Structure and Function of Bacterial Kid-Kis and Related Toxin-Antitoxin Systems

Kamphuis, Monique B.; Monti, Maria Chiara; Heuvel, Robert H. H. van den; López-Villarejo, Juan; Dı́az-Orejas, Ramón; Boelens, Rolf

doi:10.2174/092986607779816096

Cited by 31 publications

(28 citation statements)

References 90 publications

(179 reference statements)

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“…It was found that the most active TA interaction disruption peptides (III and V), having a predicted helical conformation and ability to bind to Site 2, showed a slight inhibition of the ribonuclease activity of the PemK. This was expected because the antitoxin-binding site of the structurally similar Kid partly overlaps with the RNA-binding site at Site 2 (43). However, the inhibition of ribonuclease activity with these peptides could only be attained at a 60-fold higher concentration as required for the disruption of TA interaction.…”

Section: Discussionmentioning

confidence: 96%

PemK Toxin of Bacillus anthracis Is a Ribonuclease

Agarwal

Mishra

Bhatnagar

et al. 2010

Journal of Biological Chemistry

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Bacillus anthracis genome harbors a toxin-antitoxin (TA) module encoding pemI (antitoxin) and pemK (toxin). This study describes the rPemK as a potent ribonuclease with a preference for pyrimidines (C/U), which is consistent with our previous study that demonstrated it as a translational attenuator. The in silico structural modeling of the PemK in conjunction with the site-directed mutagenesis confirmed the role of His-59 and Glu-78 as an acid-base couple in mediating the ribonuclease activity. The rPemK is shown to form a complex with the rPemI, which is in line with its function as a TA module. This rPemIrPemK complex becomes catalytically inactive when both the proteins interact in a molar stoichiometry of 1. The rPemI displays vulnerability to proteolysis but attains conformational stability only upon rPemK interaction. The pemI-pemK transcript is shown to be up-regulated upon stress induction with a concomitant increase in the amount of PemK and a decline in the PemI levels, establishing the role of these modules in stress. The artificial perturbation of TA interaction could unleash the toxin, executing bacterial cell death. Toward this end, synthetic peptides are designed to disrupt the TA interaction. The peptides are shown to be effective in abrogating TA interaction in micromolar range in vitro. This approach can be harnessed as a potential antibacterial strategy against anthrax in the future. Toxin-antitoxin (TA)3 modules in prokaryotes consist of two adjacent open reading frames encoding a stable toxin and a cognate labile antitoxin (1, 2). The vulnerability of antitoxin to proteolysis demands a constant de novo synthesis of antitoxin to maintain a steady-state level that can sequester the toxin and circumvent its deleterious activity (3). A negative feedback homeostatic loop also exists, wherein antitoxin acts as a transcriptional repressor in conjunction with the toxin as a co-repressor (4). A multitude of functions have been ascribed to the chromosome-encoded TA loci, primarily as stress managers (5-7). It has been envisaged that these modules respond to intracellular stress facilitating phenotypic switching of the bacterial cell to a quasidormant stage amenable to survival under extreme conditions (8 -12). However, a recent study demonstrates that TA loci do not impart selective advantage to the cells grown in stress, indicating that these genes can innocuously be deleted from the genome (13).Several families of two component TA loci have been identified on bacterial chromosomes, namely relBE, higBA, mazEF, ccdAB, vapBC, parDE, phd-doc, and yoeB-yefM. The toxins of these families share sequence and structural similarity to some extent, but the corresponding antidotes do not, thus indicating their distinct evolutionary origin (2). However, despite their structural similarity and common ancestral origin, their downstream targets are quite different. CcdB and ParE function as gyrase poison, whereas MazF, Kid, YdcE, and YoeB are potent ribonucleases (14 -22). VapC toxins represent a structurally dist...

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Section: Discussionmentioning

confidence: 96%

PemK Toxin of Bacillus anthracis Is a Ribonuclease

Agarwal

Mishra

Bhatnagar

et al. 2010

Journal of Biological Chemistry

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“…Antitoxins are thought to be dimeric and bind DNA via an N-or C-terminal dimerization domain folding as an RHH, helix-turn-helix (HTH), PhD-like-, or AbrB-like domain (37). The antitoxin dimer binds one, two, or four toxin monomers (33,36,38). HicB3 assembles as a dimer of dimers.…”

Section: Discussionmentioning

confidence: 99%

Functional and Structural Analysis of HicA3-HicB3, a Novel Toxin-Antitoxin System of Yersinia pestis

et al. 2014

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The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis. Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNAbinding motif.

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“…The complex interacts discontinuously with the DNA, with alternating stretches of occupation and absence (10). The tertiary structures of numerous antitoxin proteins have been elucidated recently, either as part of a complex with the cognate toxin and/or in the unbound form or in association with operator site DNA (19,20,22,24,28,31,32,35,36,43,53). These structures have revealed significant diversity in the folds that antitoxins use to bind to their regulatory sites.…”

Section: Discussionmentioning

confidence: 99%

“…Toxin-antitoxin (TA) loci are widely distributed among eubacteria and archaea, with many species possessing tens of TA cassettes that can be grouped into distinct evolutionary families (14,16,17,22,44). The most prevalent TA complexes comprise two small proteins, a stable toxin and a labile antitoxin, that associate tightly so that the toxin remains inert.…”

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confidence: 99%

Influence of Operator Site Geometry on Transcriptional Control by the YefM-YoeB Toxin-Antitoxin Complex

Bailey

Hayes

2009

J Bacteriol

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YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5 of yefM-yoeB in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The yefM-yoeB operator site arrangement is exemplified in Escherichia coli: a pair of palindromes with core hexamer motifs and a centerto-center distance of 12 bp overlap the yefM-yoeB promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.

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Structure and Function of Bacterial Kid-Kis and Related Toxin-Antitoxin Systems

Cited by 31 publications

References 90 publications

PemK Toxin of Bacillus anthracis Is a Ribonuclease

PemK Toxin of Bacillus anthracis Is a Ribonuclease

Functional and Structural Analysis of HicA3-HicB3, a Novel Toxin-Antitoxin System of Yersinia pestis

Influence of Operator Site Geometry on Transcriptional Control by the YefM-YoeB Toxin-Antitoxin Complex

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