Structural and Functional Analysis of the Kid Toxin Protein from E. coli Plasmid R1

Hargreaves, David; Santos-Sierra, Sandra; Giraldo, Rafael; Sabariegos-Jareño, Rosario; Cueva-Méndez, Guillermo de la; Boelens, Rolf; Dı́az-Orejas, Ramón; Rafferty, John B.

doi:10.1016/s0969-2126(02)00856-0

Cited by 78 publications

(96 citation statements)

References 34 publications

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“…The overall octamer topology of the Rv0301-Rv0300 complex is similar to the N. gonorrhoeae FitAB, 9 S. flexneri VapBC, 30 and R. 4 hexamer when unbound to DNA based on size exclusion chromatography and multiangle light scattering. 26 However, structures of FitAB, S. flexneri VapBC, and the Rv0301-Rv0300 complex have been observed only as octamers bound or unbound to DNA, so these most likely exist as stable octamers in vivo.…”

Section: The Functional Unit Of Rv0301 Toxin Is a Homodimermentioning

confidence: 85%

“…2 The first toxin structure to be determined was the E. coli F plasmid CcdB toxin. 3 The structures of many toxins, antitoxins, and TA complexes have been determined since, including E. coli plasmid R1 Kid toxin, 4 E. coli MazE-MazF TA complex, 5 broad-hostrange low-copy-number plasmid pSM19035 from Streptococcus pyogenes e 2 f 2 TA complex, 6 E. coli YoeB toxin and YefM2-YoeB TA complex, 7 archaeal Pyrococcus horikoshii aRelE-aRelB TA complex, 8 Neisseria gonorrhoeae FitAB TA complex, 9 solution structure of the broad host range low-copy-number plasmid pRK2/RP4 ParD antitoxin, 10 Mycobacterium tuberculosis (Mtb)…”

Section: Introductionmentioning

confidence: 99%

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The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

et al. 2012

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VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg 21 ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg 21 ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

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Section: The Functional Unit Of Rv0301 Toxin Is a Homodimermentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

et al. 2012

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“…The crystal structure of Kid (PemK) protein has been determined as a homodimer (11,45). Although the structure of MazF has not been determined, Kamada et al (24) have reported the crystal structure of the MazE-MazF complex (MazF 2 -MazE 2 -MazF 2 ), which was formed by two MazF homodimers and one MazE homodimer.…”

Section: Discussionmentioning

confidence: 99%

Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

Zhang

Zhu

et al. 2004

Journal of Biological Chemistry

122

149

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In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5 or 3 side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.In Escherichia coli, programmed cell death is proposed to be mediated through the system called "addiction module," which consists of a pair of genes encoding a toxin and an antitoxin (1). The addiction module has the following properties. (a) The toxic protein is stable, whereas the antitoxin is a labile protein. (b) The toxin and the antitoxin are coexpressed from an operon and interact with each other to form a stable complex. (c) Their expression is auto-regulated either by the toxin-antitoxin complex or by the antitoxin alone. When the co-expression is inhibited under stress conditions, the antitoxin is degraded by proteases, enabling the toxin to act on its target. In E. coli, some extrachromosomal elements are known to contain addiction modules causing the bacterial programmed cell death by the so-called postsegregational killing effect. The most studied extrachromosomal addiction modules are the phd-doc system on bacteriophage P1 (2-5), the ccdA-ccdB system on factor F (6 -9), the kis-kid system on plasmid R1 (10 -13), and the pemIpemK system on plasmid R100 (14 -17). Interestingly, the E. coli chromosome also contains several addiction module systems, such as the relBE system (18 -21), the mazEF system (22-25), and the chpB system (26 -28).The cellular effects of the toxins in the addiction modules have been studied quite extensively. CcdB, the toxin in the ccdA-ccdB system, interacts with DNA gyrase to block DNA replication (7,29), and RelE, the toxin in the relBE system, is not able to degrade free RNA but cleaves mRNA in the ribosome A site with high codon specificity (21). Recently, it was demonstrated that the A-site mRNA cleavage can occur in the absence of RelE (30). The exact mechanism of the A-site mRNA cleavage is still unknown. It has been proposed that MazF (ChpAK)...

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“…Although CcdB and MazF proteins are structurally related (44,45), their activities are divergent; CcdB inhibits gyrase through binding to its A subunit, although MazF is a ribonuclease. Still key features of how the antitoxin regulates activity of the toxin are paralleled.…”

Section: Structural Similarities Suggest That the Functional Divergenmentioning

confidence: 99%

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

Zorzini

Mernik

Lah

et al. 2016

Journal of Biological Chemistry

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Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78 -Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

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Structural and Functional Analysis of the Kid Toxin Protein from E. coli Plasmid R1

Cited by 78 publications

References 34 publications

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

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Structural and Functional Analysis of the Kid Toxin Protein from E. coli Plasmid R1

Cited by 78 publications

References 34 publications

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

Interference of mRNA Function by Sequence-specific Endoribonuclease PemK

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

Contact Info

Product

Resources

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The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site