Processing of DNase Domain during Translocation of Colicin E7 across the Membrane of Escherichia coli

Liao, Chen Chung; Hsiao, Kuo Cheong; Liu, Yu Wen; Leng, Po Huang; Yuen, Hanna S.; Chak, Kin‐Fu

doi:10.1006/bbrc.2001.5016

Cited by 26 publications

(15 citation statements)

References 27 publications

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“…This allowed us to localize precisely the cleavage site between two basic residues K451 and R452 in agreement with the consensus for OmpT-dependent cleavage sites [29] and conclude that the SC form carries the C-terminal DNase domain of colicin E2. It is noticeable that this cleavage site is the same as that previously found with periplasmic extracts [14], [15] and is therefore irrelevant for the import of DNase colicins. We note that the authentic in vivo processed form of colicin E2 (PF) is 3.5 kDa heavier, than the OmpT-cleaved SC form.…”

Section: Resultssupporting

confidence: 69%

“…Processing in the periplasm was previously considered as being an essential step for nuclease-colicin import [14], [15]. We showed that in fact, the catalytic enzyme of this cleavage is the outer-membrane protease OmpT [40], [41] (Figure S2), although its presence in the periplasmic extracts is unexpected and most probably due to some contamination.…”

Section: Discussionmentioning

confidence: 77%

“…It had been previously reported that incubation of colicin E7-Imm7 with periplasmic extracts liberated a small C-terminal peptide due to a cleavage between positions K446/R447, although it required extreme conditions with incubation times of 8–12 h. Similarly sized fragments of the DNase domain were also detected in vivo , so that it was claimed that this cleavage was required for colicin import, [14], [15]. As deduced from peptide sequence comparison of three DNase colicins, the cleavage site in colicin E7 is equivalent to positions K451/R452 in colicin E2 and K452/R453 in colicin E9.…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage of DNase colicin E7 in the presence of periplasmic extracts was previously reported, but the catalytic enzyme was not identified [14], [15]. Analysis of point mutations introduced at the in vitro determined cleavage site of colicin E7 or E2 led to a decrease or loss of colicin toxicity and seemed to prevent the cleavage of the colicin in vivo , so that the authors claimed that the periplasmic cleavage was responsible for the colicin processing, required for colicin import and cell killing [15], [16].…”

Section: Introductionmentioning

confidence: 95%

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In Vivo Processing of DNase Colicins E2 and E7 Is Required for Their Import into the Cytoplasm of Target Cells

Mora

Zamaroczy

2014

PLoS ONE

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DNase colicins E2 and E7, both of which appropriate the BtuB/Tol translocation machinery to cross the outer membrane, undergo a processing step as they enter the cytoplasm. This endoproteolytic cleavage is essential for their killing action. A processed form of the same size, 18.5 kDa, which corresponds to the C-terminal catalytic domain, was detected in the cytoplasm of bacteria treated with either of the two DNase colicins. The inner-membrane protease FtsH is necessary for the processing that allows the translocation of the colicin DNase domain into the cytoplasm. The processing occurs near residue D420, at the same position as the FtsH-dependent cleavage in RNase colicins E3 and D. The cleavage site is located 30 amino acids upstream of the DNase domain. In contrast, the previously reported periplasm-dependent colicin cleavage, located at R452 in colicin E2, was shown to be generated by the outer-membrane protease OmpT and we show that this cleavage is not physiologically relevant for colicin import. Residue R452, whose mutated derivatives led to toxicity defect, was shown to have no role in colicin processing and translocation, but it plays a key role in the catalytic activity, as previously reported for other DNase colicins. Membrane associated forms of colicins E2 and E7 were detected on target cells as proteinase K resistant peptides, which include both the receptor-binding and DNase domains. A similar, but much less proteinase K-resistant form was also detected with RNase colicin E3. These colicin forms are not relevant for colicin import, but their detection on the cell surface indicates that whole nuclease-colicin molecules are found in a stable association with the outer-membrane receptor BtuB of the target cells.

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Section: Resultssupporting

confidence: 69%

Section: Discussionmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

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In Vivo Processing of DNase Colicins E2 and E7 Is Required for Their Import into the Cytoplasm of Target Cells

Mora

Zamaroczy

2014

PLoS ONE

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“…The C-terminal nuclease domain of colicin E7 (NColE7, residues 446-576) kills the attacked cell by nonspecific digestion of RNA and/or chromosomal DNA molecules [6][7][8][9]. The host cell protects itself by coexpression of the Im7 immunity protein forming a stable complex with NColE7 at the nucleic acid-binding site [8].…”

Section: +mentioning

confidence: 99%

Preorganization of the catalytic Zn2+-binding site in the HNH nuclease motif—A solution study

Németh

Kožíšek

Schilli

et al. 2015

Journal of Inorganic Biochemistry

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Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

et al. 2014

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The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7 5 KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK > KGNG~GGNK > GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.

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Processing of DNase Domain during Translocation of Colicin E7 across the Membrane of Escherichia coli

Cited by 26 publications

References 27 publications

In Vivo Processing of DNase Colicins E2 and E7 Is Required for Their Import into the Cytoplasm of Target Cells

In Vivo Processing of DNase Colicins E2 and E7 Is Required for Their Import into the Cytoplasm of Target Cells

Preorganization of the catalytic Zn2+-binding site in the HNH nuclease motif—A solution study

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

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