Charge requirements for proton gradient-driven translocation of anthrax toxin

Abstract

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.

FIGURE 1.

The chemical potential of the PMF is sufficient to drive LF_N translocation. Analysis of LF_N translocations driven purely by a ΔpH. Activation free energies (ΔG‡; expressed as RTlnt½/c) for individual translocations of His₆-LF_N are plotted against their respective ΔpH values and fit to Equation 1. The fit parameters are: ΔG‡_o1 = 12.2 (± 1.1) kcal mol⁻¹, ΔG‡_o2 = 2.5 (± 0.2) kcal mol⁻¹, n₁ = 4.0 (± 0.5), and n₂ = 0.22 (± 0.03). Inset, representative LF_N translocation records normalized as a fraction of the theoretical maximum of translocation under the following ΔpH values at a Δψ of 0 mV: 0.84 (black), 1.01 (red), 1.19 (green), 1.67 (blue), and 2.30 (purple). The universal bilayer buffer was consistently at a pH_cis of 5.6, and pH_trans was adjusted to form the indicated ΔpH values. To control for buffer mixing lag times, t = 0 was set as the time when 1% of maximum translocation occurred. The error bars are the means ± S.D. (n = 2–5).

FIGURE 2.

Efficient ΔpH-driven translocation requires both acidic and basic charged residues. A, design of synthetic LF_N constructs (LF_N Syn) in which the first 27 amino acids are replaced using a sequence containing both acidic and basic residues (Syn^±), neither acidic nor basic residues (Syn°), only acidic residues (Syn⁻), and only basic residues (Syn⁺). The exact sequence compositions of these constructs are shown alongside the LF_N WT sequence. B, translocation activation energy versus ΔpH results for the His₆-LF_N Syn constructs shown in A: His₆-WT (black square), His₆-Syn° (purple circle), His₆-Syn⁻ (red triangle), His₆-Syn⁺ (blue inverted triangle), and His₆-Syn^± (green diamond). These ΔpH-driven translocation experiments were conducted at a ΔpH of −0.6 to 1.0, pH_cis of 5.6, and a Δψ of 60 mV. The error bars are the means ± S.D. (n = 2–5). The fit parameters for His₆-WT and His₆-Syn^± using Equation 1: His₆-WT, ΔG‡_o1 = 1.5 (± 0.2) kcal mol⁻¹, ΔG‡_o2 = 0.8 (± 0.3) kcal mol⁻¹, n₁ = 1.9 (±0.2), n₂ = 0.1 (± 0.2); His₆-Syn^±, ΔG‡_o1 = 1.5 (±0.2) kcal mol⁻¹, ΔG‡_o2 = 0.8 (± 0.3) kcal mol⁻¹, n₁ = 1.7 (± 0.2), n₂ = 0.1 (± 0.2). His₆-Syn°, His₆-Syn⁻, and His₆-Syn⁺ were fit to a single-barrier model (ΔG‡ = ΔG‡_o − 2.3nΔpH), with the parameters: His₆-Syn°, ΔG‡_o = 3.40 (± 0.08) kcal mol⁻¹, n = 0.59 (± 0.06); His₆-Syn⁻, ΔG‡_o = 4.3 (± 0.2) kcal mol⁻¹, n = 0.0 (± 0.1); His₆-Syn⁺, ΔG‡_o = 2.28 (± 0.09) kcal mol⁻¹, n = 0.14 (± 0.07). C, maximum translocation efficiency achieved within 5 min at varying pH_cis values for the His₆-LF_N Syn constructs. These ΔpH-driven translocation experiments were conducted at a ΔpH of 2.0, pH_cis ranging from 5.0 to 5.6, and a Δψ of 0 mV. To control for buffer mixing lag times, t = 0 was set as the time when 1% of maximum translocation occurred. The legend colors are identical to those in B. The error bars are the means ± S.D. (n = 2–8).

FIGURE 3.

Charge requirements for LF_N docking. A, ensemble bilayer recordings of PA channel conductance block by LF_N WT and LF_N Syn mutants with and without their His₆ tags were obtained at symmetrical pH 5.6 and saturating concentrations of LF_N (100 nm). The relative fraction of conductance block is given for each Syn mutant construct relative to LF_N WT. The error bars represent the means ± S.D. (n = 2). B, ensemble bilayer recordings of PA channel conductance block by LF_N WT and LF_N Syn mutants at symmetrical pH 5.6 following perfusion of the cis-side of the membrane. Perfusion removes excess LF_N, allowing the channel dissociation kinetics to be recorded. Significant rapid dissociation was only observed with LF_N Syn° and Syn⁻; all His₆-tagged LF_N and the non-His₆-tagged LF_N (including WT and mutants, Syn⁺ and Syn^±) did not dissociate during the recording. C, single-channel blocking events recorded for LF_N WT and LF_N Syn mutants with and without their His₆ tags at symmetrical pH 5.6. Once a single channel inserts into the membrane, LF_N is added under a Δψ of 20 mV. Approximately 2 min of a typical blocking event is shown for each LF_N. The data are acquired at 400 Hz under a low pass filter of 200 Hz. For clarity, the displayed single-channel traces were downsampled by a factor of 10. To the right of each trace is a histogram of the current level for each recording. Gaussian functions fit to these histograms assess the percentage of the time the LF_N-channel complexes spend in the open (o), blocked (b), and partly blocked states (*). The percentages of time in the blocked and partly blocked states are given as follows: all His₆-tagged LF_N constructs as well as untagged LF_N WT, LF_N Syn⁺ and LF_N Syn^± were 100% blocked; untagged LF_N Syn° was 28% blocked; and LF_N Syn⁻ was 1.6% blocked and 3% partly blocked (*). The errors for percentage of the time spent in the block states are all better than ±1%. The partly blocked (*) state is 29 (± 2)% less conducting than the fully open state.

FIGURE 4.

Acidic residues within the folded domain of LF_N are also critical to ΔpH-driven translocation. A, the ribbon depiction of the structure of LF_N is from PDB ID 1J7N (23), where regions of sequence are colored by sequence position. The unstructured leader presequence (residues 1–32) is indicated as a thick colored line. Regions in which acidic residues were replaced with serine residues are colored as follows: residues 1–18 (red), residues 19–24 (green), residues 25–32 (purple), residues 33–46 (blue), residues 47–56 (brown), and residues 57–64 (gold). B, the difference in activation energy ΔΔG‡ for each His₆-LF_N des⁽⁻⁾ series mutant are obtained using two ΔpH-driven conditions (ΔpH = 1.0 and Δψ = 20 mV; ΔpH = 0.6 and Δψ = 40 mV) and one condition in the absence of a ΔpH (ΔpH = 0 and Δψ = 60 mV), where ΔΔG‡ = ΔG‡(ΔpH>0) − ΔG‡(ΔpH = 0). ΔΔG‡ values for each mutant (MUT) are then referenced to that of LF_N WT to give the reported ΔΔΔG‡ values plotted above. ΔΔΔG‡ = ΔΔG‡(MUT) − ΔΔG‡(WT). In all cases, pH_cis = 5.6. The error bars are the means ± S.D. (n = 2).

FIGURE 5.

Acidic residue positions in the presequence of LF_N are most critical to ΔpH-driven translocation. Acidic residues were added back into the His₆-LF_N des⁽⁻⁾_1–46 construct background. A, the sequences of the first 46 residues of LF_N WT and LF_N des⁽⁻⁾_1–46 are shown, where acidic residues in the WT sequence are shaded red. B, the relative translocation t½ times for acidic residue introductions into the His₆-LF_N des⁽⁻⁾_1–46 mutant backgrounds are given as the ratio t½(des⁽⁻⁾_1–46)/t½(MUT) for ΔpH-driven translocation. The numbers on the x axis indicate the positions in which acidic residues are reintroduced into the His₆-LF_N des⁽⁻⁾_1–46 mutant background. ΔpH-driven translocation conditions were ΔpH = 0.8, pH_cis = 5.6, and Δψ = 50 mV. The error bars are the means ± S.D. (n = 2–4). C, a correlation of relative translocation rate (given as the ratio t½(des⁽⁻⁾_1–46)/t½(MUT)) for the mutants in B versus the density of acidic residues normally found in the WT sequence. Acidic residue density in this instance is the total number of acidic residues found in the four residues amino- and carboxyl-terminal to the probed site. The linear regression fit to all of the individual measurements (filled diamonds) is significant with a p value of 0.001 for the fit function, y = a + bx, where a = 0.5 (± 0.5) and b = 0.6 (± 0.2). Because multiple observations of particular acidic residue densities were obtained in certain instances, a heavy horizontal bar (mean) and error bars (± S.D.) are also given.

FIGURE 6.

Charged residues must be located immediately before the folded domain of the substrate for efficient translocation. A, sixteen-residue inserts with either no charge (Ins°) or a mix of positive and negative charges (Ins^±) were inserted into LF_N WT or LF_N L145A. The resulting constructs are called yIns^x, where y denotes the last WT residue prior to the insert, and the superscript x represents the charge of the insert. The arrows indicate two other positions (0 and 27) where the Ins° sequence was inserted. B, the relative translocation t½ times of insertion and LF_N L145 mutants (MUT) are given as the ratio t½(MUT)/t½(WT). ΔpH-driven translocation conditions were: ΔpH = 1.06, pH_cis = 5.6, and Δψ = 0 mV. The error bars are the means ± S.D. (n = 2). C, the relative translocation t½ times of the LF_N L145A and LF_N 27Ins° L145A mutants compared with their respective (L145) counterpart given as the ratio t½(L145)/t½(LF_N L145A). Translocation conditions are as in B. D, model energy diagrams depicting the changes in energy barriers caused by the L145A and 27Ins° mutations, wherein we interpret 27Ins° as greatly increasing the unfolding barrier. The L145A mutation reduces the unfolding barrier by the same extent in the WT and 27Ins° backgrounds. However, in the WT background, the rate becomes limited by the translocation barrier, so the relative increase in speed is not as large as that observed for the 27Ins° background.

FIGURE 7.

Proton gradient-driven translocation requires that acidic and basic residues be intermixed in the substrate. A, design of constructs that separate the acidic and basic amino acids in the first 27 residues of LF_N to opposite ends of the presequence. PosNeg has a contiguous stretch of basic residues at the amino-terminal end and a contiguous stretch of acidic residues at the carboxyl-terminal end; NegPos is the inverse sequence of PosNeg. A randomized construct was also prepared (called Mix), in which the acidic and basic residues are intermixed, but no position has the same charge as its WT counterpart. B, translocation records for His₆-LF_N WT (black), His₆-LF_N PosNeg (blue), His₆-LF_N NegPos (red), and His₆-LF_N Mix (purple). ΔpH-driven translocation conditions were: ΔpH = 1.06, pH_cis = 5.6, and Δψ = 0 mV.

FIGURE 8.

The charge state Brownian ratchet model for ΔpH-driven translocation of anthrax toxin. At the time of endosome acidification, the substrate is bound to the top of the channel, with its α1 helix in the α clamp. Upon channel conversion, the unstructured amino terminus docks in the Φ clamp. The low pH of the endosomal compartment will protonate most of the acidic residues, whereas positive charges chaperone any remaining deprotonated aspartic or glutamic acids. This ensures that the translocating polypeptide will have a net positive charge, allowing it to move freely through the cation-selective channel. Because of Brownian motion, a portion of the substrate will eventually emerge in the cytosol. There, the higher pH of this region will result in frequently deprotonated acidic residues, thereby giving the emerged portion of the polypeptide a net negative charge and capturing it on the cytosolic side of the membrane. Repeated cycles of emergence from the channel through Brownian motion and capture via deprotonation allow the remaining portion of the substrate to translocate across the membrane.