Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA)

It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His6-tag to different proteins increased their binding affinity to the protective antigen (PA) PA63-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB7/8-family of toxins. His6-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to −70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA63. Cell-based experiments demonstrated that addition of an N-terminal His6-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA63. Our results revealed that addition of His6-tags to several factors increase their binding properties to PA63 and enhance the property to intoxicate cells.


Introduction
Gram-positive bacteria such as Bacillus anthracis and Clostridium botulinum synthesize as most crucial virulence factors protein toxins of the AB 7/8 type. These toxins are composed of two components which are nontoxic by themselves when added to the external media of target cells [1]. One or more Acomponents of the toxins feature intracellular enzymatic activity and are responsible for the toxicity. The B-component binds to cellular receptors or directly to the membrane and transports the enzymatic component into the cell. Anthrax-toxin from B. anthracis belongs to the AB 7/8 -type of toxins classified by a pore forming B-component, protective antigen (PA) and two enzymes, edema factor (EF) and lethal factor (LF). PA is an 83 kDa water soluble precursor, which has to be activated by cleavage of a 20 kDa N-terminal part to form the functional PA 63 -heptamers/octamers [2][3][4][5]. The proteolytic activation is performed in vivo by cell bound furin. It allows pore formation and injection of both enzymatic components EF and LF into cells [6][7][8][9]. EF is an 89 kDa Ca 2+ -and calmodulin-dependent adenylate cyclase which causes severe edema by uncontrolled increase of the intracellular concentration of cAMP. LF is a Zn 2+ -binding metalloprotease that cleaves mitogen-activated protein kinase kinases (MAPK-kinases).
C. botulinum, well known for the production of potent neurotoxins, also produces other protein toxins such as the binary C2-toxin and the single-component C3 exoenzyme [10][11][12]. The homologous pore forming component to PA 63 of C2-toxin is C2II. After proteolytic cleavage with trypsin (60 kDa) it forms heptamer/octamerss that insert into biological and artificial membranes at an acidic pH and promotes the translocation of the 45 kDa enzymatic component C2I. Similar to anthrax-toxin a receptor-mediated endocytocis of C2 is required for intoxication of the cell [13,14]. C2I acts as an ADP-ribosyltransferase on arginine177 of monomeric G-actin, causing disruption of the actin cytoskeleton of eukaryotic cells [15,16].
The toxins of the AB-type represent simple but sophisticated molecular syringes for protein delivery into target cells. This means that they could be important systems for development of new strategies for efficient injection of polypeptides into target cells. Possible Trojan Horses could be binary toxins of the AB 7/8 type such as anthrax-and C2-toxin [1]. The binding of the Nterminal ends of the enzymatic components to the heptameric/ octameric channel formed by the binding components is followed by receptor-mediated endocytosis, acidification of the endosomes and final release of the enzymatic components into the cytosol of target cells, where they exert their enzymatic activities [3,17]. Interestingly, the amino-terminal part of LF is sufficient to confer the ability to associate with PA 63 -heptamer/octamerss on LF. It can be used to drive the translocation of unrelated polypeptides fused to LF  into target cells in a PA 63 -dependent manner [18,19]. Although the enzymatic components of anthrax-and C2toxin differ considerably in their enzymatic activity and in their primary structures as well, the binding components PA and C2II share a significant overall sequence homology of about 35%, which means that they are closely related in structure and probably also in function [5,8,20,21]. There exist several structural features that are important for the binding of channel blockers and enzymes to be delivered into the target cells. One is the so-called W(phenylalanine)-clamp -F427 in PA and F428 in C2II -which is combined with two rings of seven negatively charged amino acids -E399 and D428 in PA and E398 and D427 in C2II, respectively [22,23]. These negatively charged amino acids seem to interact with the positively charged N-terminal ends of the enzymatic components [19,24,25]. Another interesting feature involved in binding and transport of truncated and full-length effectors is the alpha-clamp in PA [26,27]. This represents a big amphipathic cleft on the surface of the PA 63 heptamers/octamers, which plays an important role in oligomer formation of PA 63 and unfolding and translocation of effectors [26,27].
Blanke et al. [19] have established that addition of His 6 -tag and other polycationic presequences to diphtheria toxin A chain (DTA) allows its injection into cells by PA 63 binding component. The observation that polycationic peptides at the N-terminal end of DTA facilitate its import into the cytosol of a CHO-K1 cell line is of particular interest [19]. Here we aimed at establishing whether this effect of His 6 -tag could be generalized to other toxin enzymatic components and quantify the involvement of His 6 -tag on these toxin component interactions with PA 63 in vivo and in vitro. To study this we have investigated the influence of additional charges on the N-terminal end on binding of the enzymatic factors to the channels formed by PA 63 and C2II. First results in the field were found with polycationic peptides fused to EF, LF, LF N , EF N and DTA [19,21]. Our results suggested that the binding of LF and EF to C2II is possible and that C2I binds to PA 63 in the black lipid bilayer assay as well. The most significant result that was observed was a preferential binding of His 6 -C2I, as compared to C2I, to PA 63 . Interestingly, PA 63 is able to transport His6-C2I into target cells with a higher efficiency than C2I. We extended these finding by demonstrating that a His 6 -tag fused to the N-terminal end of the epidermal cell differentiation inhibitor (EDIN) of Staphylococcus aureus also increases its affinity to PA 63 and allows an efficient PA 63 -dependent delivery of His 6 -EDIN in target cells. In addition, the stability constant for binding of His 6 -EDIN and not that of EDIN to PA 63 -channels was found to be highly voltagedependent, which could be one important factor for efficient delivery of effectors via PA 63 into target cells.

Materials
Protective antigen encoding gene was cloned with BamHI-SacI restriction sites into pET22 (Novagen) as previously described [28]. The translocation-defective PA mutant F427A [22,29] was constructed by site-directed mutagenesis using the Quick-Change TM kit (Stratagene) according to the manufacturers instructions. The PA-gene cloned in the plasmid pET19 (Novagen) [30,31], was used as a template. The construct was confirmed by DNA sequencing. The protein was expressed with an N-terminal His 6 -tag in BL21 (DE3) (Novagen) and purified by HiTrap chelating (Pharmacia) charged with Ni 2+ ions.
C2I and C2II genes were PCR-amplified from genomic DNA of Clostridium botulinum D strain 1873 and cloned into pET22 (Novagen) and pQE30 (Qiagen) expression plasmids with BamHI-SacI restriction sites.
The plasmid coding for the chimera protein MBP-gpJ (maltosebinding-protein attached to amino acids 684-1132 of Lambda phage tail protein J) was a kind gift of Alain Charbit, Necker Enfants Malade, Paris, France. Expression and purification of MBP-gpJ was performed as described previously [32]. gpJ was obtained by treatment of MBP-gpJ bound to starch column beads (amylose-Sepharose, New England Biolabs) with factor X a (Invitrogen). His 6 -gpJ (684-1132) was obtained as described previously [33].
The DNA encoding EDIN (NCBI M63917) was cloned into pET28a vector using BamHI-EcoRI restriction site as described previously [34]. Recombinant toxins containing His 6 -tags were expressed in E. coli BL21 (DE3) and purified on a Chelating Sepharose Fast Flow column previously chelated with nickel (Amersham Biosciences) as recommended by the manufacturer. Fractions containing toxin were pooled and dialyzed over night against 250 mM NaCl and 25 mM Tris-HCl, pH 8. The Nterminal His 6 -tag was removed by incubation with thrombin. Nicked anthrax PA 63 from B. anthracis was obtained from List Biological Laboratories Inc., Campbell, CA. One mg of lyophilized protein was dissolved in 1 ml 5 mM HEPES, 50 mM NaCl, pH 7.5 complemented with 1.25% trehalose. Aliquots were stored at 220uC. Channel formation by PA 63 was stable for months under these conditions.

Immunofluorescence Studies
The experiments were performed on cells fixed in 4% paraformaldehyde (SIGMA). Actin cytoskeleton was labelled using FITC-conjugated phalloidin (SIGMA), as described in [36]. Immunosignals were analyzed with inverted microscope (EVOSfl, AMG) using a 20X object lens.

Lipid Bilayer Experiments
Black lipid bilayer measurements were performed as described previously [37]. The instrumentation consisted of a Teflon chamber with two aqueous compartments connected by a small circular hole. The hole had a surface area of about 0.4 mm 2 . Membranes were formed by painting a 1% solution of diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in ndecane onto the hole. The aqueous salt solutions (Merck, Darmstadt, Germany) were buffered with 10 mM MES-KOH to pH 5.5 to pH 6. Control experiments revealed that the pH was stable during the time course of the experiments. The binding components of the binary toxins were reconstituted into the lipid bilayer membranes by adding concentrated solutions to the aqueous phase on one side (the cis-side) of a black membrane. The temperature was kept at 20uC throughout. Membrane conductance was measured after application of a fixed membrane potential with a pair of silver/silver chloride electrodes inserted into the aqueous solutions on both sides of the membrane. Membrane current was measured using a homemade current-tovoltage converter combined with a Burr Brown operational amplifier. The amplified signal was monitored on a storage oscilloscope and recorded on a strip chart recorder.

Binding Experiments
The binding of the His-tagged proteins to the C2II-channel and the binding component PA 63 was investigated with titration experiments similar to those performed previously to study the binding of 4-aminoquinolones to the C2II-and PA 63 -channels and EF and LF to the PA 63 -channel in single-or multi-channel experiments [38][39][40]. The aqueous phase contained always 150 mM KCl, buffered with 10 mM MES-KOH to pH 5.5 to pH 6. The C2II-and PA 63 -channels were reconstituted into lipid bilayers. About 60 minutes after the addition of either activated C2II or PA 63 to the cis-side of the membrane, the rate of channel insertion in the membranes was very small. Then concentrated solutions of His-tagged proteins were added to the cis-side of the membranes while stirring to allow equilibration. The results of the titration experiments, i.e. the blockage of the channels, were analyzed using Langmuir adsorption isotherms [21,41].
The Fraction of closed channels as a function of the concentration of the enzymatic components was analyzed using Lineweaver-Burk (double reciprocal) plots.

1= Fraction of blocked channels
K is the stability constant for binding of the enzymatic components of the binary toxins to the PA 63 -or C2II-channels. The half saturation constant K s is given by the inverse stability constant 1/K.

Statistical Analysis
When assessing multiple groups, one-way ANOVA was used followed by Bonferroni post hoc test with *p,0.05. Data are presented as means 6 SEM. The statistical software used was Prism 5.0 b.

Interaction of PA 63 -pores with His 6 -C2I in Artificial Black Lipid Bilayer Membranes
We compared the binding affinity of different proteins with and without a His 6 -tag to the PA 63 -and C2II-channels. Taking into account that positive charges seem to have a huge influence in binding to the PA 63 -pore but only less to the C2II-pore [40,42], we chose the enzymatic component C2I as the first substrate. In a previous study we could show that it binds to PA 63 -pores and could even be translocated into cells albeit with very low efficiency at high C2I concentration [43]. We now addressed the question, if binding and translocation are enhanced by addition of a His 6 -tag to C2I.
The stability constants K (and the half-saturation constant K s ) for the binding of His 6 -C2I to the PA 63 -channel were measured in multichannel experiments, performed as described previously [39]. A receptor is required for the binding and oligomerization of PA 63 on the surface of mammalian cells [8]. However, this is not necessary for reconstitution of PA 63 -channels in artificial lipid bilayers, where channel formation is obtained under mildly acidic conditions [44]. 60 minutes after the addition of the protein to the cis-side of the lipid bilayer, the rate of conductance increase had slowed down considerably at an applied membrane potential of 20 mV. At that time, small amounts of a concentrated protein solution were added to the cis-side of the membrane and the PA 63induced membrane conductance decreased in a stepwise manner. Figure 1A shows an experiment of this type in which increasing concentrations of His 6 -C2I (arrows) were added to the cis-side of a membrane containing about 300 PA 63 -channels. The membrane conductance decreased as a function of the His 6 -C2I concentration. The data of Figure 1A and of similar experiments were analyzed assuming Langmuir isotherms for binding (equation (1)) [39,41,45] Lineweaver-Burk (double reciprocal) plots were used to calculate the stability constant K for binding as shown in Figure 1B for the data of Figure 1A. The resulting straight corresponded to a stability constant K of (3.9360.39)610 7 M 21 for His 6 -C2I binding to PA 63 -pores (half saturation constant KS = 25 nM).
At least three individual experiments were used to calculate the half saturation constants K S of C2I-and His 6 -C2I binding to the PA 63 -channel. The average of the half-saturation constant K s was 150 nM for C2I, whereas that for His 6 -C2I binding to PA 63channels was 16 nM] in 150 mM KCl. This means that the half saturation constant K S for binding of His 6 -C2I was roughly ten times lower than that for C2I without His 6 -tag (Table 1). Titration experiments with artificial bilayer membranes of the wildtype A-B components C2II and C2I of C2-toxin revealed a half saturation constant K S of 27 nM. Interestingly, a His 6 -tag attached to the Nterminal end had no obvious effect on binding of C2I to C2IIpores (K S = 29 nM; Table 1).

Addition of His 6 -tag to C2I Potentiates its Transfer via PA 63
In further experiments we tested if addition of His 6 -tag to C2I triggers its entry into cells via PA 63 -channels in vivo. C2I acts as an ADP-ribosyltransferase, targeting cellular G-actin [46]. Therefore, successful delivery of this enzymatic component into target cells can be detected by disruption of the cytoskeleton followed by rounding up and detachment of target cells from the extracellular matrix, defined as intoxicated cells [15]. HUVECs were intoxicated with C2I and His 6 -C2I driven by PA 63 , as indicated, and the number of intoxicated cells was directly assessed by counting ( Fig. 2A). These results were compared to that of native toxin combination C2I and C2II. We observed a cytotoxic effect with the combination of His 6 -C2I and PA 63 . No effect could be detected for C2I and PA 63 under the same conditions. The specificity of this internalization was verified by using a mutant of PA 63 : PA F427A. This mutant is competent for receptor binding and internalization, but defective in the pH dependent functions: pore formation and ability to translocate bound ligands [47]. Intoxication of cells with His 6 -C2I and PA F427A did not induce any cellular effect (Fig. 2B). Thus, the increase of affinity between PA 63 and C2I, upon addition of His 6 -tag to C2I allows His 6 -C2I to efficiently intoxicate cells via PA 63 -channels.

His 6 -tags do not Facilitate Binding of EF and LF to C2IIchannels
To examine whether the N-terminal His 6 -tag of EF and LF have a similar effect on binding kinetics to the C2II-channel, as previously shown for His 6 -EF and His 6 -LF and PA 63 [21], we omitted the cleavage of the His 6 -tag after the affinity purification and studied binding to C2II-channels. Interestingly, His 6 -EF and His 6 -LF did not exhibit any significant changes of their affinity to C2II-channels as compared to EF and LF (see Table 1). The half saturation constants K S of the interactions between His 6 -EF and His 6 -LF and the C2II-channels were 19 nM for His 6 -EF and 29 nM for His 6 -LF ( Table 1).

Binding of His 6 -gpJ and gpJ Proteins to PA 63 -and C2IIchannels
The His 6 -tag had a remarkable influence on binding of enzymatic components to the PA 63 -channel but not to the C2IIchannel. To check if this interaction was specific for the presence of the His 6 -tag we performed titration experiment with a His-tagged protein that is not related to the effectors EF, LF or C2I. gpJ is a 447 amino acids C-terminal fragment of protein J (amino acids 684-1131), which is responsible for binding of bacteriophage Lambda to LamB on the surface of E. coli K-12 [31]. His 6 -gpJ exhibited high affinity binding (block) to the PA 63 -channel. The half saturation constant K S for binding of His 6 -gpJ to PA 63 was calculated to be 5.0 nM in 150 mM KCl, 10 mM MES-KOH, pH 6.0 (mean of three measurements) ( Table 1). Similar experiments with gpJ did not exploit any binding of gpJ to the PA 63 -channel. This implies half saturation constants K s of gpJ-binding to PA 63 were much higher than 100 mM. We could not detect any substantial binding of His 6 -gpJ nor of gpJ to the C2II-channel (Table 1). Our results reveal the substantial role of the His 6 -tag at the N-terminal end of C2I and gpJ for their binding to the PA 63 -but not to the C2IIchannel.
Binding of EDIN and His 6 -EDIN to PA 63 -and C2II-channels Next, we tested whether PA 63 -pores bind and transport EDIN of Staphylococcus aureus, as well as His 6 -EDIN. EDIN normally enters host cells inefficiently by nonspecific macropinocytosis and not by delivery systems considered in this study [28]. Previously, it has been shown that LF 1-254 -EDIN can enter cells via PA 63 [28]. EDIN is a Staphylococcus aureus exoenzyme with ADP-ribosylating activity on RhoA. EDIN targets RhoA in cells for inactivation producing actin cable disruption in target cells [34]. Interestingly, we found that PA 63 -pores bound both EDIN and His 6 -EDIN with half saturation constants that were considerably lower than those reported before for the crossing over of the AB 7/8 types of toxin [43]. The half saturation constant K S for EDIN binding to PA 63 -channels was on average 2.7 mM in 150 mM KCl, whereas this constant decreased to 0.7 mM for His 6 -EDIN. The results of these experiments are summarized in Table 1 and demonstrate that EDIN without His 6 -tag bound at low transmembrane voltage (5 mV) with a roughly four-fold lower affinity to the PA 63 -channels than His 6 -EDIN. When higher voltages were applied we noticed a remarkable effect of voltage on His 6 -EDIN binding (see below). The affinity of EDIN to the C2II-channels (K S = 23 mM) was by a factor of about eight-fold lower as compared to binding to the PA 63 -channels. Surprisingly, we observed a considerable effect when the His 6 -tag was attached to the N-terminal end of EDIN. The half saturation constant dropped in this case to 0.9 mM for its binding to C2II-pores (Table 1).

His 6 -tag Promotes EDIN Internalization via PA 63 -pores
We next verified the role of His 6 -tag in the uptake of EDIN into cells. After purification the His 6 -tag was cleaved as described in the material and methods section. We verified the cleavage by immunoblotting the purified proteins using an antibody against the His 6 -tag (Fig. 3A). The efficiency of RhoA targeting by EDIN was assessed by GST-Rhotekin pull down of active RhoA (GTPbound RhoA). No effect was measured on cells challenged with His 6 -EDIN alone up 10 mg/ml (Fig. 3B).
We then intoxicated cells with His 6 -EDIN in the presence and absence of PA 63 . Strikingly, this revealed that the addition of PA 63 together with His 6 -EDIN (10 mg/ml) increased the capacity of EDIN to intoxicate cells. This led us to decipher the role of His 6tag. Cells were intoxicated with PA 63 together with EDIN or His 6 -EDIN. This clearly established that addition of His 6 -tag to EDIN in presence of PA 63 produced a 78% decrease of RhoA activation specifically (Fig. 3C). In conclusion, addition of His 6 -tag to EDIN promotes its internalization via PA 63 for cell intoxication.

Immunofluorescence Studies of HUVECs and PA 63 with EDIN and His 6 -EDIN
We next analyzed the actin cytoskeleton phenotype of cells intoxicated with PA 63 +His 6 -EDIN and PA 63 +LFN-EDIN, as well as EDIN alone or in combination with PA 63 ( Figure 4A). In all cases we observed that intoxicated cells displayed a disruption of filamentous actin and actin cables, as well as a undergo spreading, as previously described ( Figure 4A) [34,36]. In addition, intoxicated cells displayed large transendothelial cell macroaperture tunnels (TEM) (see Figure 4A). Induction of TEMs results from the dose dependent inhibition of RhoA [34,36]. We thus further determined the efficiency of cell intoxication, using different combinations of toxin components by measure of the percentage of cells displaying TEMs ( Figure 4B). Most importantly, this showed that addition of His 6 -tag to EDIN increases its capacity to intoxicate cells in combination with PA 63 at a concentration of 10 mg/ml, whereas intoxication of HUVECS with LFN-EDIN saturated already at 1 mg/ml ( Figure 4B).
The Voltage Dependency of PA 63 -channels is Changed when His 6 -EDIN is Bound to the Pore PA 63 -channels exhibit a well described voltage dependency [21]. If only added to the cis-side, PA 63 -induced conductivity decreases when applied voltage is higher than +50 mV or lower than 220 mV at the cis-side. It is also known that His 6 -EF bound to the channel changes the voltage dependency [21]. When different potentials were applied to membranes after the titration of PA 63 -pores with EDIN, there was only little change in voltage dependency of the channel (Fig. 5A). On the other hand, His 6 -EDIN bound to PA 63 -channels induced dramatic responses even at low positive voltages (Fig. 5B). Starting at +10 mV, the conductivity decreased exponentially immediately after the onset of the voltage with a voltage-dependent exponential relaxation time. Its time constant decreased with higher positive potentials at the cis-side (negative at the trans-side). This result indicated that channels, which were not blocked before by His 6 -EDIN at low voltage bound this compound and closed as a result of the higher voltage. This result suggested an increase of the stability constant of binding up to very high voltages an effect that has already been observed with full length EF [21].
The increase of the stability constant for binding could be calculated from the data of Figures 5A and 5B and similar experiments by dividing the initial current (which was a linear function of voltage) by the stationary current after the exponential relaxation and multiplying the ratio with the stability constant derived at 5 mV. Figure 6 summarizes the effect of the positive membrane potential on the stability constant K for EDIN and His 6 -EDIN binding as a function of the voltage. Starting already with 210 mV at the trans-side the stability constant K for His 6 -EDIN binding started to increase and reached with about 60 to 70 mV a maximum. At that voltage K was roughly 25 times greater than at 5 mV. For higher voltages the stability constant saturated probably because of secondary effects of the high voltage on the PA 63-channel or on His 6 -EDIN binding. Figure 6 shows also the effect of the positive membrane potential at the cis-side on the stability constant K for EDIN binding to PA 63 -pores as a function of the voltage. Interestingly, EDIN binding was only little affected by voltage as Figure 6 clearly indicated.

Discussion
His 6 -tag Addition to Several Bacterial Factors Increased the Protein Binding Affinity to PA 63 -but not to C2IIchannels Recent studies demonstrated that negatively charged amino acids in the vestibule of the PA 63 -channel play a crucial role in binding of effector molecules [40,42]. Thus, it is possible that a His 6 -tag, which adds positive charges under mildly acidic conditions to the N-terminal end of His 6 -EF and His 6 -LF affects binding and transport. This has indeed been shown for the native combinations of EF+PA 63 or LF+PA 63 and the potential ion-ion interaction discussed with EF N [21,39,48,49]. Recently, we could  . Immunofluorescence studies of HUVECs treated with EDIN and His 6 -EDIN and PA 63 . A: HUVECs were intoxicated for 24 h with a combination of PA 63 3 mg/ml, His 6 -EDIN 10 mg/ml and LF 1-254 -EDIN (LFN-EDIN) 1 mg/ml, as indicated. Cells were fixed and actin cytoskeleton was labelled using FITC-conjugated phalloidin. Bar = 10 mm. Arrows indicate transendothelial cell macroaperture tunnels (TEMs, transcellular tunnels). B: Graph shows percentage of cells with toxin-induced transendothelial cell macroaperture tunnels (TEMs, transcellular tunnels). HUVECs were intoxicated for 24 h with a combination of PA 63 3 mg/ml, His 6 -EDIN or EDIN 10 mg/ml and LF 1-254 -EDIN (LFN-EDIN) 1 mg/ml, as indicated on the graph legend. Data correspond to means 6 SEM (n = 3, 400 cells per condition). doi:10.1371/journal.pone.0046964.g004 show that C2I binds to PA 63 and may even be transported into target cells albeit at high PA 63 concentration and with very low efficiency compared with the native combination of C2I with C2II [43]. Here we studied the cross reactivity of anthrax-and C2-toxin in more detail and found a strong relation between binding affinity and the presence of a His 6 -tag at the N-terminal end of the enzymatic components. The addition of positive charges at the Nterminal end of C2I (due to the partially charged histidines) enhanced binding to and translocation into target cells via PA 63pores and agreed very well with the findings previously reported for His 6 -tags attached to EF and to LF [21,39]. Binding to PA 63channels was found to be strongly enhanced for the three enzymatic components EF, LF and C2I when they contained a His 6 -tag at the N-terminal end.
Interestingly, we did not observe major effects if these His 6tagged proteins were combined with C2II-channels. The results of binding experiments with His 6 -EF and His 6 -LF to C2II-channels suggested that the increased positive charge at the N-terminal end, due to the partially charged histidines, did not increase the binding of these enzymatic components to the C2II-channels. These results definitely imply that binding of the enzymatic components to PA 63 -channels occurs in a different way than binding to C2IIchannels.
To gain deeper insight in the influence of N-terminal His 6 -tags on binding of proteins to PA 63 -channels we choose a protein, gpJ, that was not related to any of the enzymatic components used in this study [33]. As expected, we did not observe any binding of gpJ to PA 63 -or C2II-channels (K S .100 mM). However, binding was observed when a His 6 -tag was attached to the N-terminal end of gpJ. This protein had a half-saturation constant for binding to the PA 63 -channels of 5 nM, which suggested that the affinity of His 6 -gpJ to PA 63 -channels was almost the same as that of LF and EF [21,39]. This means that the affinity increase is mainly determined by the positive charges of the His 6 -tag. It is interesting to note that His 6 -gpJ did accordingly not interact with C2II-channels; revealing again a somewhat different process for binding of His 6 -tagged proteins to PA 63 -channels than to C2II-channels. However, it is not clear if His 6 -gpJ could also be imported via PA 63 into target cells because this protein has no intracellular enzymatic activity [320].

Influence of the His 6 -tag on Uptake of EF, LF and C2I into Cells
The binding step is a prerequisite, but not sufficient for the delivery of enzymatic subunits into target cells. Thus, in order to complement the results of binding studies we went on to investigate the translocation by analyzing the enzymatic activity in a cellular system. We verified that a His 6 -tag attached to the Nterminal end of C2I increased its transport by PA 63 -channels, which correlates with the difference of 10-fold measured between the stability constants for binding of C2I and His 6 -C2I. Some difference in transport was observed by using EF or LF with or without a His 6 -tag in combination with PA 63 -channels. In particular, we could demonstrate that addition of a His 6 -tag promoted uptake of LT (PA + His 6 -LF) into HUVECs (data not shown). This result is in agreement with the increased affinity of EF and LF to PA 63 -heptamer/octamerss when they contain a His 6 -tag at the N-terminus [21,23].
Although the binding of EF N and LF N (truncated forms of EF and LF) to the PA 63 -channel is substantially weaker as compared to wild-type enzymatic components [42], these proteins interact with high affinity with the PA 63 -channels and are accordingly transported into the cell [48,50,51]. Similarly, short stretches of positively charged amino acids attached to the N-terminal end of foreign proteins can lead to a PA 63 -dependent delivery as it has been demonstrated for the addition of polycationic peptides to the N-terminus of the enzymatic A chain of diphtheria toxin (DTA; residues 1-193) or for LF 1-254 -EDIN [19,28]. Bound His 6 -EDIN or EDIN Causes a Difference in Voltagedependency of PA 63 -pores Experiments with EDIN of S. aureus were performed to gain deeper insight in the binding and translocation processes through PA 63 -channels and its His 6 -tag dependence. Surprisingly, black lipid bilayer experiments displayed that not only His 6 -EDIN but also EDIN itself bound to PA 63 -channels. The affinity of EDIN and His 6 -EDIN to the PA 63 -channels was in the same range at low trans-membrane potentials because His 6 -EDIN exhibited only a three times higher affinity for binding to the PA 63 -channels than EDIN. Under normal conditions the PA 63 -channels only close for higher negative voltages applied to the cis-side [21]. For positive potential the channels are open and do not show a voltagedependent closure until 100-150 mV [21]. However, His 6 -EDIN binding to the PA 63 -channels showed an extremely high voltagedependence when the voltage was positive at the cis-side of the membrane indicating that the potential pulled His 6 -EDIN into the channels. As a result the stability constant for binding of His 6 -EDIN to the PA 63 -channels increased at voltages of +70 mV at the cis-side (corresponding to 270 mV at the trans-side) by a factor of roughly 25 as compared to zero voltage. Bound EDIN displayed an only minor voltage-dependence. This means that the His 6 -tag is responsible for the binding EDIN and gpJ to the PA 63 -channels. Binding is essential for translocation because it is the first step of the whole process (see below).
The PA 63 -channel Transports His 6 -C2I and His 6 -EDIN into the HUVECs EDIN uptake into target cells can easily be detected because it decreases RhoA activity. Import of EDIN via PA 63 -channels could not be observed. Import was however, possible when EDIN contained a His 6 -tag. This finding demonstrated that His 6 -tag itself provides the ability for proteins to be transported into cells through PA 63 -pores. This effect was presumably promoted by the voltage-dependence of His 6 -EDIN binding to the PA 63 -channels. Biological membranes are polarized to about 260 mV to 270 mV (inside negative). This may explain the much higher effect of His 6 -EDIN compared to EDIN on cells described above. In any case it clearly indicates the potentiating effect of a His 6 -tag and applied voltage on binding and translocation of protein molecules to PA 63 -channels. Summarizing the results, there definitely exists a difference in the binding and translocation mechanism between the two very homologous binding components PA 63 and C2II of anthrax-and C2-toxin. Obviously, this distinction is induced by unequal binding surroundings inside the head region of the two protein channels.
The amino acids responsible for binding within the N-terminal end of the enzymatic components are still a matter of debate, although there is clear evidence that positively charged amino acids are involved in binding, forming salt bridges between the enzymatic components and the heptamers/octamers. In a recent study it has been shown that besides the Phi-clamp also the suchcalled alpha-clamp is also involved in effector binding, unfolding and translocation in combination with PA 63 [26,27]. This alphaclamp is composed of hydrophobic and aromatic residues, such as F202 and P205 and forms a deep amphipathic cleft on the surface of the PA 63 oligomer [26,27]. It is also possible that R178 contributes to effector binding but not to translocation. However, the alpha-clamp does not seem to be very specific because of its broad substrate specificity and non-specific polypeptide binding activity [26]. It is noteworthy that amino acids of the alpha-clamp do not appear to be preserved in C2II because PA R178, PA F202 and PA P205 correspond to C2II T169, C2II W193 and C2II K196 [26,27]. This could mean that the design of the alpha-clamp in C2II if it existed has a different structure or is absent.
The positively charged N-termini of the enzymes play presumably a crucial role, because quaternary ammonium ions and 4-aminoquinolones show PA 63 and C2II channel block in lipid bilayer experiments [38,40,44,52]. Both channels show a high selectivity for cations, i.e. cations have a strong influence on the single channel conductance as compared to anions [53,54]. This means that negative charged amino acids play a crucial role in the binding and constriction region of the PA 63 -channels, where they form two rings of seven putative negatively charged amino acids in the vestibule of this pore (E398 and D426). Similarly, the channel lumen contains additional three rings of seven possibly negatively charged groups (E302, E308 and D315). Some of these charges cannot be found in the C2II-channel lumen, resulting in minor effects of His 6 -tag on binding and transport. However, transport into cells seems to be possible with C2II-pores and when N-terminal parts of C2I are coupled to foreign proteins [1,55,56]. The most interesting result of this study was that we could use the anthrax PA 63 -channels to deliver into cells a polypeptide completely unrelated to the AB 7/8 -type of toxins. In fact, we here provide evidence that the His 6 -tag addition on EDIN allows its entry in target cells, in a PA 63 -dependent manner. On the other hand, we would like to stress the point that the natural uptake of EDIN occurs very slowly at very high EDIN concentration (100 mg/ml: see Figure 3C). Nevertheless, here our data support the idea that it seems possible to design a very simple transportation system using His 6 -tags on proteins unrelated to the AB 7/8 -family and PA 63 -channels for biological purpose.