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Structure of anthrax lethal toxin prepore complex suggests a pathway for efficient cell entry

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ABSTRACT

Anthrax toxin is a tripartite complex in which the protective antigen moiety forms a pore through which lethal factor and edema factor are translocated. Fabre et al. reveal a mechanism for efficient translocation in their structure of the heptameric protective antigen prepore bound to three lethal factors.

No MeSH data available.


Related in: MedlinePlus

Electrophysiological measurement of translocation of LF through a single pore. A horizontal lipid bilayer system was used, which separates compartments with different pH values; pH 5.6 for the upper (cis) chamber and pH 6.6 for the lower (trans) chamber as described previously (Groulx et al., 2010; see Materials and methods). (A) Translocation of LF through PA63. To test function of the proteins and verify translocation of LF through PA63, PA63 was added to the cis chamber, and once a macroscopic current at 80 mV was observed, the voltage was switched to 20 mV and LF or LFN was added (arrow). At 20 mV, LF will block the pore without being translocated. Blockage of the pore was evident by reduced current. Switching the membrane potential back to 80 mV enabled translocation of LF/LFN through the pore, and once complete, the current increased again. (B) Translocation times for analysis were determined from single-channel recordings; example traces of single translocation events are shown for LFN (top) and LF (middle and bottom). The dashed lines mark the translocation event, and the arrows indicate short (<1 s) spontaneous closings that were also observed in the absence of LF/LFN and were thus ignored in the analysis. The dotted lines indicate the blocked PA. (C) The duration of single pore closings (translocations) at various concentrations of full-length LF is shown in a cloud plot. Each point represents a single measurement such as shown in B. Pore closure times at high concentrations were statistically different to the lower concentrations of LF (P < 0.05). (D) Pore closure times for LFN showed no concentration dependence, and the data were pooled. Closure times for LFN were statistically different from those of full-length LF (P < 0.005). Statistical significance was tested with one-way ANOVA.
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fig6: Electrophysiological measurement of translocation of LF through a single pore. A horizontal lipid bilayer system was used, which separates compartments with different pH values; pH 5.6 for the upper (cis) chamber and pH 6.6 for the lower (trans) chamber as described previously (Groulx et al., 2010; see Materials and methods). (A) Translocation of LF through PA63. To test function of the proteins and verify translocation of LF through PA63, PA63 was added to the cis chamber, and once a macroscopic current at 80 mV was observed, the voltage was switched to 20 mV and LF or LFN was added (arrow). At 20 mV, LF will block the pore without being translocated. Blockage of the pore was evident by reduced current. Switching the membrane potential back to 80 mV enabled translocation of LF/LFN through the pore, and once complete, the current increased again. (B) Translocation times for analysis were determined from single-channel recordings; example traces of single translocation events are shown for LFN (top) and LF (middle and bottom). The dashed lines mark the translocation event, and the arrows indicate short (<1 s) spontaneous closings that were also observed in the absence of LF/LFN and were thus ignored in the analysis. The dotted lines indicate the blocked PA. (C) The duration of single pore closings (translocations) at various concentrations of full-length LF is shown in a cloud plot. Each point represents a single measurement such as shown in B. Pore closure times at high concentrations were statistically different to the lower concentrations of LF (P < 0.05). (D) Pore closure times for LFN showed no concentration dependence, and the data were pooled. Closure times for LFN were statistically different from those of full-length LF (P < 0.005). Statistical significance was tested with one-way ANOVA.

Mentions: We were intrigued by the LFC–LFN contacts and how they might affect translocation rates and efficiency. It has been reported that the efficiency of LF translocation is independent of the degree of LF loading onto the prepore (Zhang et al., 2004). However, quantitative measures of translocation times have only been reported for LFN (Wynia-Smith et al., 2012). We therefore used an in vitro system to quantify and compare LF and LFN translocation. In lipid bilayers, PA pores form ion-conductive (cation-selective) channels that are blocked when LF is translocating through the lumen and open again once translocation is complete. We conducted electrophysiological experiments in which a single PA63 heptamer was inserted into a synthetic lipid bilayer that separated cis and trans compartments buffered to pH values of 5.6 and 6.6, respectively. LF or LFN in solution at nanomolar concentration was then introduced into the cis compartment, and translocation was controlled by manipulating the potential difference across the membrane. With a single channel, channel blockage time served as a simple surrogate for translocation time (Fig. 6). At this protein concentration range, translocation is not limited by the diffusion rate from bulk solution to the pore, and reopening of the pore could still be observed. Under these conditions, we found that full-length LF took three-times longer to translocate than LFN (with a mean of 7.5 s vs. 2.5 s for LF vs. LFN; Fig. 6, B–D). Given that the LF sequence is approximately three-times longer than LFN, this suggests that both the N-terminal domain, LFN, and the full-length LF molecule translocate at a similar rate (per residue) under these conditions.


Structure of anthrax lethal toxin prepore complex suggests a pathway for efficient cell entry
Electrophysiological measurement of translocation of LF through a single pore. A horizontal lipid bilayer system was used, which separates compartments with different pH values; pH 5.6 for the upper (cis) chamber and pH 6.6 for the lower (trans) chamber as described previously (Groulx et al., 2010; see Materials and methods). (A) Translocation of LF through PA63. To test function of the proteins and verify translocation of LF through PA63, PA63 was added to the cis chamber, and once a macroscopic current at 80 mV was observed, the voltage was switched to 20 mV and LF or LFN was added (arrow). At 20 mV, LF will block the pore without being translocated. Blockage of the pore was evident by reduced current. Switching the membrane potential back to 80 mV enabled translocation of LF/LFN through the pore, and once complete, the current increased again. (B) Translocation times for analysis were determined from single-channel recordings; example traces of single translocation events are shown for LFN (top) and LF (middle and bottom). The dashed lines mark the translocation event, and the arrows indicate short (<1 s) spontaneous closings that were also observed in the absence of LF/LFN and were thus ignored in the analysis. The dotted lines indicate the blocked PA. (C) The duration of single pore closings (translocations) at various concentrations of full-length LF is shown in a cloud plot. Each point represents a single measurement such as shown in B. Pore closure times at high concentrations were statistically different to the lower concentrations of LF (P < 0.05). (D) Pore closure times for LFN showed no concentration dependence, and the data were pooled. Closure times for LFN were statistically different from those of full-length LF (P < 0.005). Statistical significance was tested with one-way ANOVA.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037343&req=5

fig6: Electrophysiological measurement of translocation of LF through a single pore. A horizontal lipid bilayer system was used, which separates compartments with different pH values; pH 5.6 for the upper (cis) chamber and pH 6.6 for the lower (trans) chamber as described previously (Groulx et al., 2010; see Materials and methods). (A) Translocation of LF through PA63. To test function of the proteins and verify translocation of LF through PA63, PA63 was added to the cis chamber, and once a macroscopic current at 80 mV was observed, the voltage was switched to 20 mV and LF or LFN was added (arrow). At 20 mV, LF will block the pore without being translocated. Blockage of the pore was evident by reduced current. Switching the membrane potential back to 80 mV enabled translocation of LF/LFN through the pore, and once complete, the current increased again. (B) Translocation times for analysis were determined from single-channel recordings; example traces of single translocation events are shown for LFN (top) and LF (middle and bottom). The dashed lines mark the translocation event, and the arrows indicate short (<1 s) spontaneous closings that were also observed in the absence of LF/LFN and were thus ignored in the analysis. The dotted lines indicate the blocked PA. (C) The duration of single pore closings (translocations) at various concentrations of full-length LF is shown in a cloud plot. Each point represents a single measurement such as shown in B. Pore closure times at high concentrations were statistically different to the lower concentrations of LF (P < 0.05). (D) Pore closure times for LFN showed no concentration dependence, and the data were pooled. Closure times for LFN were statistically different from those of full-length LF (P < 0.005). Statistical significance was tested with one-way ANOVA.
Mentions: We were intrigued by the LFC–LFN contacts and how they might affect translocation rates and efficiency. It has been reported that the efficiency of LF translocation is independent of the degree of LF loading onto the prepore (Zhang et al., 2004). However, quantitative measures of translocation times have only been reported for LFN (Wynia-Smith et al., 2012). We therefore used an in vitro system to quantify and compare LF and LFN translocation. In lipid bilayers, PA pores form ion-conductive (cation-selective) channels that are blocked when LF is translocating through the lumen and open again once translocation is complete. We conducted electrophysiological experiments in which a single PA63 heptamer was inserted into a synthetic lipid bilayer that separated cis and trans compartments buffered to pH values of 5.6 and 6.6, respectively. LF or LFN in solution at nanomolar concentration was then introduced into the cis compartment, and translocation was controlled by manipulating the potential difference across the membrane. With a single channel, channel blockage time served as a simple surrogate for translocation time (Fig. 6). At this protein concentration range, translocation is not limited by the diffusion rate from bulk solution to the pore, and reopening of the pore could still be observed. Under these conditions, we found that full-length LF took three-times longer to translocate than LFN (with a mean of 7.5 s vs. 2.5 s for LF vs. LFN; Fig. 6, B–D). Given that the LF sequence is approximately three-times longer than LFN, this suggests that both the N-terminal domain, LFN, and the full-length LF molecule translocate at a similar rate (per residue) under these conditions.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Anthrax toxin is a tripartite complex in which the protective antigen moiety forms a pore through which lethal factor and edema factor are translocated. Fabre et al. reveal a mechanism for efficient translocation in their structure of the heptameric protective antigen prepore bound to three lethal factors.

No MeSH data available.


Related in: MedlinePlus