The protective antigen component of anthrax toxin forms functional octameric complexes

Abstract

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy-the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-A crystal structure of the PA octamer. The octamer comprises approximately 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.

Fig. 1. Heterogeneous PA channel conductance distributions

(A) Two PA samples were analyzed: (i) PA is nicked by trypsin to make _nPA; a 20-kDa piece (PA₂₀) dissociates, allowing PA to oligomerize into the pre-channel on a Q-sepharose column, making _QPA; and (ii) _nPA is mixed with LF_N to drive oligomerization, making _nPA+LF_N. Either pre-channel oligomer forms a channel upon inserting into the membrane. (B) Example of 200-Hz-filtered, single-channel data collected at a Δψ of 20 mV, 100 mM KCl, pH 6.60; γ values computed by γ = i/Δψ are listed next to each channel insertion. (C) Normalized histograms of the estimated single-channel γ values for the _QPA and _nPA+LF_N samples. Data bins are one-pS wide, and the number of channels, n, in each sample are normalized for comparison. The samples, _QPA (n = 360; black bars) and _nPA+LF_N (n = 107; red bars), are statistically distinct by a non-parametric, lower-tailed, Whitney-Mann test (p > 0.95). (D) Histogram of all the pairwise differences, δ, between measured γ values identified within the same membrane for the _nPA+LF_N sample. The δ histogram was fit to one- (dotted line) and two-Gaussian (solid line) functions, using A(δ) = A₁/σ₁√(π/2) exp(−2δ²/σ₁²) + A₂/σ₂√(π/2) [exp(−2((δ – μ₂)/σ₁)²) + exp(−2((δ + μ₂)/σ₁)²)], with R values of 0.89 and 0.96, respectively, yielding best-fit parameters: peak area A₁ = 470 (±80), A₂ = 140 (±40); mean, μ₂ = 8 (±2) pS; and standard deviation, σ₁ = 5.5 (±0.5) pS, σ₂ = 9 (±2) pS. (See also Table S2.) (E) Single-channel current records for smaller- (black) and larger-sized (red) PA channels in 100 mM KCl, pH 6.6. Arrows indicate the two respective channel sizes. Data were acquired at 400 Hz and filtered further with a 100-point-per-σ Gaussian filter to better reveal conductance sub-states.

Fig. 2. EM studies of heptameric and octameric PA

EM images of negative-stained samples of WT PA oligomers assembled either in vitro (upper panels A-F) or in vivo on cell surfaces (lower panel). Representative class averages of octamers (left) and heptamers (right) are shown. The total number of particles assessed, n, and relative percentages of heptamers and octamers are given. The proportions of heptamers and octamers are indicated by bars colored black and red, respectively. In vitro samples include: (A) _nPA assembled on an anion-exchange column (_QPA; n = 12589; 98% heptamer; 2% octamer); (B) _nPA assembled in the presence of soluble dimeric ATR2 at a 4:1 stoichiometry (+dsATR; n = 837; 74% heptamer; 26% octamer); (C) _nPA assembled in the presence of soluble monomeric ATR2 (+msATR; n = 9401; 99% heptamer; 1% octamer); (D) _nPA assembled in the presence of LF_N; (+LF_N; n = 8409; 72% heptamer; 28% octamer); (E) _nPA assembled in the presence of EF_N; (+EF_N; n = 5363; 81% heptamer; 19% octamer); and (F) disulfide-bonded PA S170C assembled on an anion-exchange column (_QPA S170C; n = 2933; 78% heptamer; 22% octamer). Oligomers extracted from cells: (G) His6-PA assembled on cells expressing ATR2 (C-CHO; n = 4729; 74% heptamer; 26% octamer), where three classes of octamers and heptamers are shown, resulting from reference-based analysis. The 5-nm scale bar shown in panel A is consistent for all images. Percentages of oligomers are means of reference-free and crystal-structure-referenced alignments unless noted otherwise. (Specific percentages are listed in Table S2.)

Fig. 3. Mass spectrometry studies of Atx assembly

(A) NanoESI mass spectrum of _nPA co-assembled with LF_N. Multiple charge-state distributions are observed that correspond to five different PA-LF_N complexes. Charge states and molecular weights were calculated according to a prior method. The PA₇(LF_N)₃ distribution clearly has the highest relative abundance and is shown above labeled with charge states. Insets show distributions of less intense oligomers PA₇(LF_N)₂ and PA₈(LF_N)₄, and low abundances of PA₂LF_N and PA₄(LF_N)₂ that may be stable intermediates in the formation of the higher order complexes. The molecular weight was assigned from the charge-state distribution that resulted in the smallest standard deviation in calculated molecular weight. (Table S1 summarizes the observed masses and describes the solvent correction.) (B) Assembly kinetics for the 63-kDa PA monomer, PA₇(LF_N)₃, PA₈(LF_N)₄, and PA₄(LF_N)₂ from a solution of _nPA mixed with excess LF_N. Data for all but PA₄(LF_N)₂ were fit with exponential functions to guide the eye. The appearance of the oligomers, PA₇(LF_N)₃ and PA₈(LF_N)₄, coincides with the disappearance of PA monomer. Data at early times indicate a rapid increase in the abundance of PA₄(LF_N)₂ followed by slow decay for t ≥ 5 min., suggesting it is an intermediate in the formation of the higher order complexes; an interpolated line is given for PA₄(LF_N)₂ also to guide the eye. All four analytes reach steady-state levels in ∼30 min.

Fig. 4. X-ray crystal structure of PA in the octameric oligomerization state

Axial views of (A) the PA^ΔMIL octamer (PDB 3HVD) side-by-side with (B) the WT PA heptamer (PDB 1TZO; ref. 7). Monomer subunit chains are colored uniquely. The MIL is depicted with spheres in the latter structure of WT heptamer. (C) A backbone alignment of two adjacent PA monomers, chains A and B, called conformation A (red) and B (blue), showing the displacement of D4. (D) Superimposed on a surface rendering of half an octamer is the square planar arrangement of symmetrically related A and B conformers calculated from the positions of each chain's center of mass. Chains A, C, E, G are conformation A; and chains B, D, F and H are conformation B. Adjacent A-B pair center of masses are 64.3 (± 0.1) Å apart at angles of 90 (±0.1)°. Domains are colored as: D1' (magenta), D2 (green), D3 (gold), and D4 (blue). (E) An A-B oligomerization interface split apart to compare relative differences in surface area burial at the oligomerization interface of the heptamer and octamer structure among the four domains. The domains (upper panel) are colored as in panel D. The relative degrees of surface area buried (lower panel) are colored as follows: surface buried equally (i.e., to within 10%) in either structure (green); surface buried 10% more buried in the heptamer (red); surface buried 10% more in the octamer (blue); and surface buried <75% in both structures (white). All molecular graphics were rendered using CHIMERA.

Fig. 5. Octameric and heptameric PA stability and activity

Negative-stained EM class-average images of PA heptamers and octamers before and after a reduction of pH and/or change in the temperature and solvent. The initial condition in panel A and B is pH 8, 0 °C. (A) Heptameric and octameric PA^ΔMIL before (left) and after (right) exposure to pH 5.7, 7% ethanol, 4 °C. Before exposure, 25% octamer, 75% heptamer, n = 10409. After exposure, 89% octamer, 11% heptamer, n = 14516. (B) Heptameric and octameric WT PA oligomer complexes with LF_N before (left) and after (right) exposure to pH 7, 37 °C. Before exposure, 28% octamer, 73% heptamer, n = 8409. After exposure, 91% octamer, 9% heptamer, n = 1084. Relative percentages are given by the bars on the right for heptamers (black) and octamers (red). (C) Ensemble protein translocation records measured using planar lipid bilayer electrophysiology. Panels compare the relative translocase activities two types of samples: _QPA+LF_N, which is >95% heptameric (black); and _nPA+ LF_N, which had been purified and shown to contain ∼90% octamer (red). Four substrates (LF, LF_N, EF, or EF_N) were used, and each was translocated at the indicated Δψ and ΔpH conditions. Records shown are the average of a set of three repetitions. The y-axes are normalized to the fraction of substrate-blocked channels that become unblocked due to translocation. Rates are given as t½ (time for half of the substrate to translocate). LF translocated at Δψ = 30 mV, ΔpH = 1 using heptameric (t½ = 57 s) and octameric PA (t½ = 96 s). LF_N translocated at Δψ = 50 mV using heptameric (t½ = 16 s) and octameric PA (t½ = 12 s). EF translocated at Δψ = 50 mV, ΔpH = 1 using heptameric (t½ = 50 s) and octameric PA (t½ = 66 s). EF_N translocated at Δψ = 60 mV using heptameric (t½ = 137 s) and octameric PA (t½ = 135 s). (D) (Right) Single-channel translocations of LF_N at 50 mV through either a large- (red) or small-sized (black) channel. Black arrowheads on either end of the translocation indicate the beginning and end of each translocation. (Left) Histogram profiles of portions of the conductance levels of the large- and small-sized channels for comparison of the open-channel conductance levels.

Fig. 6. Heterogeneous assembly mechanism may modulate toxin activity

(A) On cells, PA may encounter dimeric ATR sites and assemble into PA₂ and PA₄ intermediates. Intermediates can combine to form either PA₈ or PA₇, which can load with EF and/or LF. (In principle, LF and EF may be involved in the mechanism as well to produce similar outcomes.) During extracellular or ATR-independent assembly, PA may encounter LF or EF, making the intermediates, PA₂LF_N and PA₄(LF_N)₂, which then form either PA₈(LF_N)₄ or PA₇(LF_N)₃. Models of LF-PA complexes are derived from a theoretical model. Toxin activity is a combination of the oligomer's stability and translocase activity; instability may lead to the formation of inactive oligomeric complexes (*).