Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel.

The translocation domain (T-domain) of diphtheria toxin contains 10 α helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transport the attached amino-terminal catalytic domain across the membrane. The TH5, TH8, and TH9 helices form transmembrane segments in the open-channel state, with TH1-TH4 translocated across the membrane. The TH6-TH7 segment also inserts to form a constriction that occupies only a small portion of the total channel length. Here, we have examined the TH5 segment in more detail, using the substituted-cysteine accessibility method. We constructed a series of 23 mutant T-domains with single cysteine residues at positions in and near TH5, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents added to the solutions on either side of the membrane. For 15 of the mutants, the reagent caused a decrease in single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also found that reaction caused large changes in ionic selectivity for some mutant channels. We determined whether reaction occurred in the open state or in the brief flicker-closed state of the channel. Finally, we compared the reaction rates from either side of the membrane. Our experiments are consistent with the hypotheses that the TH5 helix has a transmembrane orientation and remains helical in the open-channel state; they also indicate that the middle of the helix is aligned with the constriction in the channel.

Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1-TH4, translocates across the membrane to the trans side. Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6-TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6-TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.

Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain.

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.

A kinetic analysis of protein transport through the anthrax toxin channel.

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Conformational switching of the diphtheria toxin T domain.

The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

Identification of channel-lining amino acid residues in the hydrophobic segment of colicin Ia.

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of approximately 177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 alpha-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573-616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as alpha-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.

Anomalous proton selectivity in a large channel: colicin A.

Some of the bactericidal proteins known as colicins exert their toxic action by forming a large, nonselective channel in the inner membrane of target bacteria. The structure of this channel is unknown. It conducts large ions but has a much smaller conductance than would be expected for a channel of its deduced size. Here we report that the colicin channel, particularly the colicin A channel, is selective for protons over other cations (and anions) by many orders of magnitude. This was deduced from measurements of reversal potentials in pH gradients across planar lipid bilayers containing these channels. For example, in symmetric 0.1 M KCl with a pH 5/pH 8 gradient across the membrane, the reversal potential of colicin A is -21 mV, rather than 0. Such a result would be unremarkable for a narrow channel but is beyond explanation by current understanding of permeation for a channel of its diameter. For this reason, we re-examined the issue of the diameter of the channel lumen and confirmed that the lumen is indeed "too large" ( approximately 10 A) to select for protons by the amount that we measure. We are thus compelled to propose that an unorthodox mechanism is at work in this protein.

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import.

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.

Sizing the protein translocation pathway of colicin Ia channels.

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include /=90 mV, even a 26-A stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is >/=26 A in diameter, or can stretch to this value. This is large enough for an alpha-helical hairpin to fit through.