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. 2004 Nov 30;101(48):16756-61.
doi: 10.1073/pnas.0405754101. Epub 2004 Nov 17.

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

Sen Zhang  1 , Alan Finkelstein, R John Collier
Affiliations

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

Sen Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Entry of the enzymatic components of anthrax toxin [lethal factor (LF) and edema factor] into the cytosol of mammalian cells depends on the ability of the activated protective antigen (PA63) component to form a channel (pore) in the membrane of an acidic intracellular compartment. To investigate the mechanism of translocation, we characterized N-terminally truncated forms of the PA63-binding domain of LF (LFN). Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LFN across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA63 channels in planar lipid bilayers at a small positive voltage (+20 mV). Fusing a H6-tag to the N terminus of the truncated proteins restored both translocation and channel-blocking activities. At +20 mV, N-terminal H6 and biotin tags were accessible to Ni2+ and streptavidin, respectively, added to the trans compartment of a planar bilayer. On the basis of these findings, we propose that the N terminus of PA63-bound LF or edema factor enters the PA63-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction.

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Figures

Fig. 1.
Fig. 1.
N-terminal sequence of LFN and deletion mutants. Shown are the sequences of the first 40 residues of wild-type (WT) LFN and that of each of the deletion mutants tested. Acidic residues are underlined; basic residues are in boldface type. Additional sequences present at the N terminus of various constructs, including the 21-residue H6-containing tag, are described in Materials and Methods.
Fig. 2.
Fig. 2.
The effects of various N-terminal deletions on the PA63-binding and translocation activities of LFN. H6-tagged constructs (dark gray bars) and non-H6-tagged constructs (light gray bars) were tested basically as described in ref. . (A) Translocation across the plasma membrane in response to low pH. (B) PA63-dependent binding of LFN to CHO-K1 cells. CHO-K1 cells were incubated with trypsin-nicked PA (2.4 × 10–8 M) for 2 h on ice, and the cells then were washed and incubated with various 35S-LFN truncations for another 2 h. The cells were treated with buffer (pH 5.0) at 37°C for 1 min to trigger translocation and then with Pronase to digest 35S-LFN remaining at the cell surface. Cell-associated 35S-LFN was determined by scintillation counting as a measure of translocation. PA63-dependent binding was measured in identical samples in which Pronase was omitted. The fraction translocated was defined as the amount of protected 35S-LFN divided by the amount bound. (Error bars, SEM.) The results shown in A and B are the average of three independent experiments. (C) LFN-induced oligomerization of PA63 in solution. Trypsin-activated PA (3 μg) was incubated with 1 μg of each LFN truncation mutant at room temperature for 1 h, and the samples then were electrophoresed on a 4–12% polyacrylamide gel. Y236A-LFN was included as a mutant form known to be deficient in PA binding.
Fig. 3.
Fig. 3.
Effects of N-terminal deletions on the ability of LFN to inhibit PA63-dependent conductance in planar bilayers. Purified (PA63)7 (2 ng) was added to the cis compartment, containing 1 ml of buffer (100 mM KCl/25 mM succinate/1 mM EDTA, pH 5.5). (A and B) The cis compartment was held at +20 mV, and after channel formation stabilized, we added full-length LFN (A) or a truncated form, LFN Δ27 (B), to the cis compartment to give a final concentration of 20 nM. The traces in A and B are representative of 12 or 11 different trials, respectively. (C) Various concentrations of full-length or truncated forms of LFN were added to the cis compartment, and the percent current decrease was determined after 1 min. DTA, used as a negative control, did not interact with the channel. The curves in C are the average of three independent experiments, except for DTA, which was tested twice.
Fig. 4.
Fig. 4.
Effect of H6-tag on the channel-blocking activity of LFN truncations. Conditions were as in Fig. 3. (A) After channel formation stabilized, LFN Δ27 with a H6-tag at either its N or C terminus was added to the cis compartment as indicated. The trace is representative of three independent experiments, all with identical results. (B) The percentage decrease in current 1 min after LFN addition was plotted as a function of final LFN concentration. The curves are averaged from three independent experiments.
Fig. 5.
Fig. 5.
Illustration of the movement of N terminus of LFN into the PA63 channel under the influence of a transmembrane voltage of +20 mV.
Fig. 6.
Fig. 6.
Evidence that the N terminus of LFN enters the channel formed by (PA63)7.(A and B) LFN (0.6 μg) containing a biotin group disulfide-linked either to the N (A) or the C(B) terminus was incubated with 6 μg of streptavidin at room temperature for 10 min before being added to the cis compartment. The membrane-impermeant disulfide reducing agent Tris-carboxyethylphosphine (TCEP) (5 mM) was added as indicated. The voltage was held at +20 mV throughout the experiment. (C) Trans addition of Ni2+ slowed unblocking of N-terminal H6-tagged LFN. EDTA was omitted from the buffer. We added 500 μMNi2+ and 1 mM EDTA to the trans compartment at indicated time points. Although some of the channels became immediately unblocked at –20 mV, others took longer to unblock. This delay may be a consequence of the distribution of LFN penetration of channels at a given voltage. (D) Use of biotin-LFN-DTA to demonstrate penetration of the N terminus to the trans compartment. The buffer used was the same as in Fig. 3, except that 1 mM adenine was present. Eleven minutes before starting the recording, 2 ng of (PA63)7 was added to the cis compartment held at +20 mV. Then, biotin-LFN-DTA (4 nM) was added to the same compartment. At the break, the cis compartment was perfused with 10 ml of buffer, and 10 μg of streptavidin was added to the trans compartment, as indicated. The voltage was shifted to –20 mV, and TCEP (5 mM) was added to the trans compartment, as indicated. The small response at –20 mV before TCEP addition may reflect a small amount of protein not binding streptavidin. The traces shown in A, B, and C were representative of three, four, and four independent trials, respectively, all of which were essentially identical. The trace in D is representative of six trials, all but one of which were successful.
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