Correspondence to: Alan Finkelstein, Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Fax:(718) 430-8819 E-mail:finkelst{at}aecom.yu.edu.
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Abstract |
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In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine+ and NAD-, it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH2-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.
Key Words: planar lipid bilayers, histidine tags, voltage gating, single channels, monomer
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INTRODUCTION |
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Diphtheria toxin (DT)* is a single, 535amino acid polypeptide secreted by Corynebacterium diphtheriae and is responsible for the disease diphtheria. The toxin has three functional domains (see Fig 1): the NH2-terminal catalytic domain (residues 1185), the COOH-terminal receptor-binding domain (residues 386535), and the translocation, or T domain, lying between them (residues 202378). The catalytic domain is connected to the T domain by a protease-susceptible loop and by an easily reducible disulfide bridge (for reviews see
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Cellular intoxication by DT is thought to proceed by the following mechanism. Pathogenic strains of Corynebacterium diphtheriae infect a host and secrete the bacteriophage-encoded toxin as a monomer. The receptor-binding domain targets it to the surface of cells harboring a heparin-binding epidermal growth factorlike precursor. While the toxin is on the surface of the cell, a host protease nicks the loop connecting the catalytic domain to the T domain, leaving the two domains still connected by their disulfide bridge. Internalization occurs via receptor-mediated endocytosis, and the toxin now finds itself in an acidifying endosome. The low pH of the endosome induces a conformational change in the toxin that inserts the T domain into the endosomal membrane and translocates the catalytic domain across the membrane into the reducing environment of the cytosol. Here, the disulfide bond linking the two domains is reduced, releasing the catalytic domain. Subsequent ADP-ribosylation of elongation factor 2 by this domain inhibits protein synthesis, thereby killing the cell. The endosome function in this process is to provide a low pH environment; experimentally it can be bypassed by exposing toxin-treated cells to a low pH, in which case the T domain translocates the catalytic domain directly across the plasma membrane (
The T domain alone, as well as whole toxin and a mutant lacking the R domain (CRM45), form channels in planar bilayers when the pH of the cis side (the solution to which T domain constructs are added) is below 6 (70 residues of the NH2 terminus of the T domain is translocated across the membrane (
In the open channel state, the topology of the T domain consists of only three transmembrane segments (TH5, TH8, and TH9;
All of this evidence, although not definitive, seems to argue against a channel composed of multiple subunits. In fact, for most protein systems, one would probably accept the above as ample evidence of monomericity. For DT though, the improbability of this proposal demands a closer look. (How can three transmembrane segments alone create a pore large enough to conduct K+ and Cl-, let alone glucosamine+ and NAD-?) Therefore, we have designed a set of experiments to test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric.
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MATERIALS AND METHODS |
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Constructs
Details concerning plasmid construction of the T domain with an NH2-terminal hexahistidine tag (H6 tag), as well as our methods of protein expression and purification for all the constructs used, were as previously reported (
The COOH-terminal H6 tag construct was engineered by inserting the same T domain into Novagen's pET-22b plasmid. To do this, an XhoI site was introduced at the COOH-terminal end of the T domain in the pET-15b vector, using the site-directed mutagenesis kit. The purified DNA from that mutant was digested with the restriction endonucleases NdeI and XhoI, gel-purified, and ligated into the pET-22b expression vector that had been cut with the same restriction enzymes and also gel-purified. This put the sequence (His)6 at the COOH terminus of the T domain. It was found that this protein was not an effective channel blocker. Again using the site-directed mutagenesis kit, we lengthened the H6 tag by inserting GGGMGSS between the COOH terminus of the T domain and the H6 tag (primers used were CGTATAATCGTCCCCTCGAGGGAGGTGGAATGGGATCGTCGCACCACCACCACCACCAC and its reverse compliment; nucleotides inserted are shown in bold type). This was a better blocker but not sufficiently distinctive to satisfy us. We therefore inserted additional residues SSGLVPR COOH-terminal of the H6 tag (primers used were CCACCACCACCACAGCAGCGGCCTCGTCCCCAGGTGAGATCCGGCTGC and its reverse compliment). This protein blocked the T domain channel effectively. Thus, our final COOH-terminal H6 construct is P378-LEGGGMGSSH6SSGLVPR. One can see that this is most of the NH2-terminal H6 tag oriented in the reverse direction.
The construct with both H6 tags was made as follows. Purified plasmid containing the T domain with COOH-terminal H6 tag was digested with the restriction endonucleases NdeI and Bpu1102, gel-purified, and cloned into the pET-15b expression vector. This put the NH2-terminal H6 tag sequence MGSSH6SSGLVPRGSHM upstream of residue 202 in our COOH-terminal H6 construct. Thus, the final sequence of the T domain with both NH2- and COOH-terminal H6 tags is: MGSSH6SSGLVPRGSHM-I202...P378-LEGGGMGSSH6SSGLVPR.
After expression and purification (3 mg/ml; that with the COOH-terminal H6 tag was at
1.5 mg/ml; and that with both the NH2-terminal and COOH-terminal H6 tags was
3 mg/ml.
Ratios for Mixtures
The appropriate ratio of NH2-terminal to COOH-terminal H6 tag T domain in the mixtures was determined as follows. The T domains were first individually incubated at 37°C for 2 h in 40% DMSO (Sigma-Aldrich) to break up most of the preformed aggregates (
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Purification of Monomeric T Domain
After treatment with DMSO, the mixtures were run on a Superdex G-75 sizing column (Pharmacia) in 50 mM NaCl, 10 mM Tris-Cl, pH 8.0, at a flow rate of 0.75 ml/min. Samples were collected in 0.5-ml fractions and concentrated 10-fold in a Savant UVS 400 Speed Vac® plus. Fig 3 shows examples of how the peaks separated. All mixtures used for bilayer experiments were taken from fractions located to the right of the monomer peak. Purification was assayed on 15% native PAGE.
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Bilayer Experiments
Planar lipid (asolectin) bilayer membranes (70100 µm in diameter) were made by a modification of the folded film method as described by
Current generated by single channels was measured by voltage clamping the cis side of the membrane to voltages between ±60 and 80 mV. Channels entered the membrane at positive voltages (
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RESULTS |
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The experiments to be described were performed in planar lipid bilayer membranes. Their rationale came from our earlier work, which showed that a histidine tag (H6 tag) attached to the NH2 terminus of the T domain rapidly and completely blocked2 the channel at cis negative voltages, whereas when the H6 tag was attached to the COOH terminus, the channel was blocked (a high frequency flickering block, occasionally entering a prolonged blocked state) at cis positive voltages (
We reasoned that if we mixed NH2-terminal and COOH-terminal H6-tagged T domains, and if the T domain channel is a multimer, then some fraction of channels should be blocked at both positive and negative voltages. For example, if the channel is a dimer, and equal amounts of NH2-terminal and COOH-terminal H6-tagged T domains were mixed, then on average (if the T domains have an equal preference to associate with one another) one quarter of the channels should be blocked only at negative voltages (both subunits have NH2-terminal H6 tags), one quarter blocked only at positive voltages (both subunits have COOH-terminal H6 tags) and one half blocked at both negative and positive voltages (one subunit has an NH2-terminal H6 tag and the other has a COOH-terminal H6 tag). If, however, the channel is a monomer, one expects to see only channels that are blocked at either negative or positive voltages, but not at both.
One could argue that before mixing these constructs, there already exist preformed homo-multimers and that these are responsible for the channel-forming activity. Thus, when mixtures are made, one sees channels that only gate at either negative or positive voltages and falsely asserts that these channels are monomeric. We attempted to circumvent this problem in two ways. First, we dissociated dimers and higher order aggregates by incubating the mixture in 40% DMSO (
After the above protocol, we performed experiments on two sets of purified mixtures of NH2- and COOH-terminal H6-tagged T domains. We observed 75 single channels, none of which gated at both negative and positive voltages. From the first mixture, a total of eight separate bilayer experiments were conducted yielding 35 single channels. (A typical record from one bilayer is shown in Fig 5.) Of these, 17 showed N-type and 14 showed C-type H6-tagged gating characteristics, whereas 4 did not gate at either positive or negative voltages (see next paragraph). The failure to observe any channels that showed both N-type and C-type gating is, of course, which is consistent with a monomeric channel. If, for the sake of argument, it is assumed that the channel is dimeric, the probability of our not seeing a single channel that showed both N-type and C-type H6 tag gating in 35 events is 4.5 x 10-5 (see Appendix). From the second mixture, 9 individual bilayer experiments were performed giving a total of 40 observed single channels. Of these, 26 showed N-type and 9 showed C-type H6-tagged gating characteristics, and 5 did not gate at either positive or negative voltages (see next paragraph). If the channel is dimeric, the probability of our not having seen one channel that showed both N-type and C-type H6 tag gating in 40 events is 1.3 x 10-4 (see Appendix). In the combined two sets of mixtures, the probability of our not having seen even one channel that showed both N- and C-type gating is 5.9 x 10-9. If more than two T domains are required to construct the channel, then the odds of our not having seen even one channel that was blocked at both negative and positive voltages become even more minuscule.
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There were nine single channels that did not gate at all. We attribute these "nulls" to a subpopulation of the NH2-terminal H6 tag proteins that had its NH2-terminal H6 tag cleaved off by trace amounts of protease in the solution. (The region of the T domain immediately downstream of the NH2-terminal H6 tag is exposed in the water-soluble crystal structure [
We found that our extensive efforts to eliminate preformed dimers and aggregates were probably unnecessary. When we compared the activity of a sample that had a large preformed dimer population to its activity after receiving the 40% DMSO treatment (using the sample illustrated in Fig 2), we saw no difference as assayed by the rate of channel entry into the membrane (results not shown). Thus, it appears that the preformed dimers and higher aggregates are not contributing significantly to channel formation.
With this in mind, we can also include in our dataset the results from a mixture that was DMSO treated, but was not run on the sizing column, and from a mixture that was neither DMSO treated nor run on the sizing column. In the former case, 21 single-channel events were recorded on six separate bilayers; of these events, 11 showed N-type gating, 9 showed C-type gating, and 1 did not gate at all. In the latter case, of 19 single-channel events observed on five bilayers, 9 showed N-type gating and 10 showed C-type gating. Combining these data with the results from the previous mixtures, we observed a total of 115 single channels in 28 bilayers and never saw a channel that showed both N-type and C-type gating.
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DISCUSSION |
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Associated with the translocation of diphtheria toxin's catalytic domain across a planar lipid bilayer, the toxin's T domain forms a channel. The T domain in this channel, which is permeable to ions as large as glucosamine+ and NAD- (
What are some possible arguments against this conclusion? It might be contended that the channels were generated from dimers or multimers that preexisted in the NH2-terminal and COOH-terminal H6-tagged T domain solutions, and so naturally we would not see any channels with both N-type and C-type H6 tag gating. This is a very unlikely possibility on two grounds. First, after incubating a T domain solution that had a relatively large dimer content for 2 h at 37°C in 40% DMSO, which converted almost all of the dimers and higher aggregates to monomers (Fig 2), we found the channel-forming ability of the solution unchanged, indicating that preformed dimers and higher multimers are not a significant source of channels. Second, in two sets of experiments, we went to the extreme of removing residual preformed dimers and multimers from mixtures of NH2-terminal and COOH-terminal H6-tagged T domains that had undergone the DMSO treatment, by running the mixtures on a molecular sizing column and using, for the experiments, only fractions from the trailing half of the monomer peak (Fig 3).
Even though preexisting dimers and multimers are not the source of channel-forming activity, this does not preclude that T domain monomers can come together on or within the membrane to form a multimeric channel. For this to account for our failure to see even one channel out of 115 that manifested both N-type and C-type H6-tagged gating, however, one would have to assume that NH2-terminal H6-tagged T domains and COOH-terminal H6-tagged T domains associate exclusively with themselves; i.e., heteromultimer formation is precluded. Although this is logically possible, we can think of no justification for this assumption. In fact, if one argues that somehow a T domain with a COOH-terminal H6 tag cannot associate with one that has an NH2-terminal H6 tag, then why would T-domains containing both an NH2-terminal and COOH-terminal H6 tag associate in the postulated multimeric channel? One would have to invoke a special attraction of NH2-terminal H6 tags and/or COOH-terminal H6 tags with themselves; we can see no physical justification for this. One might also argue that even though NH2-terminal H6-tagged T domains can form heteromultimers with COOH-terminal H6-tagged T domains, for some reason these do not form functional channels. Again, it is difficult to reconcile this position with the ability of T domains that have both an NH2-terminal and a COOH-terminal H6 tag to form channels. Thus, it seems to us that to believe that the T domain channel is a multimer, one must argue that heteromeric channels are formed by NH2-terminal and COOH-terminal H6-tagged T domains, but some of these exhibit only N-type gating, whereas others exhibit only C-type gating. Asymmetric structures can be envisioned that fulfill this condition, but this leads us into a fantasyland that is best avoided.
Throughout the analyses and discussions in this paper, we have tacitly assumed that only a single H6 tag is required for channel blocking. If this is not the case, one can imagine new scenarios in which the channel is multimeric, and yet no channels formed from our mixture exhibit both N-type and C-type gating. For example, if the channels were a trimer and required at least two NH2- or COOH-terminal H6 tags to get NH2- or COOH-terminal gating, respectively, we would observe only N-type and C-type gating channels, never channels showing both types of gating. In fact, in general, if the channel is formed by an odd number (n) of subunits and requires (n + 1)/2 NH2- or COOH-terminal H6 tags to get N- or C-type gating, respectively, the above statement would hold. (If n is even, the equivalent assumption that (n/2) + 1 H6 tags are required for blocking predicts a significant number of channels showing neither N- nor C-type gating.) We think that the requirement of multiple H6 tags to affect channel blocking is a priori unlikely; we know of no precedent for this in the channel literature. Moreover, if more than one H6 tag were required to completely block the channel, we would anticipate substates (partial block) when less than one H6 tag entered the channel. We have never observed this.
This paper presents what we feel are compelling arguments for the monomeric nature of the T domain channel formed in planar lipid bilayers. The channels formed by whole toxin in planar bilayers are indistinguishable from these (
There is a feeling in the literature that in real life (i.e., the cell) the translocation of the catalytic domain across the endosomal membrane into the cytosol is accomplished through an oligomer of DT (
There are also data using liposomes that suggest that oligomerization is involved in DT pore formation. For example,
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Footnotes |
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* Abbreviations used in this paper: DT, diphtheria toxin; DTT, dithiothreitol; MTS, methanethiosulfonate.
1 To avoid continually having to use the expressions "C-type H6-tagged gating channel" or "N-type H6-tagged gating channel" we mercifully shorten these to "C-type channel" or "N-type channel."
2 It is not definitely known whether the H6 tag sterically blocks the channel or binds externally and induces a conformational change that closes the channel. We think the former is much more likely, as the latter would require two separate external allosteric binding sites, one on the cis side and one on the trans side.
3 We cannot preclude that there is a contaminant, common to the different preparations and purification procedures of T domain, CRM45, and whole toxin, which contributes to the channel structure.
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Acknowledgements |
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We thank Jean-Claude Schwartz for his help with the size exclusion columns, Drs. Karen Jakes, Paul Kienker, Stephen Slatin, and Myles Akabas for their helpful discussions and readings of the manuscript, and Dr. Paul Kienker for his penetrating comments and critique of the Appendix.
This work was supported by National Institutes of Health grants T-32-GM07288 (to M. Gordon) and GM-29210 (to A. Finkelstein).
Submitted: 10 July 2001
Revised: 6 September 2001
Accepted: 11 September 2001
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Appendix |
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We wish to determine how unlikely it would be for us to observe in our mixing experiments no channels that show both N- and C-type gating, if the T domain channel was a multimer. We assume that all T domain monomers have an equal preference to associate with one another, independent of the H6 tag's location or absence. Suppose, for concreteness, that the channel is a dimer.
Let fN, fC, and fO be the fraction of monomers in the mixture with NH2-terminal H6 tag, COOH-terminal H6 tag, and no H6 tag, respectively. More precisely, fN, fC, and fO can be considered as the probabilities for each type of monomer to contribute to a dimer. It follows that,
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(1) |
Then the probability that a dimer channel has at least one NH2-terminal H6 tag but no COOH-terminal H6 tag (i.e., the probability that it shows only N-type gating) is:
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(2a) |
Likewise, the probabilities of a dimer channel showing only C-type gating, no gating, and both N- and C-type gating, respectively are:
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(2b) |
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(2c) |
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(2d) |
(We assume that a channel shows N-type gating if at least one of its subunits has an NH2-terminal H6 tag, and shows C-type gating if at least one of its subunits has a COOH-terminal H6 tag.)
Let [N], [C], and [O] be the number of channels observed in a set of experiments that show N-type, C-type, or no gating, respectively. (No channels were observed that showed both N- and C-type gating; i.e., [NC] = 0.) To use the observed numbers of channels (with [NC] = 0) to estimate the fractions of each type of monomer, and from this P(NC), which does not equal zero, we use the ratio of the probabilities:
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(3a) |
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(3b) |
Combining Equation 1, Equation 2a, Equation 2b, Equation 2c, and Equation 3a and Equation 3b, we obtain
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(4a) |
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(4b) |
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(4c) |
We can now substitute fN and fC from the above formulae into Equation 2d to calculate P(NC) from our data.
In our first set of experiments (RESULTS), we had: [N] = 17, [C] = 14, and [O] = 4. (Total number of events = 35.) From Equation 3a and Equation 3b we then have:
and substituting these into Equation 4aEquation 4bEquation 4cc we get:
Therefore,
This is the probability of a dimeric channel showing both N-type and C-type H6 tag gating. The probability of our not seeing one such channel in our 35 events is:
In our second set of experiments: [N] = 26, [C]=9, and [B]=5 (total number of events = 40), and by the same calculations as above:
and therefore
The probability of our not seeing one channel in our 40 events that shows both N-type and C-type H6 tag gating is:
Thus, if the channel were a dimer, the probability of our not having seen even one channel in our two sets of experiments that showed both N-type and C-type H6 gating is
If the channel were a trimer, tetramer, or larger multimer, similar calculations would give probabilities much smaller than even this.
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