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

Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain Anne Chassaing 1 , Sylvain Pichard 1 , Anne Araye-Guet 1 , Julien Barbier 1 , Vincent Forge 2 and Daniel Gillet 1 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. 4516 FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS 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 conformational 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 CT W50 ⁄ 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 CT W206 ⁄ 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. A. Chassaing et al. Membrane interaction of diphtheria toxin FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS 4517 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 (k max ) 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 k max , 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), CT W206 ⁄ 281F (orange), CT W50 ⁄ 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 CT W50 ⁄ 153F . Fig. 3. Conformational changes of C, T, CT W206 ⁄ 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: CT W206 ⁄ 281F . Open blue circles: CT W50 ⁄ 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, pK 1 ⁄ 2 = 5.3, and a Hill coefficient of 5. For CT W50 ⁄ 153F , the parame- ters are: initial k max = 334 nm, final k max = 341.5 nm, pK 1 ⁄ 2 = 5.4, and a Hill coefficient of 2.8. For C, the parameters are: initial k max = 338 nm, final k max = 343 nm, pK 1 ⁄ 2 = 4.1, and a Hill coeffi- cient of 3.6. For CT W206 ⁄ 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. Membrane interaction of diphtheria toxin A. Chassaing et al. 4518 FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS Trp residues to the solvent. The k max of C shifted from 338 to 343 nm between pH 4.8 and 3.9 (pK 1 ⁄ 2  4.2) (Fig. 3, closed red triangles). The k max of T shifted from 336 to 341 nm between pH 5.5 and 4.5 (pK 1 ⁄ 2  5.3) (Fig. 3, closed black circles). Interest- ingly, the transition of the C domain within CT W206 ⁄ 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 (pK 1 ⁄ 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 CT W50 ⁄ 153F was very similar to that of T (Fig. 3, open blue circles). The pK 1 ⁄ 2 was nearly identical. The k max 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 k max 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 binding of T over C. CT W50 ⁄ 153F and CT W206 ⁄ 281F bound 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 (pK 1 ⁄ 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 CT W50 ⁄ 153F (pK 1 ⁄ 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 CT W206 ⁄ 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 A. Chassaing et al. Membrane interaction of diphtheria toxin FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS 4519 consequence, in the case of the C domain (both C and CT W206 ⁄ 281F ), the pH dependences of the quenching could not be fitted for estimation of the pK 1 ⁄ 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 k max 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 k max 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 CT W50 ⁄ 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: pK 1 ⁄ 2 = 5.8 and a Hill coefficient of 1.9. (B) Quenching of Trp fluorescence of C, T, CT W206 ⁄ 281F and CT W50 ⁄ 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: pK 1 ⁄ 2 = 4.9 and a final F ⁄ F 0 of 48%. For CT W50 ⁄ 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 CT W50 ⁄ 153F , the data could be fitted with the pK 1 ⁄ 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 k max is 335.5 nm, the intermediate k max is 344.5 nm, and the final k max is 329.4 nm. For CT W50 ⁄ 153F , the initial k max is 334 nm, the intermediate k max is 341 nm, and the final k max is 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). Membrane interaction of diphtheria toxin A. Chassaing et al. 4520 FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS buffer, and the second transition to penetration into the bilayer. The k max of CT W50 ⁄ 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 k max of CT W50 ⁄ 153F was 3 nm lower than that of T. The first transition found for CT W50 ⁄ 153F correlated 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 k max found for CT W50 ⁄ 153F as compared with T (Fig. 4C). Indeed, the first transition of the T domain within CT W50 ⁄ 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 k max was the same for T and CT W50 ⁄ 153F , i.e. 329 nm. In both cases, the pH depen- dence of k max could be fitted with the two values of pK 1 ⁄ 2 obtained from the partition (Fig. 4A) and fluo- rescence quenching (Fig. 4B) experiments (Fig. 4C, continuous lines). The k max of CT W206 ⁄ 281F shifted 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 k max of CT W206 ⁄ 281F was 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 addition, 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. A. Chassaing et al. Membrane interaction of diphtheria toxin FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS 4521 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]. Membrane interaction of diphtheria toxin A. Chassaing et al. 4522 FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS 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 NdeIandSalI 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, CT W50 ⁄ 153F and CT W206 ⁄ 281F were expressed at 37 °CinEscherichia 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 NH 4 HCO 3 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 37 °C. 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 A. Chassaing et al. Membrane interaction of diphtheria toxin FEBS Journal 278 (2011) 4516–4525 ª 2011 The Authors Journal compilation ª 2011 FEBS 4523 2 h at room temperature before measurements were per- formed (excitation wavelength of 292 nm). Maximum emis- sion wavelength (k max ) 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)ina5mm 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 37 °C in the presence of 1 lm protein and 500 lm LUVs in 5mm citrate buffer at the indicated pH values. The fluores- cence extinction of Trp was evaluated with the ratio F ⁄ F 0 , where F and F 0 are the fluorescence intensities in the pres- ence or in the absence 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 after 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 (V 0 ) was deduced from the slope at the origin of the curves. Acknowledgements We thank A. Lecoq for help with protein folding. 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