Báo cáo khoa học: Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain" doc

The C domain Keywords diphtheria toxin; membrane interaction; molten globule; protein folding; translocation Correspondence D.. Previous work suggested that the T domain acts as a chaper

diphtheria toxin translocation domain towards the

catalytic domain

Anne Chassaing1, Sylvain Pichard1, Anne Araye-Guet1, Julien Barbier1, Vincent Forge2and

Daniel Gillet1

1 Commissariat a` l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service d’Inge´nierie Mole´culaire des Prote´ines (SIMOPRO), Gif sur Yvette, France

2 Commissariat a` l’Energie Atomique (CEA), Institut de Recherche en Technologies et Sciences pour le Vivant (IRTSV), Laboratoire de Chimie Biologie des Me´taux (LCBM), Grenoble, France

Introduction

Diphtheria toxin is a protein secreted by

Corynebacte-rium diphtheriae as a single polypeptide chain of

58 kDa [1] During cell intoxication, it is cleaved by

furin into two fragments, the A chain, corresponding

to the catalytic (C) domain, and the B chain,

corre-sponding to the translocation (T) and receptor-binding

domains The C and T domains remain covalently

linked by a disulfide bond Following binding to its cell surface receptor, diphtheria toxin is internalized through the clathrin-coated pathway The acidic pH in the endosome triggers a conformational change lead-ing to the insertion of the toxin in the membrane The C domain is then translocated across the en-dosomal membrane into the cytosol The C domain

Keywords

diphtheria toxin; membrane interaction;

molten globule; protein folding; translocation

Correspondence

D Gillet, Commissariat a` l’Energie Atomique

(CEA), Institut de Biologie et Technologies

de Saclay (iBiTecS), Service d’Inge´nierie

Mole´culaire des Prote´ines (SIMOPRO),

F-91191 Gif sur Yvette, France

Fax: +33 1 69 08 90 71

Tel: +33 1 69 08 76 46

E-mail: daniel.gillet@cea.fr

(Received 15 November 2010, revised 20

January 2011, accepted 15 February 2011)

doi:10.1111/j.1742-4658.2011.08053.x

During cell intoxication by diphtheria toxin, endosome acidification trig-gers the translocation of the catalytic (C) domain into the cytoplasm This event is mediated by the translocation (T) domain of the toxin Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the

C domain during the successive steps by which these domains interact with

a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane To this end, we compared, for each domain, isolated

or linked together in a CT protein (the toxin lacking the receptor-binding domain), each of these steps The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the

C domain is greatly affected by the presence of the T domain All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain, by 0.5–1.6 pH units The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process The results unambiguously demonstrate that the T domain acts as a specialized pH-dependent chaperone for the C domain Interest-ingly, this chaperone activity acts on very different states of the protein: in solution, membrane-bound, and membrane-inserted

Abbreviations

Br-PC, 1-palmitoyl-2-stearoyl(6,7)dibromo-sn-glycero-3-phosphocholine; EPA, phosphatidic acid; EPC, L -a-phosphatidylcholine; k max , maximum emission wavelength; LUV, large unilamellar vesicle; MG, molten globule; SRB, sulforhodamine B.

ADP-ribosylates elongation factor 2, blocking protein

translation and leading to cell death

The translocation process by which the C domain

crosses the membrane remains poorly characterized

Several models have been proposed [1,2] One suggests

that the C domain is translocated through a pore

formed by the B chain Other studies have shown that

both the C and T domains are in contact with the

bilayer, and suggest that the hydrophilic surfaces of

the C domain are hidden from the hydrophobic core

of the membrane by its unfolding or⁄ and by the

B chain [3], without translocating through the ion

channel formed by the T domain Most studies have

focused on the pH-dependent conformational changes

of the isolated T or C domains [1–9], or the entire

toxin [10,11], and their propensity to penetrate into the

bilayer It has been proposed that the T domain acts

as a chaperone for the C domain [12–15] Indeed, the

T domain at acidic pH in solution or in membranes

was shown to bind proteins in a molten globule (MG)

state or hydrophobic peptides [14,15] However, it was

concluded that the chaperone model had not been

for-merly demonstrated [15] Also, only limited pH

condi-tions were explored instead of a continuous range of

pH values; the latter is indispensable for monitoring

all of the successive steps and structural transitions of

the toxin domains leading to membrane penetration

In the present study, our aim was to determine step

by step how each of the C and T domains influences

the membrane interaction and the associated

confor-mational changes of the other domain We compared

the pH sensitivities and the membrane interactions of

the C and T domains, isolated or within the protein

CT, in which the C domain is covalently linked to the

T domain To this end, two CT proteins were

pro-duced, mutated at both Trp positions of either the

T domain or the C domain [11] It was shown

previ-ously [11] that these mutations introduced into the

whole diphtheria toxin do not affect the native

confor-mation of the toxin or its ability to bind ApUp in its

catalytic site In addition, low-pH conformational

changes and membrane insertion were only marginally

affected Here, the conformational changes of the CT

proteins in solution and upon interaction with lipid

vesicles were measured as a function of pH, by

fluores-cence spectroscopy, as well as membrane binding and

penetration into the acyl chain regions of the lipid

bilayer

The data showed that the T domain, by its own

con-formational changes, stabilizes the concon-formational

changes of the C domain that are responsible for its

membrane binding and penetration into the lipid

bilayer

Results

Recombinant proteins Five recombinant proteins were used in this study: C and T, corresponding to the isolated C and T domains,

CT, corresponding to a truncated diphtheria toxin lacking the R domain, and two mutant forms of CT in which the Trp residues of either the T or C domain were mutated to Phe [7] These mutant CTs were pro-duced for Trp fluorescence experiments CT contains four Trp residues Trp50 and Trp153 are located in the

C domain, in b-strand CB3 and just after strand CB7, respectively, according to the crystal structure of diph-theria toxin [16–18] (Fig 1) Trp206 and Trp281 are located in the T domain in helices TH1 and TH5, respectively Mutant CTW50⁄ 153F, in which the Trp residues of the C domain were replaced by Phe, allowed monitoring of the conformational changes of the T domain within CT Mutant CTW206⁄ 281, in which the Trp residues of the T domain were replaced by Phe, allowed monitoring of the conformational changes of the C domain within CT

Within the CTs, the C and T domains were folded

at basic pH and adopted their known MG state at acidic pH

We studied, by CD spectropolarimetry in the far-UV and near-UV, the secondary and tertiary structures of the five recombinant proteins at pH 7.2 At this pH, the toxin is considered to be in its native state [19] The CD spectra obtained for C in the far-UV, featuring low

Fig 1 Structure of CT (left) extracted from diphtheria toxin Protein Data Bank file 1F0L (right) Red: C domain Gray: T domain Green: receptor-binding domain Blue: connecting loop The Trp residues are indicated in yellow.

signals at 190, 210 and 222 nm as compared with the

other proteins, indicated a mixed content of a-helices

and b-sheets (Fig 2A, red curve) The far-UV CD

spec-tra of T (Fig 2A, black curve) indicated a secondary

structure mainly composed of a-helices, also in

agree-ment with the crystal structure of the toxin The spectra

of CT and its two mutants were identical, and showed a

mixed content of a and b structures compatible with a

contribution of the C and T domains

In the near-UV, the CD spectra of C showed a small

positive signal between 280 and 300 nm, which can be

attributed to Trp constrained in a rigid environment

Similarly, a double peak in the 260–270-nm region of

the spectra can be attributed to Phe side chains The

spectra of T showed a strong peak at 292 nm,

attrib-uted to Trp, as described previously [4,5] The spectra

of CT and its mutants exhibited both the signals of

Phe from C and of Trp from C and⁄ or T

The secondary and tertiary structures of the five

proteins were then studied at pH 3.5, at which both C

[13,14] and T [1,4,5] are known to adopt an MG state

In the far-UV, the spectra of C (Fig 2C, red curve)

appeared to be modified, with a loss of signal at

222 nm This suggested some loss of a-helical content,

in agreement with previous observations [20] The

spectra of T (Fig 2C, black curve) were similar to that

recorded at pH 7.2 [4,5,21], with a slight loss of

a-heli-cal content The spectra of the three CTs were mainly

unchanged, except for a small difference for the non-mutated CT, probably because of some aggregation

In the near-UV, the signals found at pH 3.5 were greatly reduced (Fig 2D) This indicated a release of the tertiary constraints on the aromatic residues of the proteins

Altogether, the data suggested that all five recombi-nant proteins were folded at pH 7.2 and exhibited a native-like structure At acidic pH, the loss of tertiary structure signals in the near-UV region of the CD spectra together with the mainly unchanged secondary structure signals in the far-UV confirmed that C [13] and T [4,5,21,22] adopted an MG conformation at acidic pH This was also the case within the CTs, and showed that the mutations introduced did not alter this behavior

The T domain favored the acid-induced MG transition of the C domain in solution when the domains were covalently linked together The maximum emission wavelength (kmax) of the Trp fluorescence was recorded to monitor the acid-induced conformational changes of the recombinant proteins (Fig 3) All proteins showed a pH-dependent transi-tion towards higher kmax, indicating exposure of their

Fig 2 Far-UV (A, C) and near-UV (B, D) CD spectra of C (red), T

(black), CT (light blue), CTW206⁄ 281F (orange), CTW50⁄ 153F (dark blue)

and C mixed with T (green) in solution at pH 7.2 or 3.5; h is the

molar ellipticity in degÆcm 2 Ædmol)1 For far-UV spectra, h is the

mean residue molar ellipticity When the light blue curve

corre-sponding to CT cannot be seen, it is overlapped by the dark blue

curve corresponding to CTW50⁄ 153F.

Fig 3 Conformational changes of C, T, CTW206⁄ 281F and

CT W50 ⁄ 153F monitored by Trp fluorescence as a function of pH Closed red triangles: C Closed black circles: T Open pink triangles:

CTW206⁄ 281F Open blue circles: CTW50⁄ 153F The best fit for each transition is represented (continuous lines) For T, the fitting para-meters are: initial k max = 335.8 nm, final k max = 341.2 nm,

pK1⁄ 2= 5.3, and a Hill coefficient of 5 For CTW50⁄ 153F, the parame-ters are: initial kmax= 334 nm, final kmax= 341.5 nm, pK1⁄ 2 = 5.4, and a Hill coefficient of 2.8 For C, the parameters are: initial

kmax= 338 nm, final kmax= 343 nm, pK1⁄ 2= 4.1, and a Hill coeffi-cient of 3.6 For CTW206⁄ 281F , the parameters are: initial

k max = 333 nm, final k max = 340.5 nm, pK 1 ⁄ 2 = 5.05, and a Hill coefficient of 1.3.

Trp residues to the solvent The kmaxof C shifted from

338 to 343 nm between pH 4.8 and 3.9 (pK1⁄ 2 4.2)

(Fig 3, closed red triangles) The kmax of T shifted

from 336 to 341 nm between pH 5.5 and 4.5

(pK1 ⁄ 2 5.3) (Fig 3, closed black circles)

Interest-ingly, the transition of the C domain within

CTW206⁄ 281F was profoundly modified as compared

with C (Fig 3, open orange triangles) The

fluores-cence shifted from 333 to 341 nm between pH 5.9 and

3.9 (pK1⁄ 2 5.1) This indicated that the Trp residues

of the C domain were in a less polar environment

within CT than when C was isolated This could be

explained by the proximity of the two domains in CT

Most of all, the transition of the C domain towards

the MG state occurred at a pH that was 0.9 units

higher than when it was isolated and was less

coopera-tive In contrast, the transition of the T domain within

CTW50 ⁄ 153Fwas very similar to that of T (Fig 3, open

blue circles) The pK1 ⁄ 2 was nearly identical The kmax

in the native state was lower by 2 nm, indicating that

the Trp residues were less exposed to the solvent,

probably because of the proximity of the C domain

The fluorescence transitions monitored for the

non-mutated CT and for a mix of C and T were more

difficult to interpret (not shown) This was because of

the concomitant measurement of the fluorescence of

four Trp residues, each contributing differently in

terms of kmax and fluorescence intensity [8] In the

case of C and T mix, two separate transitions could

be seen, corresponding roughly to the respective

transition of each domain monitored separately In

the case of CT, two overlapping transitions were

detected The second transition, occurring at the

low-est pH values and probably corresponding to the

C domain, was shifted towards higher pH, as

com-pared with that of C

Altogether, the results indicated that, within CT, the

native to MG transition of the T domain was similar

to that of the isolated T, whereas the native to MG

transition of the C domain was shifted to 0.9 pH units

higher than when it was isolated Thus, the T domain

enabled the transition of the C domain to occur at

higher pH This effect was possible only if the C and

T domains were covalently linked Also, the results

strongly suggested that the two domains interacted

during the transitions

The T domain favored the interaction of the

C domain with the membrane

We then studied the interaction of the recombinant

proteins with anionic large unilamellar vesicles (LUVs)

as a function of pH (Fig 4) Binding was monitored

according to physical partition between the LUVs and the solvent, by centrifugation and Trp fluorescence measurements

C and T bound to the LUVs from pH 6.0 to 4.5 and from pH 6.8 to 6.0, respectively (Fig 4A), indicat-ing preferential bindindicat-ing of T over C CTW50⁄ 153F and

CTW206⁄ 281Fbound to the LUVs from about pH 7.0 to 5.0 Thus, they started their binding transition at about the same pH as T, but it occurred over two pH units instead of one, showing reduced cooperativity How-ever, for these proteins, one cannot determine from these data which domain bound first to the membrane:

T, C, or both

These results indicated that isolated C bound the membrane at about one pH unit lower than T In con-trast, the presence of the T domain covalently linked with the C domain favored the interaction of C with the membrane (at least through the binding of T), at a

pH higher than when it was isolated

The T domain facilitated the insertion of the

C domain in the membrane

To better characterize the environment of the Trp resi-dues of the C and T domains within CT during the interaction with the membrane, we measured the quenching of the Trp fluorescence of T, C and the CT mutants by use of anionic LUVs containing

brominat-ed phospholipids as a function of pH (Fig 4B) We used 1-palmitoyl-2-stearoyl(6,7)dibromo-sn-glycero-3-phosphocholine (Br-PC) lipids with bromine atoms covalently bound at positions C6 and C7 of the oleoyl chains

The Trp fluorescence of C was slightly quenched below pH 4.6, and by up to 12% at pH 3.8 (Fig 4B, closed red triangles) This may indicate weak pene-tration of C in the hydrophobic layer of the mem-brane In contrast, the fluorescence of T was strongly quenched as the pH decreased below 6, by up to 48%

at pH 3.8 (pK1⁄ 2 4.9) (Fig 4B, closed black circles) This confirmed the results of similar experiments [8] indicating deep penetration of T into the bilayer Very similar values were obtained for CTW50⁄ 153F (pK1⁄ 2 4.7) (Fig 4B, open blue circles), strongly sug-gesting that the T domain reached the same depth into the hydrophobic layer of the membrane, isolated or within CT

An intermediate situation was found with

CTW206 ⁄ 281F (Fig 4B, open pink triangles) Significant fluorescence quenching was observed below pH 5.4, by

up to 25% at pH 3.8 This is twice the effect measured for C alone at the same pH Unfortunately, no plateau was detected at the pH investigated here As a

consequence, in the case of the C domain (both C and

CTW206⁄ 281F), the pH dependences of the quenching

could not be fitted for estimation of the pK1⁄ 2 and the

maximum of quenching However, it is clear, in the

pH range we explored, that the C domain penetrated

deeper inside the bilayer when it was covalently linked

to the T domain, and that this transition occurred at

higher pH than when it was isolated

The T domain favored the structural transitions

of the C domain during interaction with the membrane

In order to investigate the structural transitions under-gone by the C and T domains during interaction with the membrane, we monitored the fluorescence of the four proteins in the presence of anionic LUVs as a function of pH The kmax of C shifted from 338 nm to

343 nm between pH 4.9 and 4.1, and then from 343 to

340 nm between pH 4.1 and 3.5 (Fig 4C, closed red triangles) These two successive transitions have been observed previously [3] The increase of the kmax observed during the first transition could be attributed

to increased exposure of the Trp residues of C to the aqueous buffer Thus, this first transition could corre-spond to a partial unfolding of C, as is the case for T [4,5,8] The second transition, indicating burial of the Trp residues in an apolar environment, could corre-spond to the penetration of C in the membrane [3], as

is the case for T [4,5,8]

T interacted with the LUVs according to the two-step process described previously [4,5,8] (Fig 4C, closed black circles) The first transition was attributed

to the binding of T to the membrane and its unfolding with exposure of its N-terminal Trp residues to the

Fig 4 (A) Partition of C (closed red triangles), T (closed black cir-cles), CT W206 ⁄ 281F (open pink triangles) and CT W50 ⁄ 153F (open blue circles) between the buffer and LUVs as a function of pH, studied

by ultracentrifugation The best fit for each transition is represented (continuous lines) For T, the fitting parameters are: pK 1 ⁄ 2 = 6.4 and a Hill coefficient of 3.2 For CTW50⁄ 153F, the parameters are:

pK 1⁄ 2 = 6.25 and a Hill coefficient of 2.0 For C, the parameters are: pK 1⁄ 2 = 5.15 and a Hill coefficient of 2.7 For CT W206 ⁄ 281F , the parameters are: pK1⁄ 2= 5.8 and a Hill coefficient of 1.9 (B) Quenching of Trp fluorescence of C, T, CTW206⁄ 281F and CTW50⁄ 153F

by LUVs containing Br-PC The results are expressed as the relative quenching efficiency as compared with the Trp fluorescence at

pH 7 The lower the value, the closer the Trp from the quencher In the case of T and CT W50 ⁄ 153F , the data could be fitted with Micha-elis–Menten equations (continuous lines) For T, the fitting parame-ters are: pK1⁄ 2= 4.9 and a final F ⁄ F 0 of 48% For CTW50⁄ 153F, the parameters are: pK 1 ⁄ 2 = 4.7 and a final F ⁄ F 0 of 49% (C) Trp fluo-rescence of C, T, CT W206 ⁄ 281F and CT W50 ⁄ 153F in the presence of anionic LUVs as a function of pH In the case of T and CTW50⁄ 153F, the data could be fitted with the pK1⁄ 2 values obtained from (A) and (B) in order to estimate the k max of the various states of the domain (continuous lines) For T, the initial kmaxis 335.5 nm, the intermediate kmaxis 344.5 nm, and the final kmaxis 329.4 nm For

CT W50 ⁄ 153F , the initial k max is 334 nm, the intermediate k max is

341 nm, and the final kmaxis 329 nm (D) Permeabilization of anio-nic LUVs by C, T and CT W50⁄ 153F CT permeabilized LUVs as effi-ciently as T and CT W50 ⁄ 153F CT W206 ⁄ 281F permeabilized LUVs slightly less efficiently (not shown).

buffer, and the second transition to penetration into

the bilayer

The kmax of CTW50⁄ 153F shifted from 334 to 340 nm

between pH 7.1 and 6.0, and then from 340 to 333 nm

between pH 6.0 and 4.3 (Fig 4C, open blue circles),

thereby indicating two transitions very similar to those

of the isolated T (Fig 4C, closed black circles)

Between these two transitions, the kmax of CTW50⁄ 153F

was 3 nm lower than that of T The first transition

found for CTW50⁄ 153Fcorrelated with membrane

bind-ing (Fig 4A, open blue circles) Notably, this

mem-brane binding transition was less cooperative than that

of T (Fig 4A, closed black circles) This may explain

the decreased kmax found for CTW50⁄ 153F as compared

with T (Fig 4C) Indeed, the first transition of the

T domain within CTW50⁄ 153F was not completed when

the second transition started The second transition

correlated with the insertion in the membrane, which

was also monitored by fluorescence quenching

(Fig 4B) The final kmax was the same for T and

CTW50 ⁄ 153F, i.e 329 nm In both cases, the pH

depen-dence of kmax could be fitted with the two values of

pK1⁄ 2 obtained from the partition (Fig 4A) and

fluo-rescence quenching (Fig 4B) experiments (Fig 4C,

continuous lines)

The kmax of CTW206⁄ 281Fshifted from 335 to 339 nm

between pH 6.4 and 5.0, and then from 339 to 336 nm

between pH 5.0 and 3.6 (Fig 4B, open orange

trian-gles) Thus, although the C domain within CT

fol-lowed two transitions similar to those of the isolated

C, these transitions occurred at higher pH This

sug-gested that the T domain favored the interaction of

the C domain with the membrane when C was

cova-lently linked to T The kmaxof CTW206 ⁄ 281Fwas about

4 nm lower than that of C Again, this suggested

proximity or contacts between the C and T domains

within CT, limiting exposure of the Trp residues to the

environment

Overall, both domains underwent two structural

transitions upon binding and penetration into the

membrane For T, the first transition corresponded to

binding and the second to membrane penetration, but

this is less obvious for C Within CT, the presence

of the C domain did not affect the transitions of the

T domain, but the presence of the T domain favored

the transitions of the C domain at higher pH

Anionic LUV permeabilization

Anionic LUV permeabilization was shown to be an

indicator of penetration of the T domain into the

membrane [4,5,23] Whereas C did not permeabilize

LUVs significantly, CT permeabilized LUVs at least as

efficiently as T (Fig 4D) This confirmed that the

T domain within CT was fully capable of penetrating the lipid bilayer

Discussion

Figure 5 summarizes the data collected in the present study Various methods were used to probe the inter-actions of the C and T domains of diphtheria toxin with the membrane, alone or covalently linked together Binding to the membrane was revealed

by centrifugation experiments (Figs 4A and 5, pink arrows) Conformational changes of the C and T do-mains were monitored by Trp fluorescence of CTs mutated on the Trp of the C or T domains (Figs 4B and 5, transitions C1, C2, and T1, T2) Penetration into the fatty acid region of the bilayer was revealed

by quenching of the Trp fluorescence of the mutant CTs by Br-PC (Figs 4C and 5, green arrows) Permea-bilization of the membrane, which mainly coincided with membrane penetration, was detected by fluores-cent dye release from LUVs (Fig 4D) On the basis of all of the data, we describe the succession of steps leading to membrane binding and membrane penetra-tion of both domains of the protein In addipenetra-tion, we

Fig 5 Schematic representation of the successive steps followed

by C and CT when interacting with anionic LUVs, as a function of

pH The binding and membrane-penetration transitions indicated as pink and green arrows are from the curves of Fig 4 The different shapes named C1, C2, T1 and T2 symbolize the conformational changes of the C and T domains associated with these transitions Binding (pink) data are from Fig 4A Penetration (green) data are from Fig 4B Permeabilization data from Fig 4D (not shown on this scheme) mainly coincide with membrane penetration (green) Con-formational changes of the protein domains observed by Trp fluo-rescence are from Fig 4C The only difference found for T (not shown in the scheme) as compared with CT is that the binding transition is more cooperative and ends at pH 6.

show that the T domain behaves relatively

indepen-dently from the C domain, in solution (Fig 3) and

during membrane interaction (Fig 4), whereas the

C domain is highly influenced by the presence of the

T domain (Figs 4 and 5)

The T domain drives the successive steps by

which the C domain binds and penetrates the

membrane

From pH 7 to 5, CT binds to the membrane (Fig 5)

The T domain is responsible for initiating binding,

because binding of T and CT starts at the same pH,

whereas binding of C starts at one pH unit lower

Monitoring of the conformational changes C1 and T1

(Figs 4C and 5) associated with binding (Fig 4A,B)

led to the same conclusion (see next section)

From pH 6 to 4 or below, both domains of CT

pen-etrate into the membrane (Figs 4C and 5, green

arrows) Again, the T domain leads the way for the

C domain It is not influenced by the presence or

absence of the C domain, whereas the C domain is

clearly influenced by the presence of the T domain

The T domain favors the conformational changes

adopted by the C domain during binding to, and

penetration into, the membrane

The structural behavior of the T domain interacting

with the membrane as a function of pH is quite similar

whether it is isolated or linked to C The only

differ-ence found is that binding is less cooperative for CT

than for T (Fig 4A) As a result, binding seems to

overlap both the unfolding of T (Fig 5, structural

transition T1) and its rearrangement in the membrane

corresponding to penetration [4,8] (Fig 5, structural

transition T2) However, in fact, a fraction of bound

molecules already starts to rearrange in the membrane

(T2) while a fraction of molecules have not fully

unfolded yet, owing to the decreased cooperativity of

the reaction

In contrast, the structural behavior of the C domain

during interaction with the membrane is very different

when it is isolated or connected with T When it is

iso-lated, its unfolding (Figs 4B and 5, structural

transi-tion C1) does not coincide with binding (Figs 4A and

5, pink arrow) This indicates that the C domain binds

first to the membrane without undergoing

conforma-tional change, and then unfolds in about the same pH

range as in solution (Fig 3) Then, it progressively

penetrates the membrane to a shallow position

(Figs 4B and 5, green arrow), finishing unfolding

(Figs 4C and 5, transition C1) before a second

rear-rangement of its structure occurs (Figs 4C and 5, tran-sition C2)

When the C domain is connected with the T domain, the T domain clearly favors unfolding of the

C domain in solution (Fig 3) and during binding to the membrane (Fig 4A,C and 5, structural transi-tion C1 and pink arrow) Thus, the T domain favors the interaction of the C domain with the membrane because it stabilizes its partially unfolded state (Fig 5, C1) This strongly suggests that the C domain binds to the membrane concomitantly with the T domain or at

a pH not more than 0.5 U lower than that driving the binding of T Then, the T domain helps the C domain

to penetrate into the hydrophobic core of the mem-brane During this step, the C domain finishes its conformational change C1 (Figs 4C and 5), and then undergoes conformational change C2 (Figs 4C and 5), corresponding to deeper penetration into the acyl chain layer of the membrane (Fig 4B and 5, green arrow), than in the absence of the T domain

The T domain but not the C domain is specialized to permeabilize the membrane The T domain permeabilizes the membrane (Fig 4D) during the membrane-penetration step (Fig 4B and 5, green arrow) The deeper the T domain is inserted, the stronger is the permeabilization The results clearly show that the T domain is absolutely required for permeabilization, C alone being incapable of doing so (Fig 4D) Interestingly, the penetration of the C domain in the membrane does not impair its permeabilization by the T domain This suggests that the C domain does not plug the passageway formed

by the T domain in the bilayer One cannot state, however, whether or not this passageway is taken by the C domain to cross the membrane Nevertheless, these results indicate that the T domain is specialized

to permeabilize the membrane but the C domain is not, even though it is embedded in the bilayer In other words, the membrane is not destabilized by the insertion of C

The T domain acts as a chaperone for the

C domain

It has been proposed that the T domain acts as a chaperone for the C domain, enabling its passage through the membrane at acidic pH [12–15] Indeed, T

at acidic pH in solution or in membranes was shown

to bind proteins in an MG state or hydrophobic pep-tides [14,15] However, it was concluded that the chap-erone model had not been formerly demonstrated [15]

The present work demonstrates that the T domain in

its various pH-dependent conformations, in solution,

membrane-bound, and membrane-inserted, stabilizes

partially unfolded states of the C domain In doing so,

the T domain favors membrane binding and

mem-brane penetration of the C domain By definition, the

activity of a chaperone is the stabilization of a

par-tially folded (or unfolded) state of another protein

Thus, we demonstrate that the T domain acts as a

chaperone for the C domain A remarkable feature of

this chaperone activity is that it stabilizes at least three

different partially folded states of the C domain, each

corresponding to one of the successive steps of the

ini-tiation of translocation: conformational change in

solution, membrane binding, and membrane insertion

How does the T domain exerts its chaperone

activ-ity on the C domain? The T domain adopts an MG

state displaying hydrophobic surfaces [4,21,22] These

hydrophobic surfaces may offer an environment that

is propitious for the interaction with the hydrophobic

surfaces of the C domain, which are exposed only in

its MG state Thus, the T domain in its MG state

must greatly displace the native to MG state

equilib-rium of the C domain in favor of the MG state The

T domain favors the interaction of the C domain

with the membrane, because it brings the C domain

in its MG state into the vicinity of the bilayer, the

MG state of both domains being propitious for

mem-brane insertion and⁄ or translocation [4,13,14,21] The

membrane itself may also have a destabilizing effect

on the C domain: the interfacial pH is lower than in

the solvent and the hydrophobic acyl chains may

interact with hydrophobic regions of the protein

Finally, the T domain imposes its rule on the C

domain because it is more sensitive to pH Indeed, it

has an elaborate system for reacting to a wide range

of acidic pH values, starting just below pH 7,

involv-ing its six His residues [5]

It has been shown previously that, after

transloca-tion, the C domain and only the 63 N-terminal amino

acids of the T domain are present on the trans side of

the membrane [24,25] The remaining 124 amino acids

of the T domain are left in the membrane However, a

cytoplasmic chaperone, Hsp90, is involved in

extrac-tion of the C domain from the membrane and its

refolding [26] The T domain seems to be no longer

needed for the last stages of translocation

Our findings emphasize the importance of the

physi-cochemical properties that a protein should have in

order to interact with, and penetrate into, a

mem-brane They should be taken into consideration to

evaluate or adapt the capacity of proteins to bind or

cross a membrane

Experimental procedures

Recombinant proteins

Expression and purification of the recombinant T domain containing mutation C201S (native diphtheria toxin number-ing) has been described previously [21,23] Two DNA sequences coding for residues 1–380 of the native toxin (C and

T domains) and residues 1–193 (C domain) were prepared by PCR and cloned into the pET-28a(+) vector (Novagen, Mad-ison, WI, USA), using the NdeI and SalI restriction sites The two resulting plasmids, CTpET-28a(+) and CpET-28a(+), encoded CT and C preceded by an N-terminal His tag sequence Cys186 in the C domain protein was mutated in Ser Mutations W50F and W153F, or W206F and W281F, were introduced by PCR mutagenesis into plasmid CTpET-28a(+) The sequences were checked by DNA sequencing Production and purification of recombinant C was per-formed as described for T [21,23] CT, CTW50 ⁄ 153F and

CTW206 ⁄ 281F were expressed at 37C in Escherichia coli strain BL21(DE3) as inclusion bodies The inclusion bodies were solubilized in 8 m urea, 0.1 m Tris⁄ HCl, and 0.1 mm EDTA (pH 8), and the proteins were purified by immobi-lized-nickel affinity chromatography The proteins were folded by dialysis against a 20 mm sodium phosphate buffer

at pH 8 The proteins were further purified on a Hi Load Superdex 26⁄ 60 size exclusion column (GE Healthcare, Orsay, France), and, finally, the buffer was exchanged with

NH4HCO3 on a G25SF column before lyophilization and storage at) 20 C

Lipid vesicles

l-a-phosphatidylcholine (EPC), phosphatidic acid (EPA) and Br-PC were from Avanti Polar Lipids (Alabaster, AL, USA) Suspensions of anionic lipid bilayers at a lipid con-centration of 20 mm were prepared in 5 mm citrate buffer (pH 7.2) at an EPC⁄ EPA molar ratio of 9 : 1 LUVs and small unilamellar vesicles were prepared as described in [8]

In the presence of brominated lipids, the EPC⁄ Br-PC ⁄ EPA ratio was 5 : 4 : 1, and the LUVs were prepared at 37C

CD spectropolarimetry

CD experiments on all of the recombinant proteins were performed on a J-815 spectropolarimeter (Jasco, Tokyo, Japan) as described previously [21], at pH 7.2 and pH 3.5 Spectra were treated as previously described [21]

Fluorescence spectroscopy

Fluorescence measurements were performed with an FP-750 spectrofluorimeter (Jasco) as described previously [4] Pro-teins (1 lm) were added to 5 mm sodium citrate and 200 mm NaCl at the indicated pH, and samples were incubated for

2 h at room temperature before measurements were

per-formed (excitation wavelength of 292 nm) Maximum

emis-sion wavelength (kmax) represents the average of three values

obtained from emission spectra that were corrected for blank

measurements For experiments with LUVs, proteins (1 lm)

were mixed with LUVs (500 lm) in a 5 mm citrate buffer at

the indicated pH values The pH was always checked after

measurements Physical binding measurements were

moni-tored as described in [4] The control was obtained by

centri-fugation of the proteins at 350 000 g for 1.5 h without LUVs

Fluorescence extinction in the presence of

brominated lipids

LUVs containing EPC, Br-PC and EPA (Avanti Polar

Lip-ids) at a 5 : 4 : 1 molar ratio were incubated for 2 h at

37C in the presence of 1 lm protein and 500 lm LUVs in

5 mm citrate buffer at the indicated pH values The

fluores-cence extinction of Trp was evaluated with the ratio F⁄ F0,

where F and F0are the fluorescence intensities in the

pres-ence or in the abspres-ence of LUVs containing brominated

lip-ids, respectively The results represent the average of five

measurements

LUV leakage assay

LUVs containing 50 mm sulforhodamine B (SRB)

(Molecu-lar Probes, Eugene, OR, USA) were prepared in 5 mm

cit-rate buffer at pH 7.2 Unencapsulated SRB was removed

by size exclusion chromatography on a Sephadex G-25

col-umn equilibrated with 5 mm citrate and 50 mm NaCl buffer

(pH 7.2) Release of SRB was monitored by measuring the

increase in fluorescence on a Jasco FP-750

spectrofluorime-ter afspectrofluorime-ter addition of 9 nm protein to a 1.5-mL suspension

of 9 lm LUVs in 5 mm citrate buffer at different pH values

(excitation, 565 n; emission, 586 nm) with stirring SRB

was selected as fluorescent probe because of its high

quan-tum yield independently of the pH Fluorescence was

nor-malized as previously described [27] The initial rate (V0)

was deduced from the slope at the origin of the curves

Acknowledgements

We thank A Lecoq for help with protein folding This

work was supported by the Commissariat a` l’ Energie

Atomique (Signalization and Membrane Transport

Program of the Life Science Division) The authors

dedicate this work to the memory of A Me´nez

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