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The ɛ2ζ2 Antitoxin/Toxin System

Danny Iwamoto '10 and Basil Kahwash '10


I. Introduction

The low-copy-number pSM19035 plasmid, isolated from the pathogenic bacterium Streptococcus pyogenes, contains genes for erythromycin resistance, as well as the ω-ε-ζ (omega-epsilon-zeta) operon in its SegB region, which is responsible for encoding the ε-ζ antitoxin/toxin system {Reference 3}. This functions as a postsegregational killing (PSK) system that induces programmed cell death (PCD) if the pSM19035 plasmid is ever unsuccessfully copied into each daughter cell during cell division. This process is illustrated in this pop-up diagram .  This ensures the plasmid is inherited and maintained through subsequent generations of the prokaryote, otherwise resulting in cell death {2}.  This is also known as a plasmid addiction system (PAS), since any newly born cells  lacking the plasmid will die {3).

The gene encodes for the unstable antitoxin ε and the stable toxin ζ proteins.  Proteases are thought to be responsible for the eventual breakdown of each protein, though the ζ toxin has been shown to significantly outlast the ε antitoxin in the cytoplasm {1}. A stable heterotetramer is formed by the binding of two toxin proteins and two antitoxin proteins, forming a harmless and inactive complex in the cell cytosol.  By continuously and excessively producing antitoxin, the system's toxic effects are counteracted as long as the plasmid is maintained.  Loss of the plasmid, damage of the antitoxin gene, or unsuccessful copying into a new daughter cell results in decreasing amounts of antitoxin and the liberation of toxin proteins in the cytoplasm which then become active and inhibit cell growth, cause filamentation, and eventually induce PCD {2,3}.

II. General Structure

The complete inactivation complex is a heterotetramer composed of 754 amino acids. The complex consists of two ζ proteins and two ε proteins , which form a dumbbell-shaped structure so that the two ζ proteins are bound to opposite sides of the ε2 dimer {2}.

The ε protein contains 90 amino acids , which form three alpha helices , interacting as a coiled coil and protecting the majority of the non-polar residues by internalizing them within the protein through hydrophilic reactions. Two ε proteins form the ε2 dimer , which further protects non-polar residues by sandwiching them between the ε proteins {2}.

The ζ protein contains 287 amino acids , arranged into 11 alpha helices and 6 beta sheets . The beta sheets are centrally located within the ζ protein, and all are parallel, except for the antiparallel final sheet . The two C-terminal helices are not strongly bound to the main body of the protein, forming an exposed helix-loop-helix appendage , though it does not seem to serve a function in the toxicity of the protein.  The ζ protein also contains a phosphate-binding  loop (or P-loop), an important region for the toxin's function ... {2}

III. The Functions of the Zeta and Epsilon Proteins

The ζ protein is the toxic protein of the pair, although the exact toxic mechanism of the protein is unknown. The toxin, however, does contain a Walker ATP/GTP binding motif  , also known as a P-Loop, indicating that the ζ protein exhibits similar folding patterns to phosphotransferases, suggesting a similar function. However, the substrate for this binding site is unknown, though ATP is involved as the phosphoryl group donor. Thus, the ζ protein probably acts as a phosphotransferase to remove a single phosphoryl group from ATP, and bind it to the substrate; this is the ζ protein's toxic activity {2}.

The ε protein, as the antitoxin protein, therefore blocks this binding site sterically by forming the inactivation complex heterotetramer {2}. 

Studies have shown in vivo and in vitro stability for the ζ protein as over 60 minutes, but the ε protein is stable for less than 18 minutes {1}. Therefore, the ε  antitoxin must be produced often in order to counteract the ζ toxin. If, during cell replication, the pSM19035 plasmid is not carried by a daughter cell, proteases will degrade the existing toxin and antitoxin proteins. However, the antitoxin proteins will be degraded over three times as quickly, resulting in a stoichiometric excess of toxin proteins, which then begin to induce PCD {1,2}.

IV. The Zeta Activity Sites

The ζ protein contains two binding sites: one for binding ATP , and the other for binding the unknown substrate . These sites are close in proximity, since one of ATP’s phosphoryl groups must be transferred to the substrate . ATP and the substrate and held in place by a number of stabilizing reactions involving the residues of the ζ protein {2}.

ATP, once it enters the ζ protein, is held in place by the P-loop: residues 40-47 of the ζ protein . Lys-46 in particular of the P-loop plays a major role by forming hydrogen bonds to one oxygen atom each of ATP’s β- and γ-phosphates. In addition, other nearby residues such as Arg-158 and Arg-171 bend towards ATP to stabilize it, also by forming hydrogen bonds to ATP’s phosphoryl groups {2}. 

Once ATP is bound, the ζ protein is opened into a conformation so that the substrate can bind. Since the substrate’s molecular structure is unknown, it is unclear as to what particular residues play important roles in stabilizing the substrate. However, assuming no dramatic conformation changes occur upon substrate binding, the substrate must be a small molecule, or a solvent exposed segment of a protein or nucleic acid {2}.  The substrate is likely to be held near the ATP binding site, between a beta sheet and two alpha helices of the ζ protein , a region also filled with numerous water molecules which aid in ATP hydrolysis. The Mg2+ ion is an important atom also involved in ATP-hydrolysis, though this atom could not be identified in electron density mapping of the complex {2}. It is assumed that Glu-116  binds this catalytically important ion, which neutralizes the developing negative charge during ATP hydrolysis. Meanwhile, Asp-67 deprotonates the substrate, which allows the substrate to perform a nucleophilic attack on the γ-phosphate of ATP.  Lys-46, Arg-158 and Arg-171, of the ATP binding region, help stabilize the resultant pentacovalent transition state of the γ-phosphate through hydrogen bonding to the β- and γ-phosphates.  Collapse of the pentacovalent transition state yields the phosphorylated substrate and ADP, completing the phosphotransfer {2}.

V. Inactivation Complex

The stable inactivation complex heterotetramer is formed when two toxic ζ proteins bind to the ε2 dimer . The fact that ε is unstable means that ε will almost always be found in the ε2ζ2 complex {2}. Bonding between ε and ζ occurs in such a way that ATP is prevented from binding to ζ, also effectively preventing the substrate from binding to ζ. Most toxin-antitoxin interactions involve a single helix of ε {2} . Residues on the N-terminal of this helix make contacts with the residues of the ζ protein . For example, the carboxylate groups of Glu-12 and Glu-16 of ε form hydrogen bonds to the amino group of Arg-158 of ζ, pulling it away from where it is needed in the ATP binding site.  Other residues on ε serve to sterically impede the binding of ATP to ζ, such as  Tyr-5 and Phe-9 , which block the ATP binding site of ζ (the P-loop, Lys-46, Arg-158, and Arg-171) {2}. As a result, the ζ protein is inactivated through steric hindrance by the ε upon formation of the stable inactivation complex heterotetramer . This final button shows the complete inactivated heterotetramer, and the key amino acid residues involved. 

VI. References

1. Camacho, A. G., R Misselwitz, J. Behlke, S. Ayora, K. Welfle, A. Meinhart, B. Lara, W. Saenger, H. Welfle, and J.C. Alonso. 2002. In vitro and in vivo stability of the epsilon2zeta2 protein complex of the borad host-range Streptococcus pyogenes pSM19035 addiction system. Biol. Chem. 383:1701-1713.

2. Meinhart, Anton, Juan C. Alonso, Norbert Sträter, and Wolfram Saenger. 2003. Crystal structure of the plasmid maintenance system ε/ζ: Functional mechanism of toxin ζ and inactivation by ε2ζ2 complex formation. PNAS 100:1661-1666.

3. Zielenkiewicz, Urszula and Piotr Cegłowski. 2005. The Toxin-Antitoxin System of the Streptococcal Plasmid pSM19035. Journal of Bacteriology 187:6094-6105.

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