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* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts; and Department of Physiology and Biophysics and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York
Correspondence: Address reprint requests to Prof. Alan Finkelstein, Albert Einstein College of Medicine, Physiology and Biophysics, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3169; E-mail: alfinkel{at}aecom.yu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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), but in other cases (e.g., diphtheria toxin, i.e., DT, and botulinum toxin, i.e., BT), a portion of the B component is directly involved in the process. In fact, all of the translocation machinery for DT and BT is built into their B portion, so that the A portion can be translocated across a planar lipid bilayer without the aid of any cellular components (Oh et al., 1999; Koriazova and Montal, 2003). A part of the B portion of these self-translocating toxins forms channels in planar lipid bilayer membranes at low pH values mimicking the conditions of acidic cellular vesicles (e.g., Hoch et al., 1985), but whether the channel is a conduit for the A portion or is a "discarded wrapper" (Misler, 1984) is not clear. Equally unclear is the structure of these channels and the possible role of lipid in their architecture (Gordon and Finkelstein, 2001).
Anthrax toxin is also classified as an AB toxin, but differs from the others in that it has two A portions instead of one: edema factor (EF) and lethal factor (LF). Also, unlike DT and BT, in which the A and B portions are covalently linked, EF (89 kDa), LF (90 kDa), and PA (83 kDa) (the protective antigen B component) are separate molecules. As with the other AB toxins, the B portion (PA) first binds to a cell receptor. A furin-like protease then cleaves a 20-kDa fragment from the N-terminal end of PA, leaving PA63 attached to the membrane, which then oligomerizes and thereby generates and exposes binding sites for EF and LF. After endocytosis of PA63 with its bound EF and/or LF, these enzymes are translocated across the membrane of an acidic vesicle into the cytosol (for a general review of anthrax toxin see Collier and Young, 2003). In a manner analogous to the B portions of DT and BT, PA63 forms channels in planar bilayer membranes under mildly acidic conditions (Finkelstein, 1994). One can therefore ask 1), can EF and LF, like the enzymatic portions of DT and BT, be translocated across a planar bilayer in the absence of any cellular components, and if so, 2), does the channel act as a conduit for this process?
There are two advantages that anthrax toxin has over DT and BT in addressing this second question. First, channel formation can be separated from translocation; that is, one can first form the channel with PA63 and then add EF or LF. Thus, if under these circumstances translocation occurs, it is clear that the channel is not a discarded wrapper. Of course, one can also first form the channel from the B portion of DT or BT and then add their respective A portions, but there is little chance of observing translocation of A, even if it did occur, under these circumstances. This is because there is no receptor-binding site on the DT and BT channels for their respective A portions, as there is on the PA63 channel of anthrax toxin. Once EF or LF has bound to the PA63 channel (or, as in the case of DT and BT, the A component is already covalently linked to the B channel), the enzyme's concentration is orders-of-magnitude larger than can be achieved by adding free A to solution. The second advantage of anthrax toxin over DT and BT is that there is structural information about the PA63 channel. It is a heptameric, 14-stranded ß-barrel with a limiting diameter of 
12 Å somewhere in its length of 100 Å (Petosa et al., 1997; Blaustein and Finkelstein, 1990; Nassi et al., 2002), and there are good reasons to believe that its structure resembles in many ways that of the channel formed by Staphylococcal 
-toxin (Song et al., 1996). The binding site for EF and LF spans the interface between two subunits, and the channel can bind up to three molecules of EF and/or LF (Cunningham et al., 2002; Mogridge et al., 2002).
In this article we report our investigations of the interactions in planar lipid bilayers of the PA63 channel with LFN, the 263-residue N-terminal piece of LF that contains the region that binds to the channel. At positive voltages (
40 mV) LFN is translocated across the membrane, and does so by going through the channel N-terminal end first.
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MATERIALS AND METHODS |
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; Cunningham et al., 2002). Trypsin-nicked PAG323C (nPAG323C) was made as described by Benson et al. (1998). LFN was prepared as described by S. Zhang, A. Finkelstein, and R.J. Collier (unpublished). Its N-terminal H6-tag was removed by thrombin cleavage, leaving four exterior residues, Gly-Ser-His-Met, at the N-terminus. Biotinylation of LFN was done as described by S. Zhang, A. Finkelstein, and R.J. Collier (Unpublished). LFN-DTA (LFN with diphtheria toxin A chain attached to its C-terminus) was kindly provided by Dr. Steve Juris. Protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA).
Bilayers were formed at room temperature by the brush technique of Mueller et al (1963) across either a 0.5-mm hole in a Teflon partition or a 0.2-mm diameter hole in a Delrin cup (Warner Instruments, Hamden, CT). Membranes separated two compartments of either 3 ml or 1 ml containing symmetric solutions of 100 mM KCl, 25 mM potassium succinate, and 1 mM EDTA, pH 5.5, which could be stirred by small magnetic stir bars. The membrane-forming solution was 3% diphytanoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in n-decane. After the membrane formed, (PA63)7 was added to the cis compartment, to a final concentration of 1 ng/ml (2 pM), and a steady-state current level was reached (at a transmembrane voltage of +20 mV) after several minutes. LFN, or modified forms of it, were then added to the cis compartment to a final concentration of 3 nM. The contents in this cis compartment could be slowly perfused to remove LFN by the simultaneous addition and removal of solution through a coupled pair of syringes; the exchange of >6 volumes was accomplished in several minutes. Assuming complete mixing of cis compartment contents by the continuous stirring, this brought the LFN concentration down to <0.25% of its original value. All experiments were done under voltage-clamp conditions; voltages are those of the cis solution (to which protein was added) with respect to that of the trans solution, which was held at virtual ground. Current responses were filtered at 10 Hz and recorded either on a Narco physiograph chart recorder (Houston, TX) or filtered at 100 Hz and recorded on a computer with Axograph 4.0 software (Foster City, CA).
The sulfhydryl-specific reagents N-(ß-D-glucopyranosyl)-n'-[(2-methanethiosulfonyl)ethyl]urea (MTS-5-glucose), 2-aminoethyl methanethiosulfonate hydrochloride (MTS-EA), and [2 (trimethylammonium)ethyl] methanethiosulfonate bromide (MTS-ET) were purchased from Toronto Research Chemicals (North York, Canada).
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RESULTS |
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). The quantitative aspects of this voltage gating in terms of magnitude and timescale can vary greatly from membrane to membrane, and its mechanism is not understood. In some membranes there may be very little gating between ±70 mV, whereas in others there can be significant gating between ±20 mV. In the experiments reported here, this intrinsic voltage gating of (PA63)7 channels was small over the voltage range examined and in any case was trivial compared to the effects produced upon addition of LFN. When LFN was added to the cis solution (final concentration 0.3–4 nM) with the transmembrane voltage held at +20 mV, there was a precipitous 20- to 50-fold decline of conductance over several seconds (Fig. 1 A). If the voltage was now stepped to –20 mV there was a rapid increase of conductance (although not back to the original value before LFN addition), and if the voltage was then returned to +20 mV, this increased conductance rapidly fell back to its previous level (Fig. 1 A). (See also S. Zhang, A. Finkelstein, and R.J. Collier, Unpublished.) Larger positive voltages gave faster declines of conductance to even lower values, sometimes followed by a subsequent rise in conductance (Fig. 1 B). We attribute the declines in conductance to the entry of LFN into the (PA63)7 channels with resultant channel block, and the increases in conductance to the exit of LFN from the channels, which unblocks them. It is not possible, however, to obtain any quantitative insight into these processes from such experiments, as they are confounded by the continual presence of LFN in the cis solution; this LFN can reblock channels from which LFN has exited, and thereby attenuate or completely eliminate the conductance rise associated with LFN exit from the channels.
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+40 mV), LFN is driven all the way through the channel and out into the trans solution, thus unblocking it. When the voltage in this case is returned to +20 mV, there is no LFN still attached to the channel to reblock it. The rise in current for a few seconds upon the return of the voltage to +20 mV represents channels that become unblocked as LFN continues to pass through them to the trans solution.
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+40 mV), the conductance rise was S-shaped, and its rate increased relatively steeply with voltage. For example, the half time of unblocking was 36 s at +40 mV and 11 s at +50 mV.
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Biotin attached to the C-terminal end of LFN (LFN-biotin)
With streptavidin prebound to LFN-biotin, channels were still blocked at +20 mV (S. Zhang, A. Finkelstein, and R.J. Collier, Unpublished), indicating that LFN does not enter the channel C-terminal end first. Furthermore, these channels were not unblocked at larger positive voltages (Fig. 5), consistent with streptavidin anchoring the C-terminal end of LFN to the cis side of the membrane. This interpretation was confirmed by the addition of tris-carboxyethylphosphine to the cis solution, which reduced the disulfide bond linking biotin (with its attached streptavidin) to LFN, and hence now allowed LFN to be translocated to the trans side (Fig. 5).
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), so that adenine in the cis solution should anchor the C-terminal end of LFN-DTA to the cis side of the membrane and thereby inhibit unblocking of LFN-DTA at large positive voltages. This is precisely what we saw (Fig. 6). As expected, when adenine was removed from the cis solution, LFN-DTA was now translocated to the trans solution and unblocking occurred (Fig. 6). LFN-DTA was as active as LFN in blocking (PA63)7 channels, but the unblocking rate was slower (compare the unblocking rate at +60 mV in Fig. 6 with that in Fig. 4).
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Translocation of LFN through the channel should be slowed or prevented by any manipulation that narrows the channel lumen. The former indeed happened when we reacted the cysteines lining the lumen of channels formed by PA63G323C (Benson et al., 1998) with MTS-5-glucose; the unblocking rate at +50 mV was dramatically slowed after the reaction (Fig. 7). A similar slowing of unblocking rate was seen after reaction with MTS-EA and MTS-ET (data not shown).
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DISCUSSION |
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We found that when LFN was added to the cis solution in the presence of open (PA63)7 channels, with the transmembrane voltage held at +20 mV (with reference to the trans solution), there was a large decrease in the PA63-induced conductance, which we interpreted as a blocking of the (PA63)7 channels (Fig. 1; see also S. Zhang, A. Finkelstein, and R.J. Collier, Unpublished). When LFN was perfused out of the cis compartment (with the voltage held at +20 mV) and the voltage was then stepped to –20 mV, there was a rapid unblocking of the channels, but when the voltage was returned to +20 mV, most of the channels became reblocked (Fig. 2). We interpreted this to mean that LFN was driven out of the channels back to the cis side by the –20 mV, but it remained attached to the channels; that is, the dissociation of LFN from its binding site on the channels is slow (Fig. 3). In contrast, if the voltage was stepped to +40 mV or higher, the channels were unblocked, as at –20 mV, but remained unblocked when the voltage was returned to +20 mV (Fig. 2). (In fact, if the voltage was returned to +20 mV before all of the channels were unblocked, channels continued to unblock for several seconds—a phenomenon we comment upon later.) We interpreted this to mean that LFN was translocated across the membrane into the trans solution (Fig. 3).
Consistent with, and extending this interpretation, are the following:
All of these results are consistent with LFN binding to its cognate sites on the (PA63)7 channel and entering the channel N-terminal end first, thereby blocking the channel; negative voltages drive the LFN out of the channel back to the cis side, whereas large (40 mV) positive voltages drive LFN all the way through the channel into the trans solution (Fig. 3). Introducing bulky groups in the channel lumen slowed translocation (Fig. 7), thus confirming that the translocation pathway is through the channel lumen.
There are several aspects of LFN translocation through (PA63)7 channels that deserve comment.
Mechanism
The simplest translocation mechanism to explain our results is that positive voltages electrophorese LFN through the channel. There is, however, a problem with this proposition. Although the N-terminal two-thirds or so of LFN has a net positive charge, and therefore can be driven into the channel by positive voltages, the C-terminal one-third has a net negative charge, which would act to prevent its translocation under an applied positive voltage (Bragg and Robertson, 1989). (The disordered 26 residues at the N-terminal end of LFN—Pannifer et al., 2001—with its net positive charge, may make it particularly suitable for the N-terminus to be driven into the channel). This problem disappears if the pK values of the aspartates and glutamates of LFN are one or two pH units higher when LFN is within the channel, in which case its C-terminal third would not bear a net negative charge. Other factors, however, may be involved in the translocation of LFN, one of these being the refolding energy. Since the diameter of the (PA63)7 channel is only 12 Å somewhere in its length (Blaustein and Finkelstein, 1990), LFN must unfold to traverse the lumen—certainly losing its tertiary structure and possibly also its secondary structure. Once part of the N-terminal region has been translocated to the trans solution, one can envision that its refolding acts as a driving force for the translocation of the rest of the molecule. This could explain why translocation and unblocking continue to proceed when the voltage is stepped back down from +40 to +20 mV (Fig. 2). We have recently found that at pH 6.6 (instead of our usual pH 5.5), channels that are blocked by LFN are not unblocked at large positive voltages. This effect of pH on translocation could be due to either now imparting a net negative charge to the C-terminal third of LFN or inhibiting unfolding of LFN.
In considering the mechanism of protein translocation through the (PA63)7 channel, we have confined ourselves in this article to LFN translocation across a membrane separating symmetric solutions at pH 5.5. We have not dealt with the more biologically relevant molecules, whole LF or EF. Nor have we addressed translocation in the presence of a pH gradient across the membrane (e.g., pH 5.5/pH 7.2), a situation germane to that faced by LF and EF in an acidic vesicle. Finally, we have not considered the role that chaperones may play in translocating LF and EF into the cytosol. These are topics for future publications. The purpose here has been to show that the (PA63)7 channel functions as a conduit for protein translocation that can be driven by transmembrane voltages. The magnitude of the transmembrane potential of acidic vesicles is difficult to assess, but there is no question of its sign (e.g., Van Dyke and Belcher, 1994); namely, the interior of the vesicle is positive with respect to its surroundings, which is the same polarity that drives translocation in our experiments.
Kinetics
There are two aspects of the kinetics of translocation that are noteworthy. One, is their sigmoidal nature (Fig. 4). This must mean that there are multiple (more than one) blocked states in series that have to be traversed before LFN exits the channel. This could be a consequence of there possibly being two or three LFNs bound per channel in our experiments (Mogridge et al., 2002), in which case unblocking would not occur until the last one has passed through. Experiments with mutant channels that have only one LFN-binding site, however, continue to display S-shaped kinetics (unpublished results), which means that these kinetics are intrinsic to the translocation of a single LFN molecule. Moreover, the continued translocation that occurs for several seconds when the voltage is stepped back to +20 mV from a larger positive voltage (Fig. 2) indicates that there is more than one kinetic barrier.
A second relevant aspect of the kinetics of translocation is the timescale of several seconds. Unless one assumes an extremely slow diffusion of LFN within the channel, translocation of this 263-residue protein should occur on a millisecond or submillisecond timescale. Indeed, translocation of RNA through the staphylococcal -toxin channel, which bears many similarities to the (PA63)7 channel, occurs in microseconds (Akeson et al., 1999). As we mentioned earlier, for LFN to get through the channel lumen, the folded molecule must become unfolded under the applied voltages, and it is perhaps this step, or steps, that is rate-limiting.
The effect of sulfhydryl-specific reagents on LFN translocation
When the cysteines in channels formed by PA63G323C were reacted with MTS-5-glucose, MTS-EA, or MTS-ET, the rate of unblocking of LFN at +50 mV was dramatically slowed (Fig. 7). Although this provides convincing evidence that the pathway for LFN translocation is through the (PA63)7 channel lumen, it is surprising that introduction of a relatively rigid, bulky group such as glucose did not completely block translocation. One possible reason for this is sulfhydryl exchange, which would still narrow the channel lumen by forming disulfide bonds within it, but would, in the process, expel glucose. We suspect this, because in single-channel experiments we saw up to four conductance steps (i.e., reactions) with MTS-5-glucose (unpublished results), and it is difficult to believe that the channel could accommodate four glucoses at the level of residue 323. It is more likely that each pair of reactions (two pairs giving the observed four reactions) represents the formation of R-S-S(CH2)2 glucose followed by sulfhydryl exchange, leaving R-S-S-R in the channel, where R = PA63.
Comparison to protein translocation in other systems
As we stated in the Introduction, diphtheria toxin and botulinum toxin can also translocate their respective enzymatic domains across planar lipid bilayers (Oh et al., 1999; Koriazova and Montal, 2003), and parts of their B portion form channels (Hoch et al., 1985). But the role of these channels in the translocation process is unknown, as is their structure. More relevant to the translocation mechanism we have described for anthrax toxin is that of the autotransporters (Henderson et al., 1998). In particular, it has been shown that the translocation domain of the autotransporter NalP from Neisseria meningitides forms a 12-stranded ß-barrel pore of diameter 10 x 12.5 Å that could act as a conduit for its passenger domain (Oomen et al., 2004); it also forms channels in planar bilayers (Oomen et al., 2004). The ß-barrel of OmpF has also been proposed as the translocation pathway for colicin E3 across the Escherichia coli outer membrane, with BtuB acting as the receptor (Kurisu et al., 2003). Supporting this hypothesis is the finding that OmpF channels in planar bilayers are blocked by colicin E3 (Kurisu et al., 2003). In this context it is worth noting that several protein-conducting cellular channels have been incorporated into planar bilayers and shown to be blocked by proteins or peptides that are presumed to go through them (e.g., TiC 110 of the chloroplast inner envelope membrane, Heins et al., 2002; TOM 40 of the outer mitochondrial membrane, Hill et al., 1998); in one instance the voltage-dependent nature of the blockade by a 13-residue peptide suggested that the peptide was translocated through the channel (Thieffry et al., 1992).
Our present study of the translocation of LFN through the (PA63)7 channel is the first, to our knowledge, that presents a semiquantitative treatment of protein translocation through a channel. This system lends itself as a model for future studies of the parameters important in translocation, such as protein length, charge distribution, and the energetics of voltage-driven protein unfolding. The last mentioned is of particular interest and relevance, since the membrane potential across the inner mitochondrial membrane apparently drives protein unfolding, and moreover does so by unraveling the protein from its positively charged N-terminal end (Huang et al., 2002).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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This work was supported by National Institutes of Heath grants GM 29210 and AI 22021.
Submitted on July 30, 2004; accepted for publication September 8, 2004.
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REFERENCES |
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R. A. Melnyk, K. M. Hewitt, D. B. Lacy, H. C. Lin, C. R. Gessner, S. Li, V. L. Woods Jr., and R. J. Collier Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen J. Biol. Chem., January 20, 2006; 281(3): 1630 - 1635. [Abstract] [Full Text] [PDF]
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J. T. Wolfe, B. A. Krantz, G. J. A. Rainey, J. A. T. Young, and R. J. Collier Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current J. Biol. Chem., November 25, 2005; 280(47): 39417 - 39422. [Abstract] [Full Text] [PDF]
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K. M. Halverson, R. G. Panchal, T. L. Nguyen, R. Gussio, S. F. Little, M. Misakian, S. Bavari, and J. J. Kasianowicz Anthrax Biosensor, Protective Antigen Ion Channel Asymmetric Blockade J. Biol. Chem., October 7, 2005; 280(40): 34056 - 34062. [Abstract] [Full Text] [PDF]
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L. Movileanu, J. P. Schmittschmitt, J. M. Scholtz, and H. Bayley Interactions of Peptides with a Protein Pore Biophys. J., August 1, 2005; 89(2): 1030 - 1045. [Abstract] [Full Text] [PDF]
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 B. A. Krantz, R. A. Melnyk, S. Zhang, S. J. Juris, D. B. Lacy, Z. Wu, A. Finkelstein, and R. J. Collier A Phenylalanine Clamp Catalyzes Protein Translocation Through the Anthrax Toxin Pore Science, July 29, 2005; 309(5735): 777 - 781. [Abstract] [Full Text] [PDF]
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 M. Qa'dan, K. A. Christensen, L. Zhang, T. M. Roberts, and R. J. Collier Membrane Insertion by Anthrax Protective Antigen in Cultured Cells Mol. Cell. Biol., July 1, 2005; 25(13): 5492 - 5498. [Abstract] [Full Text] [PDF]
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R. G. Panchal, K. M. Halverson, W. Ribot, D. Lane, T. Kenny, T. G. Abshire, J. W. Ezzell, T. A. Hoover, B. Powell, S. Little, et al. Purified Bacillus anthracis Lethal Toxin Complex Formed in Vitro and during Infection Exhibits Functional and Biological Activity J. Biol. Chem., March 18, 2005; 280(11): 10834 - 10839. [Abstract] [Full Text] [PDF]
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