingerprint-ing analyses correspond to proteins known orpredicted to be periplasmic. We then constructed arecombinant protein, called GB1-Fha15–133-His6,containing an N-terminal portion of the FHA TPSdomain expected to remain in an extended yetsoluble conformation (Fig. 1b). The B1 domain of thestreptococcal G protein (GB1) and a His6-tag were

Fig. 2. Par27 PPIase activity using an RCM-T1 refold-ing assay. Refolding of denatured RCM-T1 was initiatedby dilution in 2.0MNaCl. Refolding kinetics was followedby the change of fluorescence at 320 nm at 15 °C as afunction of RCM-T1 concentration. Initial velocities weremeasured in the presence and in the absence of Par27. Thecontribution of uncatalyzed folding increases linearly withRCM-T1 concentration, and the initial velocities in thepresence of Par27 were corrected for the uncatalyzed con-tribution. A value of 43,000±2000 M−1 s−1 was obtainedfor kcat/Km from the slope of the line.

416 Par27 as Prototype for a New Group of Parvulins

fused at the N-terminus and the C-terminus of theFHA fragment, respectively. After purification bynickel affinity chromatography, the recombinantprotein was immobilized on immunoglobulin G(IgG) beads and incubated with the periplasmicextract from B. pertussis BPDR, a strain deficient forFHA and pertussis toxin. The proteins eluted off thebeads were analyzed by SDS-PAGE and identifiedby mass fingerprinting analyses (Fig. 1c). One pro-tein of approximately 27 kDa specifically bound tothe chimera and was identified by mass spectro-metry as a putative PPIase encoded by theB. pertussisgene BP3561. The analysis of the amino acidsequence predicted an N-terminal signal peptide,with a putative cleavage site between residues Ala19and Gln20. Its presence is consistent with the proteinbeing isolated from the periplasm. The matureprotein is predicted to be composed of 239 aminoacid residues with a calculated molecular mass of27,034 kDa. Sequence alignments using the ClustalWsoftware confirmed the presence of a parvulin box inthis protein, which we consequently named Par27,for 27-kDa parvulin.

Par27 is the prototype of a new group ofparvulins

Sequence analyses using BLAST and ClustalWshowed that Par27 differs from previously des-cribed parvulins. Par27 is 27.9% and 19.8% identicalwith E. coli SurA and PpiD, respectively, and is aslittle as 15.1% and 13.6% identical with Par10 andhuman Pin1, respectively. Apart from the parvulincore, Par27 harbors N- and C-terminal extensionsthat are different from those of known parvulins.The fairly polar 113-residue-long N-terminal exten-sion of Par27 has an isoelectric point of 8.1 and ispredicted to essentially form α helices. Its 48-residue-long C-terminal extension has an isoelectricpoint of 5.6 and is also predicted to be α helical. ABLASTP search on currently available bacterialgenomes has revealed a number of highly similarputative PPIases harboring comparable extensions.Their sequences are more than 40% identical withthat of Par27. Interestingly, many of these proteinscarry an acidic N-terminal extension and a basic C-terminal extension or vice-versa, suggesting thatthese extensions might interact with one another.Par27-like PPIases are found in the Proteobacteriaphylum and nearly exclusively among β proteo-bacteria, particularly in Burkholderiales. More dis-tant homologues were also found within the classesof α, γ, and ε proteobacteria that contain evenlonger N- and C-terminal extensions.

Characterization of the Par27 peptidyl–prolylcis/trans isomerase activity

Par27 was produced as a recombinant protein witha C-terminal His6-tag in E. coli and purified. Theactivity of Par27 as a proline isomerase was assessedusing RNase T1 as a substrate.24,25 RNase T1 containstwo cis proline residues (Pro39 and Pro55), and its

refolding rate is limited by the slow cis/trans iso-merization of Xaa–Pro peptide bonds.26–28 RNase T1also contains two disulfide bridges, but the reducedand carboxymethylated form of the protein (RCM-T1) retains its ability to fold into its native state athigh salt concentrations. It refolds faster than theoxidized form and has its Xaa–Pro peptide bondsaccessible for catalysis by cis/trans isomerases.24

After the unfolded protein was diluted in 2 M NaCl,refolding of reduced carboxymethylated RNase T1(RCM-T1) was followed by spectrofluorometry at320 nm. The refolding kinetics of RCM-T1 wasbiphasic, as shown previously.24 In the presence ofPar27, only the major slow phase (amplitude N90%)was observed in manual-mixing experiments. Aftercorrecting for the contribution of spontaneousrefolding, the initial velocity of the Par27-catalyzedrefolding increased linearly with increasing RCM-T1concentrations up to 1 μM (Fig. 2), suggesting thatthe Km is larger than 1 μM. The initial velocity alsoincreased linearly with increasing Par27 concen-trations up to 250 nM (data not shown). Assuming asimple Michaelis–Menten mechanism, a kcat/Kmvalue of 43,000±2000 M−1 s−1 was calculated fromthe slope of the graph shown in Fig. 2. This value is ofthe same order of magnitude as the value of30,000 M− 1 s− 1 measured with Par10.29 Theseobservations indicate that Par27 is indeed a PPIase.The Par27-catalyzed isomerization of the Xaa–Pro

bonds contained in a soluble 16-mer peptide (VYKS-P5-VVSGDTS-P13-RHL) was directly followed byNMR spectroscopy to confirm its PPIase activity.30Both Ser–Pro bonds exhibited a conformationalheterogeneity, with cis conformers representing3.0% and 4.6% of the Pro5 and Pro13 populations,respectively. In the presence of Par27, two exchangepeaks related to Hα cis/Hα trans interconversion of

417Par27 as Prototype for a New Group of Parvulins

Pro5 and Pro13 residues were observed in an 1H–1HEXSY spectrum acquired at a 300-ms mixing time(MT) (Fig. 3a, circled peaks). These interconversionsignals were only detected when Par27 was addedto the peptide sample.The Par27 PPIase activity was quantified by the

acquisition of 1H–1H EXSY NMR spectra at variousMTs. The exchange-rate constants for the Ser4–Pro5and Ser12–Pro13 peptide bonds in the presence ofPar27 were determined to be 0.98 and 0.41 s−1,respectively (Fig. 3b). The Par27 isomerase activitywas thus 2.5-fold greater for the Ser4–Pro5 bondthan for the Ser12–Pro13 bond, although the latterexhibited higher cis content. It is likely that theprimary sequence surrounding the Ser–Pro bondinfluences the isomerization rate rather than therelative populations of cis and trans conformers.

Chaperone activity of Par27

Several known PPIases, including parvulins, dis-play chaperone activities.13,16,31,32 To test whetherPar27 has similar activities, we performed anaggregation assay using an FHA derivative calledFha30 as model substrate (Fig. 1a). Fha30 is a 30-kDaprotein that is efficiently secreted in an FhaC-dependent manner and adopts a β helical fold.23 Itcorresponds to residues 1 to 304 of FhaB and, thus,totally encompasses the entire FHA fragmentinitially used as bait for Par27.The interaction between Par27 and Fha30 was

characterized by using size-exclusion chromatogra-phy (SEC) coupled to a multiangle laser lightscattering (MALLS) device and refractometry (RI),in order to measure the molecular masses of the

Fig. 3. Detection of Par27 PPIase activity by homonuclearSer–Pro motifs (S4–P5, S12–P13) was solubilized at 2 mM inAssignment of peptide resonances for both cis (signals annoconformers was performed using 1H–1H nuclear Overhauseractivity was measured at 290 K in the presence of 50 μM Par27conformers of both Pro5 and Pro13 was detected through the H(b) Exchange rate of Par27 with the 16-mer peptide substratconformation [%(cis→ trans)] for Pro5 (triangle) and Pro13 (square given.

proteins and their complexes (Fig. 4a). The mole-cular mass of Par27 was constant throughout thepeak, showing the presence of a well-defined andmonodisperse protein species. The weight-averagedmolecular mass measured within the peak of Par27was 60±5 kDa, indicating that the protein formsdimers in solution. Using seminative electropho-resis, Par27 migrated as a 55- to 60-kDa protein, inagreement with the MALLS–SEC result (data notshown). To our knowledge, Par27 is the first des-cribed dimeric parvulin.Fha30 was used either in its native conformation

(Fha30N) as a control or after denaturation in 3.5 Mguanidinium chloride and dilution of the denatur-ant (Fha30GdmCl), as described previously.19 Fha30Nhas been shown earlier to form a tail-to-tail dimer.19The two forms of the protein eluted at differentvolumes from the SEC column, and the molecularmasses estimated by SEC–MALLS–RI were 64±8and 32±1 kDa for Fha30N and Fha30GdmCl, respec-tively (Fig. 4b and c). The two forms of Fha30 werethen independently mixed with Par27, and theformation of complexes was investigated by SEC–MALLS–RI. Par27 did not form a complex withFha30N (Fig. 4b). In contrast, a higher molecularmass complex eluting at 12.9 mL was formed aftermixing Fha30GdmCl with Par27 dimers in a 1:1 ratio(Fig. 4c). The molecular mass of this complexmeasured by MALLS was 85±7 kDa and mostlikely corresponds to that of one Par27 dimer andone Fha30GdmCl molecule. No free Fha30GdmCl andPar27 were found at this 1:1 ratio, indicating thatboth proteins are entirely present within the com-plex. When the concentration of the Par27 dimerexceeded that of Fha30GdmCl, an additional peak

NMR spectroscopy. (a) A 16-mer peptide containing two50 mM sodium phosphate (pH 6.6) and 50 mM NaCl.

tated with c) and trans (signals annotated with t) prolineenhancement spectroscopy (black spectrum). The PPIase(red spectrum). Interconversion between the cis and transα cis/Hα trans exchange cross-peaks (circled resonances).e. The ratios of molecules that undergo from cis to transare) residues in the presence of 2.5% Par27 during the MT

Fig. 4. Analysis of Fha30–Par27 complexes by SEC–MALLS–RI. Proteins were analyzed by SEC, and themolecular masses of proteins and complexes were deter-mined by combining online MALLS detector and RI. Theweight-averaged molecular masses are given above theelution peaks. (a) Par 27 alone. Red crosses show themolecular masses of Par27 determined across the elutionpeak. Par27 forms dimers in solution. (b) Interaction ofPar27with native Fha30 (Fha30N).WhenPar27 and Fha30Nwere injected individually, they eluted at 14.2 and 15.0mL,respectively (blue and red curves, respectively). Anequimolar mixture of Par27 and Fha30N was eluted astwo peaks at 14.2 and 15.0 mL (black curve). (c) Complexformation of Par27 with denatured Fha30 (Fha30GdmCl).Fha30GdmCl eluted at 13.5 mL, as a monomer (black curve),when injected individually. The (Par27)dimer:Fha30GdmClratios were as follows: 1:2 (cyan), 1:1 (red), 2:1 (blue), and4:1 (green). Twohighermolecularmass complexes (I and II)were eluted at 12.0 and 12.9 mL. The molecular masses ofcomplexes I and II suggest that they consist of a dimer ofPar27 bound to one molecule of Fha30 and two dimers ofPar27 bound to one molecule of Fha30, respectively.

418 Par27 as Prototype for a New Group of Parvulins

was detected at an elution volume of 12.0 mL, whichcontains a complex of 153±11 kDa. Par27 alone atthe same concentration eluted as a single peak at14.3 mL (data not shown), suggesting that thepresence of this additional peak corresponds to acomplex of two Par27 dimers per molecule ofFha30GdmCl. These data thus show that Par27binds exclusively to a nonnative form of Fha30and that the complexes formed between the twoproteins are stable enough to be detected by SEC.We next investigated the chaperone activity of

Par27 by testing its ability to prevent the aggrega-tion of Fha30GdmCl. The time course of aggregationfollowing dilution of Fha30GdmCl was monitored bythe increase in light scattering. The intensity and therate of the aggregation signal decreased when Par27was present in the dilution buffer (Fig. 5a). Intrigu-ingly, substoichiometric amounts of Par27 dimerswere sufficient to prevent Fha30GdmCl aggregation,indicating that only a fraction of Fha30GdmClaggregated under these conditions. Bovine serumalbumin was used as a control in a 1:1 ratio withFha30GdmCl and did not prevent aggregation (datanot shown).Finally, we also investigated whether Par27 pre-

vents the aggregation of lysozyme, applying a testpreviously used with other chaperones (Fig. 5b).33,34

The aggregation of lysozyme was prevented by theaddition of Par27, but total prevention required thepresence of a fourfold excess of Par27.In addition to preventing aggregation of their

substrates, molecular chaperones can protect themfrom proteolytic degradation. We thus tested whetherPar27 has the capacity to protect Fha30GdmCl againsttrypsin digestion. Fha30GdmCl carrying a C-terminalStrep II-tag19 was mixed with Par27 or with glu-tathione S-transferase (GST), a dimeric control pro-tein of similar mass, before adding trypsin to eachsample. The degradation of Fha30GdmCl over timewas assessed by SDS-PAGE and immunoblottingusing an anti-Strep II antibody (Fig. 5c). The pres-ence of Par27 delayed the proteolysis of Fha30, in-dicating that Par27 has protective activity on itssubstrate. The protective effect of Par27 was rathersmall, because denatured proteins are highly sensi-tive to proteolysis and Par27 most likely protectsonly a limited region of Fha30GdmCl. Thus, Par27exerts two activities in vitro, acting both as a PPIaseand as a chaperone.

Role of Par27 in vivo

In an attempt to identify a role for Par27 in vivo, thecorresponding gene, BP3561, was interrupted inthe chromosome of B. pertussis BPSM by insertionof a suicide plasmid, yielding B. pertussis BPHH2.BPHH2 did not appear to be defective for FHAsecretion, since the level and the rate of secretion asassessed by pulse-chase analysis were similar tothose of the isogenic parent BPSM (data not shown).However, the growth rate of BPHH2was slightly butreproducibly slower than that of BPSM (Fig. 6a). Thissuggests that, rather than being a PPIase chaperone

Fig. 5. Chaperone activity of Par27. (a) Aggregation of Fha30GdmCl was monitored by light scattering in the absence ofPar27 or in the presence of increasing concentrations of Par27. From top to bottom, the curves thus represent theaggregation behavior of Fha30GdmCl at a final concentration of 2 μM in the absence of Par27 and in the presence of Par27 atconcentrations of 25 nM, 50 nM, 100 nM, and 2 μM. (b) The aggregation of lysozyme was monitored by light scattering inthe presence of Par27. From top to bottom, the curves represent the aggregation behavior of lysozyme at a finalconcentration of 2 μM in the absence of Par27 and in the presence of Par27 at concentrations of 2, 4, and 8 μM. (c) Theability of Par27 to protect Fha30GdmCl against proteolytic degradation was tested in the presence of trypsin. Fha30GdmClwas incubated with a fourfold stoichiometric excess of Par27 or of a control protein, GST. The reaction was initiated byincubating the mixes at 37 °C and adding trypsin at 700 ng/mL. Aliquots were taken at the indicated times, and theproteins were analyzed by SDS-PAGE followed by immunoblotting using anti-Strep II antibody that specifically detectsFha30GdmCl.

Fig. 6. In vivo role of Par27. (a) Effect of par27 inactivation on B. pertussis growth. BPHH2 (par27) and BPSM (wt) weregrown in modified SS medium for 36 h at 37 °C, and the absorbance of the cultures was measured over time at 600 nm.Filled squares, BPSM; filled circles, BPHH2. (b) Par27 binds to proteins rich in amphipathic β structure. B. pertussis cellswere fractionated between soluble, Triton-X-100-soluble, and Triton-X-100-insoluble fractions. The proteins in all threefractions were separated by SDS-PAGE and Coomassie blue stained (left panel) or transferred onto a nitrocellulosemembrane for the overlay assay. The membrane was incubated with Par27, and the binding of Par27 to target proteinswas revealed by immunoblotting with an anti-His antibody (right panel). Lanes 1, 2, and 3 contain soluble, Triton-soluble, and Triton-insoluble proteins, respectively. Molecular masses of protein standards are indicated on the left. Thearrows on both panels indicate several proteins recognized by Par27 as identified by mass fingerprinting analyses.MOMP represents the major outer membrane porin of B. pertussis.

419Par27 as Prototype for a New Group of Parvulins

420 Par27 as Prototype for a New Group of Parvulins

specific for FHA, Par27 might assume a more gen-eral, albeit nonessential role in the periplasm.An overlay analysis was performed in order to

identify B. pertussis proteins that might be the targetsof Par27. The bacteria were fractionated betweensoluble, Triton-soluble, and Triton-insoluble frac-tions. The proteins contained in these fractions wereelectrophoretically separated and transferred ontonitrocellulose, and the membrane was incubatedwith Par27. Par27 bound selectively to only a fewproteins of the Triton-insoluble membrane fraction(Fig. 6b). These putative Par27 targets were identi-fied by mass fingerprint analyses of the correspond-ing Coomassie-blue-stained proteins from a controlelectrophoresis gel. They were OMPs, including twoporins, the major outer membrane porin (coded byBP0840) and OmpA (BP0943), as well as the trans-locator domains of three autotransporters, SphB1(BP0216), BrkA (BP3494), and Tcf (BP1201). All fiveproteins are integral β-barrel proteins of the outermembrane. Thus, Par27 appears to have affinity forproteins rich in amphipathic β structure.

Discussion

In this work, a new parvulin was identified by itsaffinity for a fragment of FHA, a large secretoryvirulence protein of B. pertussis. This 27-kDa peri-plasmic protein, named Par27, has a conservedsignature of PPIases of the parvulin family. How-ever, Par27 is different from other known parvulinsby several aspects. Firstly, it contains N- and C-terminal extensions not found in known parvulins.Secondly, it forms dimers in solution, whereas otherknown parvulins are described as monomers. Themode of dimerization of Par27 remains to be deci-phered. Par27 is not ubiquitously distributed in thebacterial kingdom. BLASTP analyses have shownthat Par27-like putative proteins are present exclu-sively in the Proteobacteria phylum and mainlywithin the Burkholderiales order in the β proteo-bacteria class.The PPIase activity of Par27 was demonstrated

using two distinct in vitro assays. Par27 acceleratesthe refolding of RNase T1. Its kcat/Km for thatsubstrate is of similar magnitude as those of otherparvulins, such as Par10 and SurA.16,29 Furthermore,the PPIase activity of Par27 was directly demon-strated on a peptide substrate byNMR spectroscopy.The exchange-rate constants determined in thatassay were found to be very similar to those ofother PPIases like cyclophilin35 or the human andArabidopsis thaliana Pin1 parvulins.36,37

As a molecular chaperone, Par27 is able to preventaggregation of two proteins and to provide Fha30with some protection against proteolytic degrada-tion. Par27 was also shown to form complexes withan unfolded form of Fha30 but not with the sameprotein in its native conformation. The stability ofthese complexes suggests that Par27 might act as a“holding chaperone” for Fha30 under these con-ditions.38 Chemically unfolded Fha30 refolds slowly

after dilution of the denaturant (our unpublisheddata). Therefore, the extended structure of thedenatured protein is likely to be exposed for aprolonged period of time. A stable Par27–Fha30interaction may partially overcome this deficiency.When present in excess, Par27 dimers increment

Fha30–(Par27)2 complexes. The Fha30-dependentformation of larger complexes suggests that thesecond Par27 dimer interacts with Fha30 rather thanwith the first Par27 dimer. This interaction probablytakes place via a lower affinity site. The β helix ofFha30 contains a number of amphipathic β strandspotentially forming more than one binding site.With its highly repetitive structure, FHA is expectedto contain an even greater number of sites withsimilar characteristics.Since Par27 has a narrow distribution among

Proteobacteria, we attempted to define its specificrole in vivo as a prototype for this new parvulingroup. Although Par27 can chaperone an FHAderivative in vitro, the par27 knockout strain is notdefective for FHA secretion. It is conceivable that thein vivo effect of the par27 knockout is masked bypartially redundant functions exerted by other peri-plasmic chaperones and/or PPIases. Indeed, threeother PPIaseswere detected in the periplasm extractsof B. pertussis, one of which is a parvulin predictedto be a SurA orthologue (coded by BP3330).Our attempts to inactivate BP3330 in B. pertussis

were unsuccessful, suggesting that it encodes anessential protein for this bacterium. In E. coli, SurA isimportant for OMP biogenesis, which is linked to itschaperone function.16 The specificity of SurA forOMP folding intermediates has been well docu-mented.39–41 Although Par27 is not essential inB. pertussis, it displays affinity for several integralOMPs predicted to form β barrels. A common fea-ture of the putative Par27 substrates including FHAis their propensity to form amphipathic β structures,similar to SurA targets.Because the pathogenicity of B. pertussis relies

heavily on the secretion of virulence proteins, proteintrafficking in the cell envelope is of paramountimportance for the fitness of the bacterium. A num-ber of virulence proteins and of proteins involved inthe secretion of these proteins adopt β-barrelstructures and are targeted to the outer membrane.42

Heavy protein trafficking to the outer membranemay explain why two proteins with partially re-dundant functions, Par27 and SurA, are necessary inthat bacterium. However, because only SurA seemsto be essential, the two chaperones must have non-fully overlapping target sets. The fact that thebacterial growth rate of B. pertussis is only slightlyaffected by the genetic inactivation of par27 indicatesa lesser role for the protein in the fitness of thebacterium.A recent microarray analysis of the B. pertussis

transcriptome found par27 overexpressed in theabsence of glutamate,43 a major nutrient for thatbacterium. This suggests a role for Par27 to copewith metabolic and probably other types of stress,in agreement with our hypothesis that it may con-

421Par27 as Prototype for a New Group of Parvulins

tribute to chaperoning several proteins in the peri-plasm. In the same transcriptomic analyses,43 par27was overexpressed twofold in a B. pertussis strain“locked” in the virulent phase. By way of a tran-scriptional fusion with the par27 gene, we have alsoobserved that it is weakly coregulated with thevirulence regulon (our unpublished data). This iscompatible with a role for Par27 in the trafficking ofvirulence proteins or of components of their secre-tion machineries. Indeed, Par27 displays affinity forthe predicted β-barrel domains of three virulencefactors, BrkA, Tcf, and SphB1, which are all auto-transporter proteins. Autotransporters are secretoryproteins composed of an N-terminal passengerdomain that carries a specific function, followed bya β-barrel-forming “translocator” domain involvedin passenger secretion across the outer membrane.18

Par27 might thus escort such proteins in the peri-plasm, thereby possibly contributing to B. pertussispathogenicity.All known parvulins are 4 to 58 kDa larger than

Par10, and their additional regions were shown orhypothesized to be involved in substrate specifi-city.39,44,45 For instance, eukaryotic Pin1-like PPIasescontain an N-proximal WW domain that specificallybinds to phosphorylated Ser/Thr residues whenpreceding a proline.46 In SurA, the N- and C-terminal extensions form a chaperone moduletogether with the first, inactive PPIase domain ofthe protein.47 It will be interesting to elucidate therole of the N- and C-terminal extensions in Par27 forits chaperone activity.

Materials and Methods

Bacterial strains and media

The B. pertussis strains used were the Tohama I strepto-mycin-resistant derivative BPSM,48 BPDR (fhaB ptx),49 andBPHH2 (par27) (this work). B. pertussis was grown onBordet–Gengou (BG) agar supplemented by 1% glyceroland 10% defibrinated sheep blood (Biomérieux). The liquidcultures were performed in modified Stainer–Scholte me-dium (SS medium).50 The antibiotics used were as follows:100 μg/mL streptomycin, together with 10 μg/mL genta-micin for BPHH2. Nalidixic acid (30 μg/mL) was used inBG plates to counterselect E. coli SM10 after conjugation.

Plasmids

To generate pFUS2(par27::lacZ), we performed a PCRusing BPSM chromosomal DNA as a template and theprimers Par27 Up: 5′-tataaagcttcgtgcccgctttcgcccagaa-3′and Par27 Low: 5′-tataggtaccgcgctgccggggtccttggag-3′.The PCR product, which corresponds to the 5′ region ofthe par27 coding region (ID: BP3561), was restricted byHindIII and KpnI and ligated in the corresponding sites ofpFUS2,51 thus generating pFUS2::par27.pFJD125 codes for a chimeric protein that comprises the

GB1 domain and a polypeptide of FHA corresponding toresidues Gly15-Gly133 of the TPS domain, followed by a 6-His tag. It derives from pFJD101, which was constructedas follows. The corresponding fhaB gene fragment was

amplified by using the oligonucleotides MalC-UP1-SD:5′-ggatccgggaacaaggtttcccgttgt-3′ and MalC-LO1-SD:5′-aagcttcacttgccctgcttgctcgac-3′ as primers. The ampliconwas restricted with BamHI and HindIII and inserted intopMalC (New England Biolabs), yielding pFJD101. Thesequence of the cloned fragment was checked. Twocomplementary oligonucleotides, His101-UP: 5′-ggccgcca-ccaccaccaccaccacgggccctag-3′ andHis101-LO: 5′-ggccctag-ggcccgtggtggtggtggtggtggc-3′, were then annealed, andthe resulting linker was inserted into the NotI site ofpFJD101. The orientation of the linker was verified bysequencing, and the resulting plasmid was called pJD101-6His. It was then used as a template of a PCR usingthe oligonucleotides pMalC-UP (above) and SD-Lo-Eco:5′-gaattctcacttgccctgcttgctcgac-3′ as primers. The ampli-con was inserted into pCRII-TOPO and sequenced. TheDNA fragment of interest was then generated by restric-tion of the recombinant plasmid with EcoRI and BamHIand inserted into the same sites of pGEV2,52 resulting inpFJD125.pHod1 allows for the expression of N-terminally His6-

tagged Par27 in the cytoplasm of E. coli. It wasconstructed as follows. The par27 gene (ID: BP3561) wasamplified by PCR using BPSM genomic DNA as atemplate with primers Par27RECUp (5′-aagcttttactgga-tcttggcctgttc-3′) and Par27RECLo (5′-ggatcccagaacgtggc-gaccgtgaac-3′). The sequence coding for the signalpeptide of Par27 was not included. The 720-bp HindIII-BamHI amplicon was inserted into pQE30 (Qiagen),generating pHod1. pHod1-encoded Par27 contains at itsN-terminus 12 additional residues, MRGSHHHHHHGS,as compared with the chromosome-encoded protein.E. coli M15 (pRep4) was used for expression of therecombinant gene.

Inactivation of the Par27 gene (BP3561) in B. pertussis

pFUS2(par27::lacZ) was first introduced into E. coliSM10, and the recombinant strain was used as a conjuga-tion donor with BPSM as the recipient.51 The isolatedexconjugants were selected on BG plates supplementedwith streptomycin and gentamicin. The correct insertion ofthe plasmid in the par27 locus was verified by PCR. Therecombinant B. pertussis strain was called BPHH2.

Protein production and purification

Par27 was purified as follows. E. coli M15(pRep4) har-boring pHod1 was cultured at 37 °C under orbital shakingin LB broth supplemented with 100 μg/mL ampicillin and50 μg/mL kanamycin to an optical density at 600 nm(OD600) of 0.6. IPTG (1 mM) (Sigma) was then added, andthe incubation continued at 37 °C for an additional 3 h.Cells were harvested by centrifugation at 4 °C, and thebacterial pellets were stored at −20 °C until further use.Frozen cell pellets were thawed in 50 mL of 50 mM Tris–HCl (pH 8), 150 mM NaCl, 5% glycerol, 50 μg DNase I,and a tablet of protease inhibitor Complete™ ethylene-diaminetetraacetic acid (EDTA)-free (Roche). The cellswere disrupted using a French press (15,000 psi). Celldebris was removed from the crude extract by twosuccessive centrifugations at 12,000g for 30 min at 4 °C.The supernatant was diluted in 150mL of 50mMTris–HCl(pH 8.0), 150 mM NaCl, 5% glycerol, and 5 mM imidazole(purification buffer) and filtered through a 0.22-μm filter.A HiTrap Chelating Sepharose column (5 mL) (GEHealthcare) preloaded with 0.1 M CoCl2 was used forthe purification of Par27. The sample was applied onto the

422 Par27 as Prototype for a New Group of Parvulins

column preequilibrated in the purification buffer. Washingsteps of 10 column volumes each were performed withincreasing imidazole concentrations, ranging from 10 to100 mM with a 10-mM increment. Par27 was eluted in thepurification buffer supplemented with 500 mM imidazoleand then further purified on a preparative size-exclusioncolumn, Superdex75 (GE Healthcare), previously equili-brated in 10 mM Tris–HCl (pH 8.0) and 50 mM NaCl.Par27 was eluted in a single peak. The protein aliquotswere conserved at −80 °C.E. coli BL21 (DE3; pFJD125) was grown at 37 °C under

orbital shaking in 200 mL LB containing 150 μg/mLampicillin to an OD600 of 1 to produce GB1-Fha15–133-His6.The expression of the chimera encoded by pFJD125 wasthen induced by adding 1 mM IPTG to the culture, whichwas then incubated for 2 h at 37 °C. The cells werecentrifuged as above, and the pellet was resuspended in10mLTSI buffer [50 mMTris–HCl (pH 7.5), 150 mMNaCl,and 20 mM imidazole] supplemented with 10 μg/mLDNase I and a tablet of protease inhibitor Complete™EDTA-free. The cells were broken by two passages in aFrench press, and the extract was clarified by centrifuga-tion, as described above. The clarified lysate was thenapplied onto a nickel affinity column (Chelating Sephar-ose™) loaded with Ni++ cations according to the manu-facturer's instructions (Amersham Pharmacia Biotech-nologies). The column was washed with the loadingbuffer, and the protein was directly eluted in the samebuffer containing 0.5M imidazole. It was diluted in 50mMTris–HCl (pH 7.6) and 150 mM NaCl (TS buffer) beforeserving as a bait in the affinity chromatography experi-ments (see below).Fha30 and Strep-tagged Fha30 were produced and

purified as previously described.19

Bidimensional electrophoresis and proteinidentification

Periplasmic extracts from B. pertussis were resolved bytwo-dimensional electrophoresis as described elsewhere53using BDH ampholytes pH 3–10 (Pharmacia) and a 12%polyacrylamide gel. The proteins were stained withcolloidal Coomassie blue dye. The protein spots weresubjected to tryptic digestion. Peptides were extractedfrom the gel, and the mass peptide fingerprints wereobtained by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry. The mass peptidefingerprint of each protein was compared with thedatabase of putative B. pertussis proteins.

Affinity chromatography

The bait protein GB1-Fha15–133-His6 was immobilizedonto 1 mL IgG Sepharose® 6 Fast Flow (Amersham) asfollows. The beads with IgG were conditioned asrecommended by the manufacturer. One-third of resinwas removed (fraction A). The rest of the beads wereincubated with the bait protein (∼500 μg; purified asdescribed above) in 15 mL TS buffer. The beads werewashed with 10 volumes of TS buffer and divided intotwo fractions (B and C). Fractions A and B were sepa-rately incubated with the B. pertussis periplasmic extractadjusted to pH 7, for 5 min under rotation. The beadswere washed twice with 10 volumes of TS buffer. Toelute the bound proteins, we quickly mixed 200 μL ofbeads from fractions A, B, and C with 400 μL 0.5 Macetic acid (pH 3.4) in Eppendorf tubes, and the mix-tures were centrifuged softly for 2 min to pellet the

beads. The supernatants were mixed with Laemmlibuffer, and concentrated Tris–HCl (pH 9) was added tothe mixtures to bring them to neutral pH. Finally, theproteins were analyzed by SDS-PAGE and mass finger-printing spectrometry.

SEC and MALLS

SEC was performed with a Superdex S200 column (GEHealthcare). The column was equilibrated in 20 mM Tris–HCl (pH 7.5) and 150 mM NaCl. Separations wereperformed at 20 °C with a flow rate of 0.5 mL/min.Typically, 40 μL of protein solutions at concentrations of∼5 mg/mL was injected. Online MALLS detection wasperformed with a DAWN-EOS detector (Wyatt Technol-ogy Corp., Santa Barbara, CA) using a laser emitting at690 nm. For a diluted polymer solution, the amount oflight scattered is directly proportional to the product ofprotein concentration and molar mass, according toZimm's formula:54

Ru

K*C¼ MP(u)� 2A2CM2P2(u) (1)

where Rθ is the measured excess Rayleigh ratio; C is theprotein concentration (in grams per milliliter); M is themolar mass (in grams per mole); P(θ) is the form factor,which depends on the structure of the scattering particlesand describes the angular dependence of the scatteredlight; andA2 is the second virial coefficient. K* is an opticalconstant given by the following equation:

K* ¼ 1NA

2πn0E2

� �2 dndc

� �2

(2)

where NA is Avogadro's number, n0 is the refractiveindex of the solvent at the incident radiation wavelength(1.33 for a diluted aqueous buffer), dn/dc (in millilitersper gram) is the specific refractive index increment ofthe solute, and λ is the wavelength of the incident lightin void. For particles smaller than the incident wave-length, no angular dependence is observed (P(θ)=1), andfor a sufficiently diluted solution, A2 is negligible(A2=0). Equation (1) then simplifies and the Rayleighratio only depends on protein concentration and molarmass:

Ru

K*C¼ M (3)

The molar mass, M, can thus be calculated if C is known.Protein concentration was measured online by refractiveindexmeasurements using an RI2000 detector (SchambeckSFD) and refractive index increment dn/dc=0.185 mL/g.Within the elution peak, the chromatogram is divided intoslices, and for each slice, MALLS and refractive indexmeasurements are used to calculate the molar mass usingEq. (3). Weight-averaged (Mw) molar masses are obtainedfrom the molar mass distribution across the elution peak.Analysis of the data was performed with the ASTRAsoftware (Wyatt Technology Corp.).

Peptide synthesis

The peptide was synthesized on a continuous-flowsynthesizer (Pioneer) using the Fmoc temporary-pro-tection strategy. After TFA cleavage (with a TFA solu-tion containing 2.5% triisopropylsilane and 2.5% water),

423Par27 as Prototype for a New Group of Parvulins

the crude peptide was purified by RP-HPLC on a C18Nucleosil column equilibrated in 0.05% TFA (buffer A).The peptide was eluted by using a 40-min linear gradientof 0–40% acetonitrile in buffer A at 6 mL/min. Homo-geneity of fractions was analyzed by RP-HPLC andmatrix-assisted laser desorption/ionization time-of-flightmass spectrometry. The homogenous fractions werepooled and lyophilized to provide peptide purity greaterthan 95%.

Peptide assignment and direct measurement ofPPIase activity by NMR exchange spectroscopy

NMR experiments were performed on a Bruker 600-MHz DMX spectrometer (Bruker, Karlsruhe, Germany)equipped with a cryogenic triple-resonance probehead.All 1H spectra were calibrated with 1 mM sodium3-trimethylsilyl-d(3,3′,2,2′)-propionate as a reference.Standard total correlated spectroscopy and nuclear Over-hauser enhancement spectroscopy spectra with 60- and400-ms MTs, respectively, were acquired on a 2-mMpeptide solution in 25 mM phosphate buffer (pH 6.6) and50mMNaCl at 293 K to assign peptide 1H resonances. Thecontent of cis conformers was calculated with the integra-tion of the Hα/Hγ correlation peaks for both cis and transsignals in a homonuclear total correlated spectroscopyexperiment. To measure the PPIase activity, we recordedan 1H–1H EXSY spectrum at an MT of 300 ms on a 2-mMpeptide sample containing 0.050 mM (2.5%) Par27 in25 mM phosphate buffer (pH 6.6) and 50 mM NaCl in100% D2O. A series of 1H–1H EXSY spectra were thenacquired at various MTs (100, 200, 300, 400, and 500 ms) toquantify the exchange rate of the Par27 isomerase activity.Integration of theHα cis/Hα trans cross-peaks detected forboth proline residues was performed. The exchange cross-peaks were normalized with the integration value of thecorresponding diagonal peak to provide the ratio ofmolecules that underwent changes from the cis to thetrans conformation during the MT.The exchange-rate constant (kexch) was calculated by

fitting the theoretical curve obtained with the followingequation to the experimental points:

% cisYtransð Þ ¼ a1� expð�ð1þ 1=aÞkexch �MTÞ1þ expð�ð1þ 1=aÞkexch �MTÞ

where %(cis→ trans) corresponds to the fraction ofmolecules that underwent changes from the cis to thetrans conformation during the MT and a is the excess oftrans over cis conformer.

RCM-T1-based PPIase activity assay

The RNase T1 was purchased from Sigma. Thereduction and carboxymethylation of RNase T1 to gen-erate RCM-T1 were performed as previously described.24

The predicted mass of RCM-T1 was verified by massspectrometry. RCM-T1 was unfolded in 0.1 M Tris–HCl(pH 8) for at least 1 h at room temperature at a finalconcentration of 20 μM. The refolding was initiated by a40-fold dilution in 2 M NaCl. The time course of refoldingof the RCM-T1 was followed at 320 nm with a QM-4spectrofluorometer (PTI) using λexc=268 nm. For thecalculation of the kcat/Km, Par27 was used at a finalconcentration of 25 nM and RCM-T1 at concentrationsranging from 100 nM to 1 μM. All the mixes above wereprepared in a total volume of 2 mL, and the measures wereperformed in a quartz cuvette under agitation at 15 °C. For

each RCM-T1 concentration, the initial velocity valuemeasured in the absence of Par27 was subtracted from theinitial velocity value measured in the presence of Par27.RCM-T1 and Par27 were prediluted in such a manner as toadd constant volumes to each mix.

Aggregation assays

The lysozyme aggregation assay was performed asdescribed.34 The final concentration of lysozyme was2 μM. Par27 was added at final concentrations of 2, 4, and8 μM.The Fha30 aggregation was observed using guanidi-

nium-denatured Fha30, Fha30GdmCl. Fha30GdmCl wasprepared by incubation of native Fha30 in 3.5 M Gdn–HCl for at least 1 h at room temperature as described.19

The aggregation of Fha30GdmCl was obtained by itsdilution in 5 mM Mops (pH 6.5) at a final concentrationof 2 μM Fha30GdmCl and 10 mM Gdn–HCl. Par27 wasadded prior to Fha30GdmCl in the buffer to final concen-trations of 50 nM, 100 nM, 200 nM, and 1 μM. The integrityof Fha30GdmCl after 600 s of incubation in the presence ofPar27 was checked by SDS-PAGE, demonstrating thatreduction of light scattering was not caused by proteolysis(data not shown). All the measurements were performedat 24 °C in a total volume of 1.4 mL in a quartz cuvette andunder agitation.

Par27 protective effect during trypsinolysis

C-terminally Strep-tagged Fha30GdmCl was prepared asdescribed previously.19 It was diluted to 4.5 μM in 100mMTris (pH 8) and 150 mM NaCl (working buffer). Par27 orGSTwas added in fourfold excess, at a final concentrationof 18 μM in the working buffer before the addition ofFha30GdmCl. The reaction mixtures were prepared in afinal volume of 250 μL. The first aliquot was taken beforethe addition of trypsin (time 0). The digestion was initiatedby the addition of trypsin at a final concentration of700 ng/mL and an immediate incubation at 37 °C.Aliquots were taken every 30 s for 3 min and immediatelyplaced on ice. The proteins were heated for 5 min indenaturing Laemmli buffer at 95 °C and loaded onto anSDS-PAGE gel. The Fha30 degradation was analyzed byimmunoblotting using anti-Strep II antibody (IBA).

B. pertussis periplasm extraction

B. pertussis BPDR was grown in SS medium at 37 °C toan OD600 of 1. The bacteria were collected by centrifuga-tion for 15 min at 5400g at 4 °C, and the pellet wasresuspended in 0.5 M glucose, 1 mM EDTA, and 0.2 MTris–HCl (pH 8)55 with 0.2 mg/mL lysozyme and 125 μg/mL AEBSF (Roche). After 30 min of incubation on ice, thesuspension was diluted twofold with 10 mM ice-coldMgCl2. After 30 min of incubation on ice, the cells werecentrifuged for 10 min at 7800g. Contaminating outermembrane fragments were discarded from the super-natant following a 1-h ultracentrifugation at 10 °C and100,000g.

Overlay assay with Par27

The fractionation of B. pertussis BPSM cell extracts wasadapted from a previously described protocol.56 BPSMgrown in 30 mL to an OD600=1.1 was harvested after

424 Par27 as Prototype for a New Group of Parvulins

37 °C incubation by centrifugation at 8000g at 4 °C. Thecell pellet was washed in 8 mL of 100 mM Tris–HCl (pH 8)and centrifuged at 8000g at 4 °C. The cells wereresuspended in 10 mL 100 mM Tris–HCl (pH 8) and150 mMNaCl, supplemented with 10 μg/mL DNase I andComplete EDTA-free protease inhibitor. The cells werebroken by French press, and the lysate was centrifuged at8000g for 10 min at 4 °C to remove unbroken cells. Theinsoluble proteins present in the supernatant wereseparated by a 100,000g ultracentrifugation for 1 h at12 °C. The soluble fraction (fraction S) was stored at−20 °C. The insoluble proteins were first resuspended in150 μL 100 mM Tris–HCl (pH 8), and Triton X-100(prediluted to 10% in the same buffer) was then added to afinal concentration of 1%. The mixture was incubated onice for 30 min with regular vortexing. Triton-soluble(fraction TS) and Triton-insoluble (fraction TI) proteinswere separated by a 100,000g ultracentrifugation for30 min at 4 °C. The proteins in TI fraction wereresuspended in 150 μL 100 mM Tris–HCl (pH 8). TS andTI fractions were stored at −20 °C.The overlay assay was performed as follows. The

fractions S, TS, and TI were separated electrophoreticallyon an SDS gel, and proteins were then transferred onto anitrocellulose membrane. The latter was blocked inphosphate-buffered saline containing 0.1% Tween 20(PBST) supplemented with 5% skimmed milk for 40 minat 4 °C under gentle orbital agitation. The membrane wasincubated with PBST containing 6 μM Par27 for 20 minunder agitation at room temperature. Weakly boundPar27 was removed by three washing steps of themembrane in PBST. The binding of Par27 was detectedby immunoblotting using a monoclonal anti-His antibody(Roche) and an anti-mouse horseradish-peroxidase-con-jugated antibody (Promega) followed by enhanced che-miluminescence (ECL Western Blotting DetectionReagents, Amersham Biosciences).

Acknowledgements

We wish to thank Dr Hélène Valadié for her kindassistancewith light scattering andRudyAntoine forhis help and advice during this work. F. Jacob-Dubuisson is a researcher of the Centre National dela Recherche Scientifique, and H. Hodak acknowl-edges the receipt of a predoctoral fellowship from theFondation pour la Recherche Médicale and a post-doctoral fellowship from the Institut Pasteur de Lille.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2007.10.088

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