Crystal structure of the DNA-bound VapBC2 antitoxin/toxin pair from Rickettsia felis

Mate, M.J.; Vincentelli, Renaud; Foos, Nicolas; Raoult, Didier; Cambillau, Christian; Ortiz-Lombardı́a, M.

doi:10.1093/nar/gkr1167

Cited by 52 publications

(70 citation statements)

References 60 publications

(85 reference statements)

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“…This type of gene regulation has recently also been demonstrated for vapBC TA modules, in which the toxin-antitoxin complex displays a distinct molecular architecture (Winther & Gerdes, 2012). Unlike the CcdAB and Phd-Doc TA systems, the proteins encoded by vapBC modules seem to assemble into a unique globular heterooctameric complex, as has been shown for VapBC from Shigella flexneri and Rickettsia felis (Dienemann et al, 2011;Maté et al, 2012). In both cases, these complexes have been shown to aptly bind their promoter region to regulate expression of the operon.…”

Section: Resultsmentioning

confidence: 64%

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

et al. 2012

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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.

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Section: Resultsmentioning

confidence: 64%

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

et al. 2012

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“…The existing crystal structures of VapBC pairs show that the C-terminal portions of the VapB antitoxins wrap around and bind in grooves on the surface of their VapC partners (33,(35)(36)(37)(38). Secondary structure prediction indicates the presence of two ␣ helices (␣2 and ␣3) in the C terminus of VapB4 at positions which match the positions of the two helices that grip VapC5 in the VapBC5 structure (37) (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…However, the fact that inclusion of the N-terminal domain suppresses the defect caused by some of the mutations in the C-terminal domain suggests that features in the DNAbinding module might function to stabilize the interaction between the toxin and antitoxin. Indeed, crystal structures of VapBC pairs indicate that dimerization of VapB toxin-antitoxin pairs involves interactions between the DNA-binding modules of the antitoxins (35)(36)(37)(38). These contacts certainly contribute to the formation of heteromeric complexes seen in the crystal structures and may help stabilize the interaction of some of the VapB4 mutants with VapC4.…”

Section: Discussionmentioning

confidence: 99%

“…The analysis of the crystal structure of the VapBC TA complex from Shigella flexneri suggests that 4 aromatic residues in the C-terminal domain of VapB (Trp47, Trp50, Phe51, and Phe60) contact the hydrophobic core of VapC, and 2 residues (Arg64 and Gln66) interact with the conserved negatively charged amino acid residues of the PIN domain (33). Similarly, the crystal structures of VapBC complexes from M. tuberculosis, Neisseria gonorrhoeae, and Rickettsia felis suggest that multiple contacts govern the interactions between the VapB antitoxins and their cognate VapC toxins (34)(35)(36)(37)(38). These structures raise the question of how many protein-protein contacts are required for stable VapBC interaction and whether binding is likely to be sensitive to smallmolecule protein-protein interaction inhibitors.…”

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confidence: 99%

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Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

2015

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T oxin-antitoxin (TA) systems were first identified to be addictive genetic elements responsible for the maintenance of plasmid stability during cell segregation (1, 2). Subsequently, chromosomally encoded TA systems were characterized to be involved in several mechanisms of toxin action (3, 4). TA systems typically consist of two elements, a toxin protein that suppresses cell growth by targeting essential cellular metabolic pathways and an antitoxin RNA or protein that binds to and blocks the growth inhibition activity of the toxin. Different types of TA systems have been defined according to the nature and modes of action of antitoxins, and of these, the type II TA systems are the most abundant and well studied (5, 6). The type II toxins-antitoxins are typically encoded in the same operon, where the gene for the antitoxin is located upstream of that for the toxin, except in the case of higBA, which has a reversed gene order (7). The type II antitoxin is a protein that directly interacts with its cognate toxin, resulting in neutralization of toxicity. The antitoxin or the toxin-antitoxin protein complex often acts as a repressor that autoregulates the transcription of TA operons via its ability to bind to the promoter (8). During cellular stress, the antitoxin is hydrolyzed by a protease, such as Lon or Clp, to release the toxin from the protein complex and derepress transcription of the TA operon (7). These properties allow TA systems to play regulatory roles in bacterial gene expression and the establishment and maintenance of dormancy. This nonreplicative state may contribute to latency.Type II TA systems are classified into several different families on the basis of sequence similarities and the functions of the toxins (7-9). CcdB and ParE inhibit DNA replication by targeting DNA gyrase (10, 11). Doc arrests translation elongation by phosphorylation of elongation factor Tu (12, 13). Kis and HicA cleave mRNA in a ribosome-independent manner (14, 15). MazF targets various RNAs, including mRNA, 16S rRNA,. RelE inhibits translation by cleavage of ribosome-bound mRNAs at the ribosomal A site (20). HipA inhibits translation by phosphoryla-

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“…Characterizing TA interactions allows for a better understanding of how the antitoxin controls toxin activity and bacterial persistence. Crystal structures of VapBC complexes from S. flexneri (61% identity, 76% similarity to NTHi VapC2), M. tuberculosis (23% identity, 51% similarity), Neisseria gonorrhoeae (27% identity, 47% similarity), and Rickettsia felis (43% identity, 65% similarity) suggest the toxin-binding domain of VapB antitoxins bind in a cleft containing the VapC active site, including the conserved amino acid residues of the PIN domain, through multiple amino acid contacts (7,(37)(38)(39)(40)(41). Similarly, previous work in M. tuberculosis demonstrated that two amino acid mutations in the VapB4 antitoxin were required to disrupt the toxin-antitoxin interaction (42).…”

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confidence: 99%

Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

Walling

Butler

2016

J Bacteriol

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Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCEToxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae, as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.T oxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Originally discovered as addictive elements involved in postsegregational killing during cell division, they have since been identified as playing a critical role in responding to stress (1-3). TA systems are typically encoded by a single operon that produces a stable protein toxin and its cognate labile antitoxin, with the gene encoding the antitoxin located upstream of the toxin in most cases (4). Under normal growth conditions, the antitoxin, which may be a protein (types II, IV, and V) or an RNA...

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Crystal structure of the DNA-bound VapBC2 antitoxin/toxin pair from Rickettsia felis

Cited by 52 publications

References 60 publications

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

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