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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.


Purification and imaging of the (PA63)7–(LF)3 complex. (A) Superose 6 gel filtration shows a peak for the (PA63)7 heptamer that shifts to higher mobility (larger size) when incubated with increasing ratios of LF. At a (PA63)7/LF molar ratio of 1:3, saturation is somewhat <100% complete, owing to the low concentrations used. At 1:4 and 1:5 ratios, saturation is complete, and the major peak is consistent with a molecular ratio of 3:7 (LF/PA63) (the peaks in the low molecular weight region comprise excess uncomplexed LF). (B) Coomassie-stained SDS-PAGE gel of purified (PA63)7–(LF)3 used for vitreous ice cryo-EM. Densitometry confirms a PA63/LF ratio of 7:3. Molecular mass is indicated in kilodaltons. (C and D) Vitreous ice cryo-EM images of (PA63)7–(LF)3 complexes collected without (C) or with (D) pre-applying a layer of carbon to the EM grid in order to have the complexes adopting multiple orientations. (E and F) (PA63)7–(LF)3 complexes shown in two characteristic orientations, side view (E) and top view (F). The first column shows reprojection of the (PA63)7–(LF)3 reconstruction; second column, the corresponding 2-D class averages corresponding to the same orientation; third through seventh columns, examples of individual complexes (PA63)7–(LF)3 in the corresponding orientations. For viewing purposes, the contrast is inverted in the projections and averages; the raw images were low pass filtered to 40 Å.
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fig1: Purification and imaging of the (PA63)7–(LF)3 complex. (A) Superose 6 gel filtration shows a peak for the (PA63)7 heptamer that shifts to higher mobility (larger size) when incubated with increasing ratios of LF. At a (PA63)7/LF molar ratio of 1:3, saturation is somewhat <100% complete, owing to the low concentrations used. At 1:4 and 1:5 ratios, saturation is complete, and the major peak is consistent with a molecular ratio of 3:7 (LF/PA63) (the peaks in the low molecular weight region comprise excess uncomplexed LF). (B) Coomassie-stained SDS-PAGE gel of purified (PA63)7–(LF)3 used for vitreous ice cryo-EM. Densitometry confirms a PA63/LF ratio of 7:3. Molecular mass is indicated in kilodaltons. (C and D) Vitreous ice cryo-EM images of (PA63)7–(LF)3 complexes collected without (C) or with (D) pre-applying a layer of carbon to the EM grid in order to have the complexes adopting multiple orientations. (E and F) (PA63)7–(LF)3 complexes shown in two characteristic orientations, side view (E) and top view (F). The first column shows reprojection of the (PA63)7–(LF)3 reconstruction; second column, the corresponding 2-D class averages corresponding to the same orientation; third through seventh columns, examples of individual complexes (PA63)7–(LF)3 in the corresponding orientations. For viewing purposes, the contrast is inverted in the projections and averages; the raw images were low pass filtered to 40 Å.

Mentions: PA83 was expressed in Escherichia coli as a His-tagged protein and purified as previously described (Santelli et al., 2004). PA63 was generated by trypsin cleavage (nicking) of pure PA83 in vitro (1:2,500 molar ratio), and the nicked PA was loaded onto a monoQ ion exchange column (GE Healthcare) at pH 8.6 and eluted with a gradient of 0–0.6 M NaCl, which removes PA20 and leads to the spontaneous formation of (PA63)7 prepores, which are water soluble at high pH. LF was purified as previously described (Park and Leppla, 2000). To confirm the binding stoichiometry of 7:3, (PA63)7 prepores were incubated with increasing ratios of LF and run on a Superose 6 size exclusion/gel filtration column (GE Healthcare). For EM experiments, (PA63)7 (500 µl at 230 nM) was incubated with a twofold (LF/(PA63)7) excess of LF (25 µl at 10 µM), and the complex was separated on a Superose 6 column (Fig. 1 A). Peak fractions were collected, and the stoichiometry was confirmed by SDS-PAGE analysis and densitometry of Coomassie-stained bands (Fig. 1 B).


Structure of anthrax lethal toxin prepore complex suggests a pathway for efficient cell entry
Purification and imaging of the (PA63)7–(LF)3 complex. (A) Superose 6 gel filtration shows a peak for the (PA63)7 heptamer that shifts to higher mobility (larger size) when incubated with increasing ratios of LF. At a (PA63)7/LF molar ratio of 1:3, saturation is somewhat <100% complete, owing to the low concentrations used. At 1:4 and 1:5 ratios, saturation is complete, and the major peak is consistent with a molecular ratio of 3:7 (LF/PA63) (the peaks in the low molecular weight region comprise excess uncomplexed LF). (B) Coomassie-stained SDS-PAGE gel of purified (PA63)7–(LF)3 used for vitreous ice cryo-EM. Densitometry confirms a PA63/LF ratio of 7:3. Molecular mass is indicated in kilodaltons. (C and D) Vitreous ice cryo-EM images of (PA63)7–(LF)3 complexes collected without (C) or with (D) pre-applying a layer of carbon to the EM grid in order to have the complexes adopting multiple orientations. (E and F) (PA63)7–(LF)3 complexes shown in two characteristic orientations, side view (E) and top view (F). The first column shows reprojection of the (PA63)7–(LF)3 reconstruction; second column, the corresponding 2-D class averages corresponding to the same orientation; third through seventh columns, examples of individual complexes (PA63)7–(LF)3 in the corresponding orientations. For viewing purposes, the contrast is inverted in the projections and averages; the raw images were low pass filtered to 40 Å.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037343&req=5

fig1: Purification and imaging of the (PA63)7–(LF)3 complex. (A) Superose 6 gel filtration shows a peak for the (PA63)7 heptamer that shifts to higher mobility (larger size) when incubated with increasing ratios of LF. At a (PA63)7/LF molar ratio of 1:3, saturation is somewhat <100% complete, owing to the low concentrations used. At 1:4 and 1:5 ratios, saturation is complete, and the major peak is consistent with a molecular ratio of 3:7 (LF/PA63) (the peaks in the low molecular weight region comprise excess uncomplexed LF). (B) Coomassie-stained SDS-PAGE gel of purified (PA63)7–(LF)3 used for vitreous ice cryo-EM. Densitometry confirms a PA63/LF ratio of 7:3. Molecular mass is indicated in kilodaltons. (C and D) Vitreous ice cryo-EM images of (PA63)7–(LF)3 complexes collected without (C) or with (D) pre-applying a layer of carbon to the EM grid in order to have the complexes adopting multiple orientations. (E and F) (PA63)7–(LF)3 complexes shown in two characteristic orientations, side view (E) and top view (F). The first column shows reprojection of the (PA63)7–(LF)3 reconstruction; second column, the corresponding 2-D class averages corresponding to the same orientation; third through seventh columns, examples of individual complexes (PA63)7–(LF)3 in the corresponding orientations. For viewing purposes, the contrast is inverted in the projections and averages; the raw images were low pass filtered to 40 Å.
Mentions: PA83 was expressed in Escherichia coli as a His-tagged protein and purified as previously described (Santelli et al., 2004). PA63 was generated by trypsin cleavage (nicking) of pure PA83 in vitro (1:2,500 molar ratio), and the nicked PA was loaded onto a monoQ ion exchange column (GE Healthcare) at pH 8.6 and eluted with a gradient of 0–0.6 M NaCl, which removes PA20 and leads to the spontaneous formation of (PA63)7 prepores, which are water soluble at high pH. LF was purified as previously described (Park and Leppla, 2000). To confirm the binding stoichiometry of 7:3, (PA63)7 prepores were incubated with increasing ratios of LF and run on a Superose 6 size exclusion/gel filtration column (GE Healthcare). For EM experiments, (PA63)7 (500 µl at 230 nM) was incubated with a twofold (LF/(PA63)7) excess of LF (25 µl at 10 µM), and the complex was separated on a Superose 6 column (Fig. 1 A). Peak fractions were collected, and the stoichiometry was confirmed by SDS-PAGE analysis and densitometry of Coomassie-stained bands (Fig. 1 B).

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.