Assembly Dynamics and Stability of the Pneumococcal Epsilon Zeta Antitoxin Toxin (PezAT) System from Streptococcus pneumoniae

Hannes Mutschler,Jochen Reinstein,Anton Meinhart

doi:10.1074/jbc.m110.126250

Hannes Mutschler, Jochen Reinstein + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.126250

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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.

Highlights

In addition to plasmid-encoded TA systems, similar modules were discovered to be encoded from chromosomes of free
The pneumococcal epsilon zeta antitoxin toxin (PezAT) system from S. pneumoniae encoded from the chromosomal pneumococcal pathogenicity island 1 (PPI1) (15) is one of the only two ⑀/␨ systems characterized to date
The results of this study indicate that PezT gets sequestered by PezA in a quasi-irreversible manner indicating that PezAT is a unique TA system

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—PezA, PezT, and all of their variants were expressed from pET28b vectors using the E. coli BL21(DE3)/Codon Plus-RIL strain (Stratagene). To obtain the fluorescently labeled His-F-PezA variant, dithioerythritol-free His6PezA(M66C) was labeled with 1,5-IAEDANS (Invitrogen) by diluting 25 ␮M His PezA(M66C) in buffer G. For the experiments probing PezT release, PezAT was expressed as described above Both cultures expressing either PezA or PezAT were harvested at an A600 of 0.8 by centrifugation (5 min at 6,000 ϫ g, 4 °C). Salt dependence of PezAT association was characterized by rapid mixing experiments of PezA and PezT (1 ␮M and 1:1 stoichiometry) at different NaCl concentrations and by fitting of the two-step model to individual time traces. The dynamics of the PezA homodimer equilibrium were addressed by rapid dilution experiments where 5 ␮M PezA was mixed with buffer D to final ratios of 1:15, 1:12, 1:10, 1:8, 1:6, 1:5, and 1:4 in the absence and presence of stoichiometric amounts of tryptophan-free PezT(D66T,W232Y) using a SFM-400 stopped-flow device (BioLogic). Elution profiles of labeled PezAT and PezT were fit to a two-component skew-normal distribution function using CalcCenter 3.0 (Wolfram Research)

RESULTS

Our experiments show that a

Best fit values were

DISCUSSION