Toxin-antitoxin modules are small regulatory circuits that ensure survival of bacterial populations under challenging environmental conditions. The ccd toxin-antitoxin module on the F plasmid codes for the toxin CcdB and its antitoxin CcdA. CcdB poisons gyrase while CcdA actively dissociates CcdB:gyrase complexes in a process called rejuvenation. The CcdA:CcdB ratio modulates autorepression of the ccd operon. The mechanisms behind both rejuvenation and regulation of expression are poorly understood. We show that CcdA binds consecutively to two partially overlapping sites on CcdB, which differ in affinity by six orders of magnitude. The first, picomolar affinity interaction triggers a conformational change in CcdB that initiates the dissociation of CcdB:gyrase complexes by an allosteric segmental binding mechanism. The second, micromolar affinity binding event regulates expression of the ccd operon. Both functions of CcdA, rejuvenation and autoregulation, are mechanistically intertwined and depend crucially on the intrinsically disordered nature of the CcdA C-terminal domain.

Toxin–antitoxin (TA) modules are small operons involved in bacterial stress response and persistence. higBA operons form a family of TA modules with an inverted gene organization and a toxin belonging to the RelE/ParE superfamily. Here, we present the crystal structures of chromosomally encoded Vibrio cholerae antitoxin (VcHigA2), toxin (VcHigB2) and their complex, which show significant differences in structure and mechanisms of function compared to the higBA module from plasmid Rts1, the defining member of the family. The VcHigB2 is more closely related to Escherichia coli RelE both in terms of overall structure and the organization of its active site. VcHigB2 is neutralized by VcHigA2, a modular protein with an N-terminal intrinsically disordered toxin-neutralizing segment followed by a C-terminal helix-turn-helix dimerization and DNA binding domain. VcHigA2 binds VcHigB2 with picomolar affinity, which is mainly a consequence of entropically favorable de-solvation of a large hydrophobic binding interface and enthalpically favorable folding of the N-terminal domain into an α-helix followed by a β-strand. This interaction displaces helix α3 of VcHigB2 and at the same time induces a one-residue shift in the register of β-strand β3, thereby flipping the catalytically important Arg64 out of the active site.

Intrinsically disordered proteins (IDPs) are proteins that lack a unique three-dimensional structure in their native state. Many have, however, been found to fold into a defined structure when interacting with specific binding partners. The energetic implications of such behavior have been widely discussed, yet experimental thermodynamic data is scarce. We present here a thorough thermodynamic and structural study of the binding of an IDP (antitoxin CcdA) to its molecular target (gyrase poison CcdB). We show that the binding-coupled folding of CcdA is driven by a combination of specific intramolecular interactions that favor the final folded structure and a less specific set of intermolecular contacts that provide a desolvation entropy boost. The folded structure of the bound IDP appears to be defined largely by its own amino acid sequence, with the binding partner functioning more as a facilitator than a mold to conform to. On the other hand, specific intermolecular interactions do increase the binding affinity up to the picomolar range. Overall, this study shows how an IDP can achieve very strong and structurally well-defined binding and it provides significant insight into the molecular forces that enable such binding properties.

The Staphylococcus aureus genome contains three toxin–antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE.

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Escherichia coli O157 paaR2-paaA2-parE2 constitutes a unique threecomponent toxin-antitoxin (TA) module encoding a toxin (ParE2) related to the classic parDE family but with an unrelated antitoxin called PaaA2. The complex between PaaA2 and ParE2 was purified and characterized by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. It consists of a particle with a radius of gyration of 3.95 nm and is likely to form a heterododecamer. Crystals of the ParE2-PaaA2 complex diffract to 3.8 Å resolution and belong to space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 142.9, c = 87.5 Å . The asymmetric unit is consistent with a particle of around 125 kDa, which is compatible with the solution data. Therefore, the ParE2-PaaA2 complex is the largest toxin-antitoxin complex identified to date and its quaternary arrangement is likely to be of biological significance.

CcdB Vfi from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdB Vfi is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdB Vfi possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdB Vfi shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications. Toxin-antitoxin (TA)6 modules are a class of operons that are abundant on the chromosomes of bacteria and archaea (1-3). They were originally discovered on low copy number plasmids and bacteriophages (4), where they are believed to act as addiction systems. The role of chromosome-encoded TA modules, however, is heavily debated. A whole series of functions has been proposed (5) ranging from DNA parasites over stabilization of chromosomal regions to regulators of metabolism response, mediators of persister cell formation, and altruistic suicide modules. Although each potential function has its drawbacks, regulation of metabolism during periods of nutritional stress is the most widely accepted hypothesis. This results from the observations that TA modules are activated during stress response (6), that this activation at least under some circumstances is reversible (7), and that after induction metabolic activity within the cells can still be observed (8). The latter property has been exploited to design a single protein production system in Escherichia coli (8). Recent data suggest that at least one family of TA modules may be under quorum sensing control (9) and that a lone MazF TA toxin mediates programmed cell death during multicellular fruiting body development of Myxococcus xanthus (10).The F plasmid ccd operon is the oldest known TA module, encoding the toxin CcdB F and the antitoxin CcdA F . It was originally discovered as an operon that couples plasmid replication to cell division. Later it was shown that its action can be described as a plasmid addiction system that relies on the difference in lifetime of CcdA F and CcdB F . As long as the F plasmid is present in the cell, both proteins are present in low amounts. They form a complex, preventing CcdB F to exert its toxic action. This complex acts as a repressor for the ccd operon (11). CcdA F is under constant degradation by the Lon protease (12), but when the CcdA F pool becomes too small, this activates transcription from the ccd operon, leading to the establishment of a dynamic equilibrium.This ...

SummaryToxin-antitoxin (TA) modules are small operons associated with stress response of bacteria. F-plasmid CcdBF was the first TA toxin for which its target, gyrase, was identified. Plasmidic and chromosomal CcdBs belong to distinct families. Conserved residues crucial for gyrase poisoning activity of plasmidic CcdBs are not conserved among these families. Here we show that the chromosomal CcdBVfi from Vibrio fischeri is an active gyrase poison that interacts with its target via an alternative energetic mechanism. Changes in the GyrA14-binding surface of the Vibrio and F-plasmid CcdB family members illustrate neutral drift where alternative interactions can be used to achieve the same functionality. Differences in affinity between V. fischeri and F-plasmid CcdB for gyrase and their corresponding CcdA antitoxin possibly reflect distinct roles for TA modules located on plasmids and chromosomes.