The thermodynamic stability of the proteins of the ccd plasmid addiction system

Dao‐Thi, Minh‐Hoa; Messens, Joris; Wyns, Lode; Backmann, Jan

doi:10.1006/jmbi.2000.3815

Cited by 31 publications

(38 citation statements)

References 30 publications

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“…10 shows that, according to the criterion introduced by Uversky (51,52,54) and Uversky et al (53), the addiction antitoxins in the monomeric form have a high tendency to be partially unstructured, whereas the corresponding toxins tend to be structured even as monomers. The prediction of structuring based on the mean hydrophobicity and mean net charge of the protein polypeptide chain is in good agreement with our observations and with recently reported results on addiction modules (11,47,89,90,102). Moreover, the large amount of the random-coil form found in free antitoxins in solutions (11,47,89,90,102) represents high vulnerability for their proteolytic cleavage (25,101,103).…”

Section: Discussionsupporting

confidence: 91%

“…Information of this type is rather scarce, but nevertheless, some thermodynamic characteristics common to addiction modules are available (10, 11, 47, 89 -91). For example, the measured ⌬G 0 for the MazE dimer-monomer transition is very similar to the corresponding ⌬G 0 values observed for the antitoxins CcdA (89) and ParD (91). Toxins have much higher thermodynamic stability (89).…”

Section: Discussionsupporting

confidence: 73%

“…For example, the measured ⌬G 0 for the MazE dimer-monomer transition is very similar to the corresponding ⌬G 0 values observed for the antitoxins CcdA (89) and ParD (91). Toxins have much higher thermodynamic stability (89). Moreover, it is known that the dimeric antitoxin (A 2 ) has two binding sites for the dimeric toxin (T 2 ) and vice versa, so they can form complexes of the type .…”

Section: Discussionsupporting

confidence: 72%

“…ϭ A 2i T 2j (46,48,90,92). The expression of the antitoxin and toxin can be inhibited either by low affinity binding of the antitoxin to the promoter/operator DNA (10,11,47,89,90) or by binding of the antitoxin-toxin complexes to the same part of the DNA with higher affinity (10,48,90,92). According to these observations, one may expect that the strength of the specific interactions (binding affinities) within various addiction modules would be of similar magnitude.…”

Section: Discussionmentioning

confidence: 99%

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Energetics of Structural Transitions of the Addiction Antitoxin MazE

Lah

Simic

Vesnaver

et al. 2005

Journal of Biological Chemistry

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The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions. It codes for the toxin MazF and the antitoxin MazE, which interferes with the lethal action of the toxin. To better understand the role of various conformations of MazE in bacterial life, its order-disorder transitions were monitored by differential scanning calorimetry, spectropolarimetry, and fluorimetry. The changes in spectral and thermodynamic properties accompanying MazE dimer denaturation can be described in terms of a compensating reversible process of the partial folding of the unstructured C-terminal half (high mean net charge, low mean hydrophobicity) and monomerization coupled with the partial unfolding of the structured N-terminal half (low mean net charge, high mean hydrophobicity). At pH < 4.5 and T < 50°C, the unstructured polypeptide chains of the MazE dimer fold into (pre)molten globule-like conformations that thermally stabilize the dimeric form of the protein. The simulation based on the thermodynamic and structural information on various addiction modules suggests that both the conformational adaptability of the dimeric antitoxin form (binding to the toxins and DNA) and the reversible transformation to the more flexible monomeric form are essential for the regulation of bacterial cell life and death.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 73%

Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

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Energetics of Structural Transitions of the Addiction Antitoxin MazE

Lah

Simic

Vesnaver

et al. 2005

Journal of Biological Chemistry

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“…In the far UV (250 -200 nm) the predominant contribution comes from the protein moiety. Having no substantial differences in the spectra, between 320 and 250 nm, for the free and protein-bound states of a given DNA can be interpreted as a sign of no significant structural alterations in the DNA (55). Because that is the case for the distinct RepA-DNA complexes in Fig.…”

Section: Binding To Iteron Dna Dissociates Repa Dimers Into Monomers mentioning

confidence: 87%

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

Díaz-López

Lages-Gonzalo

Serrano-López

et al. 2003

Journal of Biological Chemistry

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RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.

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Identification and characterization of a novel toxin–antitoxin module from Bacillus anthracis

Agarwal

Bhatnagar

2007

FEBS Letters

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Comparative genome analysis of Bacillus anthracis revealed a pair of linked genes encoding pemK (K, killer protein) and pemI (I, inhibitory protein) homologous to pem loci of other organisms. Expression of PemK in Escherichia coli and Bacillus anthracis was bacteriostatic whereas the concomitant expression of PemI reversed the growth arrest. PemK expression effectively inhibited protein synthesis with no significant effect on DNA replication. Coexpression and interaction of these proteins confirmed it to be a Type II addiction module. Thermal denaturation analysis reflected poor conformational stability of PemI as compared to PemK. Circular dichroism analysis indicated that PemI contains twice the amount of b-sheets as PemK. Gel retardation assays demonstrated that PemI binds to its upstream DNA sequence. This study reports the first evidence of an active chromosome encoded toxin-antitoxin locus in B. anthracis.

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The thermodynamic stability of the proteins of the ccd plasmid addiction system

Cited by 31 publications

References 30 publications

Energetics of Structural Transitions of the Addiction Antitoxin MazE

Energetics of Structural Transitions of the Addiction Antitoxin MazE

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

Identification and characterization of a novel toxin–antitoxin module from Bacillus anthracis

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