Assembly dynamics and stability of the pneumococcal epsilon zeta antitoxin toxin (PezAT) system from Streptococcus pneumoniae

Abstract

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.

FIGURE 1.

In vivo and semi-in vivo stability of PezAT. A, degradation pattern of PezAT after PezAT expression and subsequent translation inhibition using chloramphenicol. E. coli DH5α cells bearing pASK5+(pezAT) (black) were induced with 0.2 μg/ml of anhydrotetracycline at an A₆₀₀ of ∼ 0.5 (blue arrow). After 1 h chloramphenicol was added leading to inhibition of protein expression (red arrow). The growth profiles of a control culture bearing pASK5+(pezA) demonstrated that growth inhibition was caused by chloramphenicol only are shown in light gray. Samples with equivalent amounts of cells were analyzed using SDS-gel electrophoresis to monitor PezA and PezT degradation (lower panel). B, growth profiles subsequent to removal of anhydrotetracycline after 1 h of expression of PezAT. E. coli DH5α cells bearing either pASK5+(pezAT) (black) and pASK5+(pezA) (gray) were induced with 0.2 μg/ml at an A₆₀₀ of ∼0.5. Fresh LB medium was inoculated to a final A₆₀₀ of ∼0.1 and cell growth was monitored. Cell cultures in which PezAT expression was abolished after removal of inducing agent show similar growth profiles the control. C, degradation of 1 μm of the antitoxin His-F-PezA in B. subtilis raw extract. His-F-PezA was either monitored by ECL Western blot analysis using anti-His₅ (left) or imaging of IAEDANS fluorescence (right). D, degradation of 1 μm of the toxin PezT(D66T) in B. subtilis raw extract monitored by Western blot analysis as in C. E, degradation of 1 μm His-F-PezA·PezT(D66T) complex in B. subtilis raw extract similar as in C.

FIGURE 2.

Urea-induced denaturation experiments. A, unfolding of 5 μm PezA in the absence (white circles) and presence (gray circles) of 5 μm PezT(D66T,W232Y) monitored by the red shift of the spectral center of mass of tryptophan fluorescence. B, PezA denaturation (5 μm) monitored either by tryptophan fluorescence (white circles) or the mean residue ellipticity at 222 nm (white triangles). C, denaturation of PezA (5 μm) in the presence of equimolar concentrations of PezT(D66T,W232Y) monitored by tryptophan fluorescence (gray circles) and denaturation of the complex by mean residue ellipticity at 222 nm (gray triangles). D, denaturation of 5 μm PezT(D66T,W232Y) alone (white diamonds) and in the presence of 5 μm PezA (gray diamonds) monitored by mean residue ellipticity at 222 nm. Note that individual values have been normalized in panel D.

FIGURE 3.

Tryptophan fluorescence experiments of PezA and PezT upon complex formation. A, fluorescence titrations of 3 μm PezT(D66T) with wild type PezA (dark gray circles) or tryptophan-free PezA(W111F) (light gray circles) or buffer (white circles). Excitation was set to 295 nm and emission recorded at 347 nm. B, ribbon representation of the PezAT heterotetramer of PezA (red) and PezT (blue). Wild type tryptophan residues Trp-232 of PezT (orange) and Trp-111 (green) are highlighted as stick models. Whereas Trp-232 enables monitoring binding of PezA, Trp-111 of PezA was shown to be sensitive to changes in the homodimer interface of the antitoxin. C, exemplary real time trace of binding of 1 μm PezT(D66T) to 1 μm PezA(W111F) (gray) measured in a stopped-flow device. Tryptophan fluorescence of PezT(D66T) was excited at 296 nm and emission recorded with a 320-nm long-pass filter. The corresponding fit according to the two-step model is shown as black line. D, similar as C but 5 μm PezT(D66T) and 5 μm PezA(W111F). E, similar as C but 3 μm PezT(D66T) and 1.5 μm PezA(W111F). Note the difference in scaling of the time axis.

FIGURE 4.

Relaxation kinetics of the PezA homodimerization measured by tryptophan fluorescence. A, exemplary time trace of a rapid 1:15 buffer dilution at 10 μm PezA (gray) and the monoexponential fit describing a one-step dimerization mechanism. B, exemplary relaxation time traces of the PezAT heterodimer/heterotetramer equilibrium measured by rapid 1:15 buffer dilution at 10 μm PezA·PezT(D66T,W232Y) complex (gray) and the corresponding fit (black). Protein fluorescence was excited at 280 nm and emission recorded with a 320-nm long pass filter.

FIGURE 5.

PezA and PezT binding is accelerated by electrostatic interactions. A, open book representation of the PezA and PezT binding interface showing complementary charged surface potentials at moderate ionic strength. Charge distributions have been calculated using the PyMOL APBPS, PDB code 2PQR at 0.2 m NaCl. B, dependence of the apparent rate constants λ₁ (dark gray) and λ₂ (light gray) on the ionic strength. The black line corresponds to the fit of λ₁ using Equation 1 with a fixed value of 6 Å for A. Best fit values were −9.5 for U/kT and 1 × 10⁶ m⁻¹ s⁻¹ for k(∞).

FIGURE 6.

Dissociation kinetics of the PezAT complex measured by exchange kinetics. 0.3 μm preassembled PezA(W111F)/PezT(D66T)_Alexa488 was incubated with 3 μm unlabeled PezT(D66T). A, the decrease of fluorescent PezAT species and increase of free, labeled PezT was monitored by analytical gel filtration. B, data after peak integration and normalization were fit in parallel with a monoexponential, yielding an apparent dissociation constant of 6.5 × 10⁻⁶ s⁻¹.

FIGURE 7.

Model of the PezAT assembly dynamics. The onset of PezAT heterotetramer formation is driven by rapid association of PezT with either PezA monomers or dimers. This initial association step is based on long range electrostatic interactions resulting in a transient PezAT complex in which both proteins are still separated by ∼7 Å. A subsequent rapid rearrangement eventually leads to the final stereospecific inhibitory PezAT complex, which is predominantly a heterotetramer due to the nanomolar affinity of the central PezA homodimer interface.