A schematic illustration of two putative mechanisms for PA63 channel-mediated LF and EF transport into the cytoplasm. One model suggests that LF and EF thread through the pore. 19,20 The results shown here suggest that anthrax toxin complexes (i.e., LF or EF bound to the PA63 channel) rupture membranes. A previous study demonstrated that LF in the complex is enzymatically active. 25
Interaction of animal-harvested anthrax toxin with artificial lipid bilayer membranes. (a) The I–V relationship of anthrax toxin was initially strongly rectifying (•). The degree of rectification decreased with increasing time after sample addition (10 min (□), and 90 min (▵). Inset: schematic illustration of LF bound to a PA63 channel in an artificial lipid bilayer membrane. (b) Ionic current time series recordings and video micrographs (inset) at V = 180 mV demonstrate that anthrax toxin harvested from infected rabbits causes the membrane to become unstable and eventually rupture (†). The grey rods in the micrographs are Ag/AgCl electrodes, and the solutions contained 100 mM KCl, 5 mM MES at pH 7.2. Capsule material was present in the isolated fractions of anthrax toxin.
The effect of pH gradients on the PA63 channel instantaneous I–V relationship in the absence and presence of LF (a)–(d). The cis and trans compartments were buffered at either pH 7.2 or 5.5, as indicated in each panel. The LF concentration in the cis chamber was either zero (□) or 1 nM (•). Normalized current for the LF-free (e) and 1 nM LF data (f) highlights relative changes due to pH. Colorization indicates the pH condition as used in (a)–(d), normalized at 10 mV and 120 mV, respectively. The error bars, which represent the standard deviation (n ≥ 7), are generally smaller than the symbols.
Time course of the PA63 channel conductance when (a) LF was removed before or (b) maintained [LF] = 1 nM during cis-side acidification. (Top) The conductance equivalent of ≈60 PA63 channels was reconstituted into a planar bilayer membrane at pHc|t 7.2|7.2 (black). Then, 1 nM LF was added to the cis chamber (blue). (Middle) The pHc|t 5.5|7.2 gradient was formed by perfusing the cis chamber with pH 5.5 buffer that contained either [LF] = 0 (red) or [LF] = 1 nM (green). (Bottom) The neutral pH condition (pHc|t 7.2|7.2) (black) was restored by perfusing the cis chamber with pH 7.2 buffer. If LF was present, the cis chamber was first purfused with pH 5.5 buffer (red) then pH 7.2 buffer (black). The ionic current was monitored for ≈20 min after each perfusion and ∼60 min after the final perfusion. The applied potential was V = +50 mV. Breaks in the current recordings correspond to ≈30 s pulses at V = −50 mV. The periodic noise corresponds to magnetic stirring during perfusion. (c) Instantaneous I–V measurements and the current rectification ratio (inset) taken at the beginning ( ) and end ( ,•) of the ionic current series confirm that the complex became essentially irreversibly bound only when maintained [LF] = 1 nM during cis-side acidification. Similar results were obtained with EF (not shown).
Rupture events of planar membranes caused by complexes of either essentially irreversibly bound recombinant (a) LF:PA63 channel and (b) EF:PA63 channel. Membrane rupture was also achieved by exposing the channel to the cis addition of the polycations (c) 28 kg/mol poly-L-lysine, and (d) 56 kg/mol poly(allylamine hydrocholoride). Chirality and cis/trans addition did not inhibit the membrane rupture capability (not shown). The membranes rupture events are denoted by (†). The solutions on either side of the membrane contained 100 mM KCl and 5 mM MES at pH 7.2. Membrane rupture with irreversibly bound recombinant proteins was also achievable at pHc|t 5.5|7.2 (not shown). The applied potential was |V| = 120 mV for recombinant proteins or |V| = 50 mV for the polycations.
Hypothetical pH-induced changes to the putative binding pocket for the PA63 channel and LF. (Left) A top-down view for part of the theoretical model of the PA63 channel 22 and the crystal structure of LF 76 oriented to illustrate the proposed binding site. The colored regions correspond to the subunits shown in the right panel. (Right) A “folded-open” representation of the LF:PA63 channel binding pocket that includes a PA63 dimer and LF. The space-fill region emphasizes the residues at the binding site. The electrostatic potentials were computed at (a) pH 7.2, (b) pH 5.5, and (c) their difference. Negative and positive electrostatic surface potentials are denoted by red and blue, respectively.
Protein topology of the disordered N-terminus of EF/LF and the lumen of the PA63 channel β-barrel. (Top) Extended chain representation of the first 30 disordered amino acids (right to left) from the N-terminus of LF. (Middle) The lumen of the β-barrel from a model of its structure. (Bottom) Extended chain representation of the first 30 disordered amino acids (right to left) from the N-terminus of EF. PyMol was used to generate the topological representation based on sequences of EF, 124 LF, 76 and whole model of (PA63)7. 22 Residues are colored by the following classifications: Marine – His, Purple – Basic, Yellow – Acidic, Grey – Hydrophobic. Blue and Red denote N and O moieties, respectively. The bar represents the membrane spanning region of the β-barrel.
The pH dependence of the LF:PA63 channel binding constant as estimated from the channel conductance at V = +70 mV. The symbols correspond to the pH conditions described in Fig. 3 (pHc|t 7.2|7.2 □; pHc|t 5.5|7.2 ○). The solid lines are the least-squares best fits of a simple binding equation for 1:1 stoichiometry (i.e., 1/(1 + [LF]/K d ), where K d is the reaction dissociation constant. (Inset) The pH dependence of K d . The pH in the trans chamber was 7.2. The dashed line is to guide the eye. The error bars represent the standard deviation from n = 3–5 membranes.
Calculated pKa values of residue side chains in the PA63 channel lumen.
Article metrics loading...
Full text loading...