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Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen*

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe427 residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe427 in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity ≥1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

How the enzymatic moieties of intracellularly acting bacterial toxins cross membranes is not well understood and is a topic of interest in protein structure and function, as well as in bacterial pathogenesis. Anthrax toxin consists of three nontoxic proteins that co-assemble to produce a series of free or cell-bound toxic complexes (1). Two of the proteins, Lethal Factor (LF) 3 and Edema Factor (EF), are enzymes that modify cytosolic targets (2,3). The third, Protective Antigen (PA), is a receptor-binding and pore-forming protein that transports LF and EF to the cytosol (4). Initially, PA is activated by cellular proteases of the furin family, or by serum proteases (5)(6)(7)(8), which cleave the 83-kDa protein into N-terminal 20-kDa and C-terminal 63-kDa fragments. The larger fragment self-associates to form ring-shaped heptamers (prepores), which bind up to three molecules of LF and/or EF, and a smaller population of octamers, which bind up to four molecules of these moieties (9,10). The resulting hetero-oligomeric complexes then undergo receptor-mediated endocytosis (11)(12)(13). Once within the acidic environment of the endosome, the PA moiety is conformationally altered to generate a membrane-inserted pore (14,15), and LF and EF unfold (16,17) and translocate across the membrane to the cytosol (18).
Recent electrophysiological studies of anthrax toxin in planar lipid bilayers have shown that the PA pores play an active role in translocating LF and EF across the endosomal membrane. The pore is a mushroom-shaped structure, with a globular cap and a 100-Å long stem with a transmembrane segment at its distal end (Fig. 1). The enzymic moieties bind to the cap via their homologous N-terminal domains (LF N and EF N ). An unstructured segment containing a high density of both basic and acidic residues is present at the extreme N terminus of LF N and EF N , and there is good evidence that this segment initiates N-to C-terminal translocation (17,19,20). The blockage of ion conductance through the pore when LF N , EF N , or their parent proteins bind is also dependent on this highly charged segment. Translocation of bound ligands through PA pores in planar bilayers may be effected by applying either a pH gradient or a cis-positive potential (e.g. ϳ50 mV) and is manifested by the relief of channel blockage (21,22). The pH gradient emulates the gradient across the endosomal membrane and may provide the major driving force for translocation in vivo (21).
A specific phenylalanine residue (Phe 427 ) within the lumen of the cap of the pore, near the junction of the cap with the stem, plays a key role in the PA transport function (Fig. 1) (23). Mutation of Phe 427 to almost any residue except Trp, Tyr, or Leu causes a loss of the ability of PA to mediate toxicity, and indeed, many Phe 427 mutations are dominantly negative, in that mixing a low ratio of mutant to wild-type PA together results largely in the formation of nonfunctional oligomers. Mutation of Phe 427 to Ser, Thr, or His inhibits the translocation function of PA without significantly affecting the conformational transition of the prepore to the pore, whereas some other mutations at this site affect pore formation (24). Inhibition of ion conductance in planar bilayers by a translocatable ligand (e.g. LF N ) also depends on Phe 427 (23,25). Binding of LF N to wild-type pore results in an almost complete blockage of current, but binding to F427A pores, for example, causes only partial blockage (23,25).
Electron paramagnetic resonance studies with PA containing a thiol-reactive EPR probe linked to Cys introduced at position 427 suggested that the side chains at this position converge during prepore-to-pore conversion and are brought into in close proximity (Յ10 Å) within the lumen of the pore (23). Single-channel conductance values of various Phe 427 mutants are consistent with the notion that this residue lies at a constriction point in the translocation pathway; the term Phe clamp (or ⌽ clamp) has been coined for this structure (23). Single-channel traces recorded in the presence of LF N suggest that the Phe clamp interacts directly with the N-terminal region of the bound ligand to form a seal against the passage of ions.
In an earlier report, we described a protocol for preparing heteroheptamers with an inhibitory mutation in only a single subunit (25). Both F427A and F425A were known to have dominant negative inhibitory effects on toxicity in PA homo-oligomers (26 -30), and isolating heteroheptamers with the inhibitory mutation in only one subunit enabled us to characterize the effects of these mutations in greater detail. In the current study we applied the same protocol to isolate a series of heteroheptamers in which Phe 427 had been replaced with a charged residue in a single subunit. The observed effects of F427D, F427E, F427K, F427R, and F427H on cytotoxicity, translocation, and other activities of the pore offer insights into the structure and function of the Phe clamp.
Expression and Purification of Proteins-Recombinant WT PA and PA mutants were overexpressed in the periplasm of E. coli BL21 (DE3) and purified by anion-exchange chromatography (14). The mutated PA proteins contained two mutations, K563C and F427X (where X ϭ a charged natural amino acid) (Fig. 1). The proteins were biotinylated using a 10-fold molar excess of N ␣ -(3-maleimidylpropionyl)biocytin at 4°C overnight and purified over avidin resin (25).
Formation and Purification of Homoheptameric and Heteroheptameric PA 63 Prepore-WT PA prepore ([WT] 7 ) and homoheptameric PA F427X/K563C prepore ([F427X] 7 ) were formed by limited trypsin digestion followed by anion-exchange chromatography (10). Heteroheptamers containing predominantly one mutant subunit were prepared as described (25). Briefly, we first mixed WT PA with biotinylated PA F427X/K563C in a 20:1 molar ratio, nicked the mixture with trypsin, and purified the resulting heptamers by anion-exchange chromatography. The resulting preparation was then passed over monomeric avidin, and bound biotinylated heptamers were eluted with 2 mM d-biotin. The product was desalted to remove free biotin and passed over a second monomeric avidin column to ensure the removal of [WT] 7 .
Cytotoxicity Assays-Cytotoxicity assays were performed as described (28,29). PA-dependent translocation of LF N -DTA was reported by the DTA domain, the enzymatic domain of diphtheria toxin, which inhibits protein synthesis. Translocation of the DTA domain into CHO-K1 cells was determined by measuring the incorporation of [ 3 H]leucine into protein after a 16-h incubation with varying concentrations of PA heptamers and 100 nM LF N -DTA at 37°C under 5% CO 2 . The data were plotted as averages of at least three experiments (n Ն 3), with standard errors shown as bars. EC 50 values were calculated from the fit of the curves (IGOR Pro Software, version 6.0.3.1; Wavemetrics, Inc., Lake Oswego, OR).
Macroscopic Electrophysiology-Planar phospholipid bilayer experiments were performed in a Warner Instruments Planar Lipid Bilayer Work station (BC 525D, Hamden, CT). Planar bilayers were painted (33) onto a 200-m aperture of a Delrin cup in a Lucite chamber, with 3% 1,2-diphytanoyl-sn-glycerol-3-phosphocholine in n-decane (Avanti Polar Lipids, Alabaster, AL). One-ml aliquots of buffer were added to the cup and the chamber, and both compartments were stirred constantly. Cis (side to which PA prepore and LF N were added) and trans compartments contained 100 mM KCl, 1 mM EDTA, and 10 mM each sodium oxalate, potassium phosphate, and MES, pH 5.5. Macroscopic current responses to steps in voltage were recorded as described (20); n Ն 3. The membrane potential, ⌬, is defined as ⌬ ϭ cis Ϫ trans , where trans ' 0 mV.
Macroscopic PA 63 Channel Formation, LF N Conductance Block, and Translocation under the Influence of ⌬pH-Once a membrane was formed in the planar lipid bilayer system, PA prepore (25 pM) was added to the cis compartment, which was held at a ϭ ϩ5 mV with respect to the trans compartment. After appropriate current increase, the cis compartment was perfused with ϳ10 ml of non-PA-containing buffer at a flow rate of ϳ3 ml/min to remove any free PA. Once the current was constant, LF N was added to the cis compartment (1 g/ml), and its binding to PA channels was monitored by the decrease in conductance. The cis compartment was perfused again after LF N addition to eliminate free ligand. Initial values of dissociation represent the fraction conductance unblocked by LF N (n ϭ 3) over time. The rate of dissociation of LF N over time was calculated based on the slopes of the fitted curves (IGOR Pro Software, version 6.0.3.1). Translocation was initiated by raising the pH of the trans compartment to pH 7.2 with 2 M KOH while maintaining the cis compartment pH at 5.5. Translocation was quantified by monitoring the half-time to complete alleviation of conductance block (t 1/2 ). Experiments for each PA protein tested were normalized to control experiments where KOH was added to the trans compartment in the absence of LF N to adjust for current changes due to salt addition alone. The kinetics of translocation were monitored at ϭ ϩ5 mV by the rise in current; n Ն 3.
Single-channel Electrophysiology-Single-channel measurements were obtained under conditions similar to macroscopic current recordings, but with a 100-m aperture membrane. The 1-ml cis and trans compartments contained 1 M KCl and 10 mM MES at pH 5.5, and current was measured over a range of applied membrane potentials. Increments of ϳ0.1 ng of PA 63 heteroheptamer prepore were added until a single channel was observed. For PA blocking experiments with LF N , the cis and trans compartments contained 100 mM KCl, 1 mM EDTA, and 10 mM each sodium oxalate, potassium phosphate, and MES, pH 5.5. After channel formation LF N was added to the cis compartment (final concentration, 1 g/ml). Data were analyzed using Clampfit version 10.0 software (Axon Instruments, Sunnyvale, CA) and Microsoft Excel. Analysis was performed on 10-s records. Single-channel conductance was calculated from Gaussian fits to current amplitude histograms; n Ն 3. Percent block of PA channel activity by LF N was determined as 1 Ϫ P o (the probability of the channel to reside in the open state).
Ion-selectivity Measurements-Anion/cation selectivity of the PA channel was measured by determining the dependence on the activity ratio of KCl in the cis versus trans compartments of the membrane potential at which the current across the membrane equals zero (E rev ). Channel activity for each of the proteins tested was elicited under symmetric conditions of 0.1 M KCl, 10 mM MES, pH 5.5, followed by serial additions of 19 l of 3.6 M KCl to the cis compartment. The E rev was measured for symmetric cis/trans solutions and after each addition. E rev was plotted against the activity ratio (activity of KCl in cis/activity of KCl in trans) and fit to the Goldman-Hodgkin-Katz equation to determine the probability to conduct K ϩ (p K )/probability to conduct Cl Ϫ (p Cl ); n Ն 3. Activity coefficients were obtained from Appendix 8.10, Table 2 in Robinson and Stokes (34); the KCl concentration was assumed to be equal to the K ϩ concentration as well as the Cl Ϫ concentration.

Effects of Single-subunit Phe 427 Mutations on Cytotoxicity-
We prepared five hetero-oligomeric forms of the prepore, each containing a positively or negatively charged residue at position 427 of a single subunit. The protocol yields predominantly heptamers and was used earlier to generate [WT] 6 [F427A] 1 and [WT] 6 [F425A] 1 (25). EC 50 values were determined in our standard cytotoxicity assay, in which LF N -DTA, the catalytic moiety of diphtheria toxin (DTA) fused to LF N , is delivered to the cytosol, inhibiting protein synthesis and causing cell death. EC 50 values of heteroheptamers containing either a basic or an acidic residue in the Phe clamp were found to be at least 3 orders of magnitude higher than that of the [WT] 7 ( Fig. 2 and Table 1  trast, incorporating either a single Asn or Gln residue into the Phe clamp caused relatively little inhibition of cytotoxicity (5-fold and 8-fold, respectively; data not shown).
Effects of LF N on Ion Conductance through Hetero-oligomeric Pores-Binding of LF or LF N , to pores formed from [WT] 7 prepore causes rapid and virtually complete (ϳ98%) blockage of ion conductance, as measured macroscopically in planar phospholipid bilayers (20,22). With pores containing a single Glu or Asp in the Phe clamp, the blockage by saturating levels of LF N was at least as rapid and complete as that observed with [WT] 7 pores. In contrast, pores containing a Lys, Arg, or His residue showed greatly reduced blockage (reduction ϳ30%) ( Table 1 and Fig. 3A). Results consistent with these findings were observed in single-channel ion-conductance recordings in the presence and absence of LF N (Table 1 and Fig. 3B). Channels with an acidic residue in the Phe clamp gave a blocking pattern by LF N (final concentration 1 g/ml) similar to that seen with [WT] 7 channels, with fast transitions to and from the open state and prolonged periods in the closed state, reflecting prolonged occlusion times by the cargo protein (35). With a basic residue, however, no transitions to the fully open state were seen, and there were fast, short lived transitions between closed and intermediate states.
Effects of Single-subunit Mutations on Dissociation of LF N -To probe the effects of single-subunit Phe 427 mutations on the interaction of the pore with LF N , we first measured the rate of dissociation of pore-bound LF N into the cis compartment. LF N was bound under symmetric acidic conditions (pH 5.5), and macroscopic current was monitored as the cis chamber was perfused with the same buffer. As shown in Fig. 3A and Table 1, with pores containing a Glu or an Asp in the Phe clamp, as with WT pores, the current increased at a low rate (Ͻ0.5%/min) over a perfusion period of 5 min, and thus the pores remained firmly blocked by LF N over this period. In contrast, with pores containing a Lys, Arg, or His in the Phe clamp, the current increased ϳ10-fold faster over the same period, reflecting much more rapid dissociation.
Effects of Single-subunit Mutations on Translocation of LF N -If, after brief perfusion of the cis compartment, we raised the pH of the trans compartment to neutrality, bound LF N was induced to translocate across the membrane. Translocation of LF N through [WT] 7 channels was complete within less than a min (t 1/2 ϳ23 s) under these conditions, whereas translocation through the mutant heptameric channels was ϳ10to ϳ440fold slower ( Fig. 3C and Table 1). The relative activities of the various heteroheptamers in this assay were similar to their activities in the cytotoxicity assay (Fig. 2). Values of t 1/2 of translocation ranged from 326 s for [WT] 6 [F427R] 1 , the most active heteroheptamer, to Ͼ10,000 s for [WT] 6 [F427E] 1 and [WT] 6 [F427H] 1 , the least active ones (Table 1).
Effects on Ion Conductance and Selectivity-We characterized the single-channel ion-conductance properties of the heteroheptamers in planar 1,2-diphytanoyl-sn-glycerol-3-phosphocholine bilayers. The cis and trans compartments were filled with 1 M KCl, pH 5.5, and traces were recorded at ϩ50 mV and Ϫ50 mV.
[WT] 7 and [WT] 6 [F427A] 1 exhibited an ohmic current response, with symmetric single-channel conductance (␥) at positive and negative potentials. In contrast, all of the heteroheptamers showed asymmetric ␥ values (Table 1). Either Glu or Asp in the Phe clamp caused an increase in ␥ at both positive and negative membrane potentials. Basic residues, on the other hand, caused a decrease at positive potentials and an increase at negative potentials ( Table 1). The [WT] 7 channel inactivates under negative membrane potentials, and the acidic residues allowed some of the WT inactivation characteristics to be maintained. However, [WT] 6 [F427R] 1 and [WT] 6 [F427K] 1 exhibited significantly reduced inactivation, and the inactivation for [WT] 6 [F427H] 1 was completely abolished (data not shown). Although the [WT] 7 channel is seen to transition to the fully closed state at least once within a 10-s time scale, no transitions to the fully closed state were observed with any of the mutant heteroheptameric channels (Fig. 4A).
We measured the ion selectivity of each heteroheptamer to determine the extent to which the cation selectivity of the wildtype PA pore had been altered. Heteroheptamers with an acidic residue in the Phe clamp displayed similar selectivity to [WT] 7 , but those with a basic residue exhibited significantly reduced selectivity ( Fig. 4B and Table 1), such that the flux of Cl Ϫ was closer in magnitude to that of K ϩ . a EC 50 values representing the cytotoxicity of the protein when combined with LF N -DTA on CHO-K1 cells were calculated from the curves shown in Fig. 2. b Percentage of total conductance blocked upon adding LF N to a single PA channel formed on planar lipid bilayers at ⌬ ϭ ϩ5 mV. LF N block was assayed over a range of applied membrane potentials for each mutant and then calculated for ⌬ ϭ ϩ5 mV as the limits of signal detection often precluded direct measurement at ⌬ ϭ ϩ5 mV. c Percentage of total conductance blocked upon adding LF N to macroscopic PA channels formed on planar lipid bilayers. d Percentage of total conductance blocked lost over time upon perfusion of excess LF N associated with macroscopic PA channels formed on planar lipid bilayers. e Translocation rate expressed in time required for half of bound LF N to pass through PA, as calculated by determining the slope of the tangent of the curves shown in Fig. 3C. f Average single-channel conductance over a range of applied positive membrane potentials (Fig. 4A). pS, picosiemens. g Average single-channel conductance over a range of applied negative membrane potentials. pS, picosienems. h Probability to conduct K ϩ /probability to conduct Cl Ϫ as calculated from the curves shown in Fig. 4B. i The values shown for EC 50 , t 1/2 , and LF N block and dissociation for ͓WT͔ 6 ͓F427A͔ 1 were calculated based on data reported previously (25).

DISCUSSION
There is evidence that translocation through the PA 63 pore is driven by a transmembrane proton gradient, and we have proposed a Brownian ratchet model of this process that depends on protonation and deprotonation of acidic residues of the translocating polypeptide (21,23,24,36). The pore is known to be strongly cation-selective, reflecting an electrostatic barrier that discriminates against the passage of negatively charged ions. This barrier would also be expected to discriminate against the passage of anionic side chains of Glu or Asp residues of polypeptides in transit through the pore. Because such side chains would spend a greater fraction of time in the protonated, uncharged state when on the acidic, endosomal side of this barrier than on the neutral, cytosolic side, their diffusion across the barrier would occur preferentially in the endosome-to-cytosol direction. Thus, ratcheting of a polypeptide through the pore from the endosome to the cytosol would be predicted, based simply on the proton gradient, the electrostatic barrier within the pore, and Brownian motion. This model has received support from demonstration that an essentially nontitratable anionic group (SO 3 Ϫ ) introduced at any of a variety of locations within LF N serves as a stop-translocation signal (36).
Translocation of polypeptides through the pore must involve a delicate balance of interactions between the transporter and the cargo. Clearly, the Phe clamp must accommodate all of the various side chain chemistries of translocatable proteins, but must not bind any particular group too tightly, lest translocation be impeded or arrested. Our working hypothesis is that the phenyl side chains of Phe 427 directly contact the translocating polypeptide chain and form a pliable surface that conforms to the various side-chain structures of the polypeptide, generating an ion-proof seal that preserves the pH gradient that drives translocation.
The exact location of the electrostatic barrier within the pore is not known, but mutating Phe 427 to Ala caused the pore to become less selective for K ϩ over Cl Ϫ and lowered the barrier to translocation of LF N bearing an SO 3 Ϫ group (36). These findings, together with the fact that two acidic residues, Asp 425 and Asp 426 , are immediately adjacent to Phe 427 , suggest that the main barrier lies at the level of the Phe clamp. Our finding that incorporating an acidic residue into the Phe clamp did not significantly affect the selectivity of the channel for cations, whereas a basic residue reduced the selectivity, is consistent with this hypothesis.
We found that replacement of Phe 427 with either an acidic or a basic residue in a single subunit of multimeric PA 63 inhibited cytotoxicity by Ն10 3 in our standard assay (Fig. 2). This remarkable effect of a single charged residue explains the dom-  6 [F427E] 1 (purple circles). The channels were blocked with 10 nM LF N , and then dissociation of LF N was monitored over time during perfusion of the cis compartment. B, representative single-channel currents of heteroheptameric PA wild type and mutant channel activity blocked by the addition of 1 g/ml LF N to the cis compartment, recorded at ⌬ ϭ ϩ20 mV in 0.11 M KCl. C and O denote the closed and open states, respectively. C, translocation of LF N through macroscopic PA channels. At time 0, translocation was initiated by adding 2 M KOH to the trans compartment to raise the pH to 7.2; there was an ϳ20-s mixing delay in this system with both compartments continuously stirred. Representative data are shown from n Ն 3 trials.
inant negative phenotype of these mutations. The importance of charge per se in causing the inhibition of pore function is indicated by the small effects on cytotoxicity (Ͻ10-fold) of incorporating an Asn or a Gln residue into the Phe clamp. It should be noted that Asn and Gln are structural analogs of His as well as Asp and Glu.
The nature of the effects observed depended on the sign of the charge introduced into the Phe clamp. With the acidic replacements, there was a 3-and 4-fold increase in single-channel conductance for Asp and Glu, respectively, whereas the native cation selectivity was maintained (Fig. 4). We attribute these observations to a significant alteration in constriction diameter based upon the pore size estimation model proposed by Hille (35). Conversely, the presence of a basic residue apparently maintained a similar architecture with altered cation selectivity (Fig. 5A). His ablated voltage-dependent inactivation of ion conductance, whereas Lys and Arg allowed at least some sensitivity to applied voltage to be maintained.
An acidic residue did not impair the ability of LF N to block ion conductance in macroscopic measurements and did not significantly alter the blocking pattern in single-channel measurements. Thus, as in the wild-type pore, the Phe clamp interacted with the polypeptide substrate to form a seal that almost completely blocked ion conductance, transitioning to the open state briefly every couple of seconds (Fig. 3, A and B); and there was minimal dissociation of the polypeptide during a severalminute perfusion of the cis compartment (Fig. 3A). In contrast, with a basic residue the ability of LF N to block ion conductance was significantly reduced (ϳ30%). Furthermore, there were major changes in the pattern of channel opening and closing in single-channel traces (Fig. 3B), and significant dissociation of LF N occurred during perfusion of the cis compartment (Fig.  3A). Thus, a basic residue prevented the formation of a tight and durable interaction of the Phe clamp with the polypeptide substrate, whereas an acidic residue did not diminish the interaction. states, respectively. The ␥ of wild type is symmetric over positive and negative potentials (154 Ϯ 3 picosiemens); however, the heteropheptamer mutations all have asymmetric ␥ with respect to membrane potential polarity (see Table  1 Table 1 for fits to data). These findings support current thinking about the interaction of the Phe clamp with LF N . The first ϳ30 N-terminal residues of LF N are unstructured and densely populated with acidic and basic residues. This segment is predicted to have a net positive charge under acidic conditions, and there is evidence that the positive charge functions to attract the N-terminal tract into the acidic lumen of the prepore or pore and mediate its interaction with the Phe clamp. Thus, if one deletes this tract, LF N loses the ability to block ion conductance through the pore, and this ability is restored when one replaces the tract with one containing a His 6 tag (20). Details of the interaction of the tract with the Phe clamp are unknown, but we hypothesize an electrostatic interaction with Asp 425 and/or Asp 426 , immediately adjacent to Phe 427 (37). There may also be a contribution from cation-interactions between positively charged residues of the tract and phenyl rings of the clamp. This contribution is not determinative, however, because the homoheptameric F427L pore functions reasonably well (23). The blockage of current by LF N suggests that the N-terminal tract threads at least part way through the Phe clamp even in the absence of a transmembrane proton gradient.
In the context of this model, we can understand how incorporating a basic residue into the Phe clamp might disrupt the clamp interaction with the cationic N-terminal tract of LF N , either by electrostatically repelling the tract or by interacting with side chains of Asp 425 and/or Asp 426 and perturbing the structure of the clamp (Fig. 5B). In contrast, incorporating an acidic residue can be envisioned to enhance the electrostatic interaction of the cationic N-terminal tract of LF N with the clamp. However, increased conductance, resulting from alteration of the native architecture of the pore, may preclude productive translocation (Fig. 5B).
Cytotoxicity mediated by the heteroheptamers correlated well with the kinetics of translocation across planar bilayers. All charged residues strongly inhibited translocation, with Glu and His showing the largest effects. We assume, therefore, that the effects on translocation are primarily responsible for the inhibition of cytotoxicity. As suggested by the lowered affinity of the substrate protein for the pore, inhibition of translocation by a basic residue in the Phe clamp may be due in large part to disruption of the interaction of the Phe clamp with the cationic N-terminal tract of LF N . For those polypeptides that manage to thread part way through the Phe clamp, a basic residue incorporated into the clamp could inhibit the passage of basic residues of the translocating polypeptide by electrostatic repulsion and inhibit the passage of acidic residues by electrostatic attraction. His may promote leakage of protons to a greater degree than Lys or Arg, thereby dissipating the proton gradient more effectively and causing a greater effect on toxicity.
An acidic residue in the Phe clamp may inhibit translocation by concurrently modifying the luminal architecture at or near the constriction point and by tightly binding the basic N-terminal region of LF N . For polypeptides that thread through the clamp, passage is inhibited by electrostatic repulsion or attraction with acidic and basic residues, respectively. The especially strong effect of Glu over Asp may result from stronger distortion of the Phe clamp constriction. In the final analysis, the effects of replacing a single Phe 427 residue in the pore with a charged residue are complex and appear to result both from perturbation of the seal against ion (proton) conductance formed by the Phe clamp and from electrostatic interactions of the introduced acidic or basic side chain with charged side chains the translocating polypeptide.