HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 33, 24239-24245, August 17, 2007

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 282/33/24239    most recent M703392200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Perier, A. Articles by Gillet, D. Search for Related Content PubMed PubMed Citation Articles by Perier, A. Articles by Gillet, D. Social Bookmarking                      What's this?

Concerted Protonation of Key Histidines Triggers Membrane Interaction of the Diphtheria Toxin T Domain^*

Aurélie Perier¹, Anne Chassaing¹, Stéphanie Raffestin, Sylvain Pichard, Michel Masella, André Ménez, Vincent Forge, Alexandre Chenal²³, and Daniel Gillet²⁴

From the Commissariat à l'Energie Atomique, Institut de Biologie et Technologies de Saclay, Service d'Ingénierie Moléculaire des Protéines, Gif-sur-Yvette F-91191, France and Institut de Recherche en Technologies et Sciences pour le Vivant, Laboratoire de Chimie Biologie des Métaux, Grenoble F-38054, France

Received for publication, April 24, 2007 , and in revised form, June 13, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His²²³-His²⁵⁷ and His²⁵¹ are the most sensitive triggers for the formation of the molten globule state in solution, whereas His³²²-His³²³ and His²⁵¹ are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The function of a number of proteins is regulated by acidic pH. This is particularly the case for a series of bacterial protein toxins with intracellular targets (1). These include, for instance, Pseudomonas exotoxin A (2), anthrax toxin (3), botulinum toxins (4) and diphtheria toxin (5). After binding to a cell surface receptor, these toxins are internalized into the endosome. The acidic pH found in this compartment triggers the insertion of a translocation moiety into the membrane, which assists the passage of a catalytic domain into the cytoplasm. There, the catalytic domain modifies a substrate, leading to perturbation of a physiological process. The purpose of the present study is to determine how pH controls the conformational changes leading to the interaction of the translocation domain (T domain)⁵ of the diphtheria toxin with membranes.

In its soluble form, the T domain of the diphtheria toxin is composed of 10 helices named TH1-TH9 and TH5', organized in a globular shape (6) (Fig. 1). Hydrophobic helices TH8 and TH9 are sandwiched by two layers of amphiphilic helices, TH1-TH4 and TH5-TH7. In solution, the acidification of pH between 6 and 5 induces a conformational change, leading to a molten globule (MG) state prone to interact with membranes (7, 8). In the presence of membranes however, the T domain binds to the phospholipid bilayer between pH 7 and pH 6. This interaction involves the C terminus of the domain, whereas the N terminus is more exposed to the solvent (9). Between pH 6 and pH 4, the protein rearranges its structure and penetrates deeply inside the membrane (8-10). The pH ranges at which these transitions occur suggest that the protonation of His side chains may be involved. Indeed, it has been shown that His residues may function as pH sensors for many molecular mechanisms involving protein-membrane interaction (11, 12) and/or MG formation (13, 14).

The T domain of diphtheria toxin contains six His residues (Fig. 1). Unfortunately, the protein aggregates around its isoelectric point at high concentration (15). This precludes probing the role of these His residues by NMR. To overcome this experimental problem, we substituted the His by Phe residues using site-directed mutagenesis and studied the behavior of the mutant T domains by a combination of biophysical approaches. The data showed that MG formation and membrane interaction involve the concerted protonation of key His side chains. However, different His residues have a preponderant role in the different steps studied: the pair His²²³-His²⁵⁷ and His²⁵¹ are important for the formation of the MG state in solution, whereas the pair His³²²-His³²³ and His²⁵¹ are the most sensitive triggers of the membrane binding step.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Recombinant Proteins—The recombinant T domain containing mutation C201S (native diphtheria toxin numbering) and referred to as "wild type" (WT) has been described previously (15, 16). His to Phe mutations were introduced by site-directed mutagenesis in plasmid pA/T C201S and checked by DNA sequencing. Protein expression was performed as described previously (15, 16) except that yields were increased by growing overnight precultures at 25 °C. The T domains were purified from the soluble cytoplasmic fraction as described (9, 15). Proteins were subjected to immobilized nickel affinity, size exclusion, and ion exchange chromatography. The purification buffer of the T domain was finally exchanged with NH₄HCO₃ on a G25SF column before lyophilization and storage at -20 °C. The molar epsilon used was 17,780 M^-1·cm^-1 at 280 nm, and the molecular mass was 21,860 g·mol^-1.

Lipid Vesicles—Suspensions of large unilamellar vesicles of anionic lipid bilayers at a lipid concentration of 10 mM were prepared in 5 mM citrate buffer at pH 7.5 with egg phosphatidylcholine and egg phosphatidic acid (Avantipolar Lipids, Alabaster, AL) at a 9:1 molar ratio by reverse phase evaporation and filtration twice on 0.4-µm and four times on 0.2-µm filters (17, 18). As described previously (5, 8), the T domain requires anionic lipids for membrane insertion at pH < 6. Zwitterionic lipids are sufficient for the binding step at pH 6. A 9:1 molar ratio of phosphatidylcholine/phosphatidic acid was chosen, because it corresponds to the minimal anionic lipid content, allowing membrane insertion of the T domain.

Circular Dichroism Spectropolarimetry—CD experiments were performed on a J-815 spectropolarimeter (Jasco, Tokyo, Japan) as described previously (15). Proteins were diluted in 5 mM sodium citrate buffer at the indicated pH 1 h before measurements. Protein concentration and pH were checked after measurements. For near UV and far UV measurements, each spectrum is the average of 20 scans and 10 scans, with proteins at concentrations of 20 and 5 µM, respectively. The spectra were corrected for the blank and were not smoothed. Spectra were treated as described previously (15).

Fluorescence Spectroscopy in Solution—Fluorescence measurements were performed with an FP-750 spectrofluorimeter (Jasco, Tokyo, Japan) as described previously (9). Proteins were diluted at a concentration of 1 µM in 5 mM sodium citrate buffer, 200 mM NaCl at the indicated pH, 2 h before measurements. pH was checked after measurements. Maximum emission wavelength (_max) and fluorescence intensity ratios at 360 nm over 320 nm (FI_360/320) represent the average of three values obtained from emission spectra that were corrected for blank measurements. Values of (pH of midtransition from the native to the MG state) were determined from curves of FI_360/320 as a function of pH fitted with a two-state model: A + nH⁺ AH_n, where n is the number of protons and A and AH represent the native and molten globule states. The dissociation constant of the transition is given by Kⁿ = [A][H⁺]ⁿ/[AH_n]. The measured fluorescence signal S depends on the species composition at each pH value according to the following equation: S = S_i + (S^f - Sⁱ) x f_A with f_A = A/(A + AHⁿ) = 1/(1 + (AHⁿ/A)) = 1/(1 + (H⁺/K)ⁿ). S is the signal used to monitor the reaction, Sⁱ and S^f are the initial and final values, respectively, [H⁺] is the proton concentration, K is the dissociation constant, and n is the Hill coefficient. Curves were fitted using Kaleidagraph 3.6 (Synergy Software, Reading, PA).

Fluorescence Spectroscopy in the Presence of LUV—The proteins at a concentration of 1 µM were mixed with LUV at a lipid/protein molar ratio of 500 in a 5 mM citrate buffer at the indicated pH. Samples were incubated for 2 h at 22 °C before measurements. pH values were always measured afterward. The two-step model we used to fit the experimental data from the mutant proteins is based on two consecutive Hill plots, using the same equation as described above. The parameters of the membrane penetration step were considered as constants (nH₂ and pK₂ from the second transition were taken from the WT T domain fitting results), whereas the parameters describing the binding step (nH₁ and pK₁ from the first transition) were fitted for each mutant. For physical binding measurements, samples were centrifuged in an L-70 ultracentrifuge (Beckman, Roissy, France) using a Ti 70.1 rotor at 60,000 rpm for 20 min at 22 °C to pellet the LUV. The fraction of bound proteins was determined as (F₀ - F)/F₀, where F₀ and F are the fluorescence intensities of the sample before and after centrifugation.

LUV Leakage Assay—Protein-induced permeabilization of LUV at pH 4 was measured using the ANTS/DPX assay. LUV were prepared in a 5 mM sodium citrate, 200 mM NaCl buffer at pH 7.5 containing 20 mM ANTS (Molecular Probes, Eugene, OR). Nonencapsulated ANTS was removed by gel filtration on a Sephadex G-25 column (Amersham Biosciences). Concentrated protein samples at pH 7.5 were diluted in a suspension of LUV in 5 mM sodium citrate, 200 mM NaCl, and 20 mM DPX (Molecular Probes) at pH 4 at 22 °C. Protein concentration was 9 nM at a lipid/protein molar ratio of 1000. The fluorescence decrease, due to the leakage of ANTS out of the LUV and its quenching by DPX, was recorded. Fluorescence measurements were performed by setting the emission at 516 nm and the excitation at 360 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Secondary and Tertiary Structures of the Mutant T Domains in the Native and MG States—The T domain contains six His residues at positions 223, 251, 257, 322, 323, and 372 (Fig. 1). In order to investigate the effect of the absence of protonation of His residues on the behavior of the protein, 16 mutants of the T domain were produced in which single or multiple His residues were replaced by Phe (Table 1). The mutant T domains are numbered thereafter according to the mutated position(s). Phe was chosen because the shape and volume of its side chain are similar to those of a neutral His. Thus, it is the best amino acid to mimic a nonprotonable form of His. Among the six His residues of the T domain (Fig. 1), two pairs are located in loops. His²²³ between helices TH1 and TH2 is stacked with His²⁵⁷ located between helices TH3 and TH4. His³²² and His³²³ are between helices TH7 and TH8. His²⁵¹ and His³⁷² are located in helices TH3 and TH9, respectively.

View this table: [in this window] [in a new window]   TABLE 1 Mutated positions, , pK1, and pK2 (corresponding to membrane binding and membrane penetration monitored by fluorescence) for all mutant T domains

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Structure (Protein Data Bank file 1F0L) and amino acid sequence of the diphtheria toxin T domain. Helices TH1-TH4 are shown in green, TH5-TH7 in blue, and TH8 and TH9 in red. His side chains are shown in orange.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Far UV (A and B) and near UV (C and D) CD spectra of a selection of mutant T domains in solution, at pH 7.5 or 3.5. Dashed line, WT; green, T251; blue, T223-257; red, T322-323; black, THis.

His to Phe Mutations Shifted the Transition to the MG State toward Lower pH Values in an Additive Manner—The acid-induced conformational change of the mutant T domains was monitored by Trp fluorescence spectroscopy (Fig. 3). The results were compared with those obtained with the WT T domain (Fig. 3A, closed circles). As described previously (8, 15), the spectra of the WT T domain in solution showed a red shift of the maximum emission wavelength (_max) from 336 nm in the native state to 341 nm in the MG state. All mutants followed a transition toward higher _max. The end of the transition corresponded to the MG state, in agreement with the results of the CD experiments described above. However, this transition occurred at lower pH for practically all mutants to various extents (Fig. 3 and Table 1). In the case of THis, in which all His residues were mutated, this transition was strongly altered with a shift of _max from 332 to 336 nm. This suggested that the Trp remained in a rather apolar environment despite the release of tertiary constraints revealed by the near UV CD spectra.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Effect of His to Phe mutations on the transition of the T domain from the native state to the MG state monitored by Trp fluorescence. A, max as a function of pH. Closed circles, WT; cross, T251; open triangle, T223-257; inverted open triangle, T322-323; open circles, THis. B, as a function of the number of mutations.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Effect of His to Phe mutations on the interaction of the T domain with anionic LUV as a function of pH. A-E, partition between the buffer and the LUV measured by ultracentrifugation (closed circles) and max of Trp fluorescence (open circles) as a function of pH. The traces are the best fits to the experimental data according to the model applied (see "Experimental Procedures" and "Results"). F, as a function of the number of mutations. All mutant T domains carrying the pair of mutations 322-323 as well as T322 and T323 are shown as squares. All other mutant T domains carrying the pair of mutations 223-257, as well as T223 and T257 are shown as circles. Triangle, T372; diamonds, T251 and T251-257. Mutations had a cumulative effect on membrane binding. However, mutation of the pair 322-323 (and of histidine 251) had an effect on membrane binding stronger than that of the pair 223-257, as shown by the correlation lines. Correlation coefficients were as follows: r = 0.95 for the squares; r = 0.38 for the circles.

The combination of mutations within the T domain had cumulative effects (Fig. 3 and Table 1). A correlation was observed between the number of mutations carried by the protein and the , the maximum effect being observed for the mutant THis with = 3.8. However, as for the proteins bearing a single mutation, the addition of any of the mutations 223, 251, and 257 had an effect on the stronger than that of the mutations 322, 323, and 372. Remarkably, the triple mutant T223-251-257 had a similar to that of THis.

These results indicated that the protonation of all of the His residues of the T domain were implicated in the formation of the MG state in solution, with a preponderant role for His²²³, His²⁵¹, and His²⁵⁷.

His to Phe Mutations Shifted Membrane Binding, but Not Membrane Penetration, toward Lower pH Values in an Additive Manner—We studied the interaction of the mutant T domains with anionic LUV as a function of pH. The interaction was monitored by centrifugation and Trp fluorescence. All mutants interacted with the membrane according to the two-step process found for the WT T domain (8, 9) (Fig. 4). A first transition corresponding to membrane binding was detected by centrifugation (Fig. 4, closed circles) and by a change of fluorescence _max (Fig. 4, open circles), from 336 to 344 nm for the WT T domain (Fig. 4A). A second transition corresponding to penetration into the bilayer was characterized by a decrease of _max (Fig. 4, open circles), from 344 to 332 nm for the WT T domain (Fig. 4A). LUV permeabilization was found to accompany membrane penetration (8), a property retained by the mutants (Fig. 5).

For almost all mutants, membrane binding appeared to be shifted to various extents toward lower pH values, whereas membrane penetration seemed not to be (Fig. 4 and Table 1). If this were the case, the alteration of the fluorescence transition pattern found for these mutants could be explained by the only shift of the first transition, which would mask the second transition. We designed a fitting model to evaluate the validity of this interpretation. For all mutants, we fitted the data of the second transition with the same parameters (pK and nH) taken from the WT T domain fitting curve (see "Experimental Procedures"). Then for each mutant, we determined the value of these parameters for the first transition. These values (Table 1) reproduced well the experimental data (Fig. 4). Thus, the model strongly supported the observation suggesting that the mutations affected membrane binding but not membrane penetration. We propose that as soon as the membrane-bound state started to be populated (i.e. at a pH below 6), the second transition occurred simultaneously and reached the level of membrane penetration found for the WT T domain at this pH value. In other words, the membrane-bound state could not be observed in these steady-state experiments.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Kinetics of permeabilization of anionic LUV by mutant T domains at pH 4. Dashed line, WT; green, T251; blue, T223-257; red, T322-323; black, THis.

The combination of mutations had cumulative effects (Fig. 4F and Table 1). Correlations were observed between the number of mutations carried by the protein and the , the maximum effect being observed for the mutant THis with = 5.2. The proteins combining the mutations 322 and 323 (Fig. 4F, squares) or the mutation 251 (Fig. 4F, diamonds) exhibited a shift of stronger than that of proteins combining mutations 223 and 257 (Fig. 4F, circles) and 372 (Fig. 4F, triangle). As illustrated in Fig. 6, a correlation was found between the and values for proteins carrying the mutations 322 and 323 (Fig. 6, squares). In contrast, the mutations 223 and 257, which had an effect on MG formation had no effect on membrane binding (Fig. 6, circles). This suggested that the protonation of these His residues was not required for membrane binding but rather for the stabilization of the MG state in solution.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Confrontation of the and the of the mutant T domains. All mutant T domains carrying the pair of mutations 322-323 as well as T322 and T323 are shown as squares. All other mutant T domains carrying the pair of mutations 223-257 as well as T223 and T257 are shown as circles. Triangle, T372; diamonds, T251 and T251-257. A correlation was found between the and , for proteins carrying the mutations 322-323 (Fig. 6, squares; correlation coefficient r = 0.89). In contrast, the mutations 223 and 257, which had an effect on MG formation had no effect on membrane binding (Fig. 6, circles, correlation coefficient r = 0.26).

Effect of Mutations on MG State Formation and Membrane Binding Is Not Linked with a Change of Stability—Two explanations may account for the effect of the mutations on MG state formation and membrane interaction. The protonation of His residues was critical to induce the conformational change, and/or the Phe substituting for the His increased the stability of the protein in the native state. To rule out these two possibilities, we studied the effect of the mutations on the equilibrium unfolding transition of the T domain in the native state (i.e. at pH 7.5).

The guanidium hydrochloride-induced unfolding reaction of the T domain was monitored with Trp fluorescence (Fig. S1 and Table S1). For all mutants, an unfolding intermediate state was detected, as characterized previously for the WT T domain (15). The free energy values calculated from the first unfolding transition (G₀₁) were used to compare the stability of the WT and mutant T domains. Besides T251, T251-257, and THis, which appeared more stable than the WT T domain, the stability of the other mutants was unchanged or diminished (Table S1). We plotted the G₀₁ of each mutant against its and (Fig. S2). No correlation was found linking stability and pK values (a similar absence of correlation was found using G₀₁ + G₀₂). For instance, the decrease of of T223-257 was not a consequence of the stabilization of the native state, the mutant being less stable than the WT T domain. This lack of correlation suggested that the effect of mutations was mainly due to the loss of protonable residue(s) rather than to a stabilizing effect of the mutation(s). This is less clear, however, for His²⁵¹.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
All His Residues of the T Domain Act as pH-sensitive Triggers, with Various Sensitivities—We studied the relative implication of the His residues of the diphtheria toxin T domain in the various steps by which it changes its conformation and interacts with the membrane as pH decreases. Our aim was to determine the involvement of His protonation in triggering these conformational changes, which are expected to occur in the endosome during cell intoxication. His residues were mutated to Phe in order to mimic a nonprotonable form of His. These mutations could also affect the stability of the protein, but guanidium hydrochloride-induced unfolding experiments showed this was not the case, except perhaps for His²⁵¹ (Figs. S1 and S2 and Table S1).

The major finding of this study is that the protonation of the His residues of the T domain participates in the pH-dependent transitions, leading to MG state formation in solution and to membrane binding. Although the accumulation of mutations shifted the transitions toward more acidic pH (Figs. 3B and 4F), the protonation of the remaining His residues or of acidic residues (Asp and Glu) could still trigger the conformational changes. In other words, all His residues are involved in a cooperative manner, but none is indispensable. Only the change of all His residues strongly shifts the pH of formation of the MG state and the capacity of the T domain to bind membranes. However, the importance of the protonation of each His side chain is different, depending on the step investigated (Fig. 7). We found that the pair His²²³-His²⁵⁷ and His²⁵¹ are the most sensitive triggers for the formation of the MG state (Figs. 3, 6, and 7), whereas the pair His³²²-His³²³ and His²⁵¹ are the most sensitive triggers for membrane binding (Figs. 4, 6, and 7).

Mechanisms of Tertiary Structure Destabilization by Protonation of His Residues—The structure of the diphtheria toxin (6, 19) shows that at neutral pH, the side chain of the His residues of the T domain are partially buried within the protein. These side chains are neutral, slightly polar. The gain of a positive charge by protonation induces a strong gain of polarity. Hence, maintaining this side chain in the rather apolar environment of the protein becomes energetically unfavorable. This should induce the solvation of the protonated His together with local destabilization of the protein. In addition, some His side chains may be involved in hydrogen bond networks. This is the case for His²⁵⁷, which shares hydrogen bonds with Glu²⁵⁹ and Ser²¹⁹. Disruption of hydrogen bond networks by protonation of the His side chain should also destabilize the protein.

Regional Role of His Protonation—The examination of the position of each His residue in the structure of the T domain (Fig. 1) may help to propose a functional role for each protonation event. Four His residues are arranged in two pairs: His²²³ and His²⁵⁷ are stacked together and located between helices TH1 and TH2 and between TH3 and TH4, respectively; His³²² and His³²³ are vicinal and located in a loop forming a hinge between helices TH7 and TH8. In each case, the mutation of one or the other or both residues had almost the same effect (Fig. 3B for His²²³-His²⁵⁷ and Fig. 4F for His³²²-His³²³). This suggests that each pair constitutes a functional unit, in which protonation (of one or both residues) plays a regional role (Fig. 7, native state).

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Schematic illustration of the pH-dependant conformational changes of the T domain. For simplicity, each of the three layers of helices of the T domain is represented as a cylinder: green for TH1-TH4, blue for TH5-TH7, and red for TH8-TH9. However, this does not exclude the possibility for movements of the helices within each layer, which are not rigid bodies, movements that certainly occur. The four states of the protein are shown at the pH at which they are accumulated. His residues are circled on the structure of the T domain, the color of each circle corresponding to the color of the arrow of the transition in which they are mostly implicated. Protonable residues other than His and involved in membrane penetration are indicated.

From our data, we propose a model describing the role of His protonation in inducing the interaction of the T domain with the membrane (Fig. 7). The protonation of His³²²-His³²³ and His²⁵¹ induces a destabilization of the tertiary structure. This gain of flexibility would favor membrane binding of the T domain, mainly via hydrophobic effects, involving its C-terminal part (9). The protonation of His²²³-His²⁵⁷ would be required to release the remaining local forces packing the N-terminal helices. Finally, a combination of hydrophobic and attractive electrostatic interactions between the lipid bilayer and the N-terminal region of the T domain allows its conformational reorganization and membrane permeabilization (8, 9).

His Residues as Triggers of Protein Conformational Changes at Physiological pH Values—When all six His residues were mutated, the and dropped to pH 3.8 and 5.2, respectively (Figs. 3B and 4F). This suggests that the protonation of Glu and Asp residues may partially take over for the missing His residue(s) at lower pH. Hence, besides the particular behavior of His upon protonation (switching from a neutral, slightly polar, to a charged polar side chain), the disruption by protonation of the electrostatic network, involving Glu and Asp residues, may also contribute to the structural rearrangements of the T domain. Nevertheless, we propose that the selection of a number of His residues rather than Glu and Asp, located in hinges of the protein, enables the T domain to function at a rather mild pH, such as the pH of the endosome, contributing to toxin efficacy (21).

	FOOTNOTES

 
^* This work was supported by the Commissariat à l'Energie Atomique (Signalization and Membrane Transport Program of the Life Science Division) and by Action Concertées Incitative-microbiology program of Fonds National de la Science Grant MIC 0325. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.

¹ These authors contributed equally to this work.

² These authors contributed equally to the elaboration and direction of this work.

³ To whom correspondence may be addressed: Unité de Biochimie des Interactions Macromoléculaires, URA CNRS 2185, Dépt. de Biologie Structurale et Chimie, Institut Pasteur, 25-28 Rue du Dr. Roux, 75724 Paris cedex 15, France. Tel.: 33-1-44-38-92-12; Fax: 33-1-40-61-30-42; E-mail: chenal{at}pasteur.fr. ⁴ To whom correspondence may be addressed. E-mail: daniel.gillet{at}cea.fr.

⁵ The abbreviations used are: T domain, translocation domain; ANTS, 8-amino-naphthalene-1,3,6-trisulfonic acid; DPX, p-xylene-bis-pyridinium bromide; LUV, large unilamellar vesicle(s); MG, molten globule; WT, wild type.

	ACKNOWLEDGMENTS

 
We thank Dr. Bernard Gilquin (Institut de Biologie et Technologies de Saclay, Commissariat à l'Energie Atomique) for fruitful discussion on the role of protonation of His residues in the conformation of proteins.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Menetrey, J., Gillet, D., and Menez, A. (2005) Toxicon 45, 129-137[Medline] [Order article via Infotrieve]
2. Mere, J., Morlon-Guyot, J., Bonhoure, A., Chiche, L., and Beaumelle, B. (2005) J. Biol. Chem. 280, 21194-21201[Abstract/Free Full Text]
3. Krantz, B. A., Finkelstein, A., and Collier, R. J. (2006) J. Mol. Biol. 355, 968-979[CrossRef][Medline] [Order article via Infotrieve]
4. Koriazova, L. K., and Montal, M. (2003) Nat. Struct. Biol. 10, 13-18[CrossRef][Medline] [Order article via Infotrieve]
5. Chenal, A., Nizard, P., and Gillet, D. (2002) J. Toxicol. Toxin Rev. 21, 321-358
6. Bennett, M. J., and Eisenberg, D. (1994) Protein Sci. 3, 1464-1475[Medline] [Order article via Infotrieve]
7. Zhan, H., Oh, K. J., Shin, Y. K., Hubbell, W. L., and Collier, R. J. (1995) Biochemistry 34, 4856-4863[CrossRef][Medline] [Order article via Infotrieve]
8. Chenal, A., Savarin, P., Nizard, P., Guillain, F., Gillet, D., and Forge, V. (2002) J. Biol. Chem. 277, 43425-43432[Abstract/Free Full Text]
9. Montagner, C., Perier, A., Pichard, S., Vernier, G., Menez, A., Gillet, D., Forge, V., and Chenal, A. (2007) Biochemistry 46, 1878-1887[CrossRef][Medline] [Order article via Infotrieve]
10. Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098[Abstract/Free Full Text]
11. Mason, A. J., Gasnier, C., Kichler, A., Prevost, G., Aunis, D., Metz-Boutigue, M. H., and Bechinger, B. (2006) Antimicrob. Agents Chemother. 50, 3305-3311[Abstract/Free Full Text]
12. Kampmann, T., Mueller, D. S., Mark, A. E., Young, P. R., and Kobe, B. (2006) Structure 14, 1481-1487[Medline] [Order article via Infotrieve]
13. Geierstanger, B., Jamin, M., Volkman, B. F., and Baldwin, R. L. (1998) Biochemistry 37, 4254-4265[CrossRef][Medline] [Order article via Infotrieve]
14. Wei, Z., and Song, J. (2005) J. Mol. Biol. 348, 205-218[CrossRef][Medline] [Order article via Infotrieve]
15. Chenal, A., Nizard, P., Forge, V., Pugniere, M., Roy, M. O., Mani, J. C., Guillain, F., and Gillet, D. (2002) Protein Eng. 15, 383-391[Abstract/Free Full Text]
16. Nizard, P., Chenal, A., Beaumelle, B., Fourcade, A., and Gillet, D. (2001) Protein Eng. 14, 439-446[Abstract/Free Full Text]
17. Rigaud, J. L., Bluzat, A., and Buschlen, S. (1983) Biochem. Biophys. Res. Commun. 111, 373-382[CrossRef][Medline] [Order article via Infotrieve]
18. Nizard, P., Liger, D., Gaillard, C., and Gillet, D. (1998) FEBS Lett. 433, 83-88[CrossRef][Medline] [Order article via Infotrieve]
19. Choe, S., Bennett, M. J., Fujii, G., Curmi, P. M., Kantardjieff, K. A., Collier, R. J., and Eisenberg, D. (1992) Nature 357, 216-222[CrossRef][Medline] [Order article via Infotrieve]
20. Wang, J., Rosconi, M. P., and London, E. (2006) Biochemistry 45, 8124-8134[CrossRef][Medline] [Order article via Infotrieve]
21. Srivastava, J., Barber, D. L., and Jacobson, M. P. (2007) Physiology 22, 30-39[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Galloux, H. Vitrac, C. Montagner, S. Raffestin, M. R. Popoff, A. Chenal, V. Forge, and D. Gillet Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain: THE BELT REGION IS A REGULATORY LOOP FOR MEMBRANE INTERACTION J. Biol. Chem., October 10, 2008; 283(41): 27668 - 27676. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 282/33/24239    most recent M703392200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Perier, A. Articles by Gillet, D. Search for Related Content PubMed PubMed Citation Articles by Perier, A. Articles by Gillet, D. Social Bookmarking                      What's this?