Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

Christopher N. Penfold; Bryan Healy; Nicholas G. Housden; Ruth Boetzel; Mireille Vankemmelbeke; Colin Kleanthous; Geoffrey R. Moore; Richard James

doi:10.1128/JB.186.14.4520-4527.2004

Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

Christopher N. Penfold, Bryan Healy, Nicholas G. Housden, Ruth Boetzel, Mireille Vankemmelbeke, Colin Kleanthous, Geoffrey R. Moore, Richard James

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil Mull receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.

Original language	English
Pages (from-to)	4520-4527
Number of pages	8
Journal	Journal of Bacteriology
Volume	186
Issue number	14
DOIs	https://doi.org/10.1128/JB.186.14.4520-4527.2004
Publication status	Published - Jul 2004

Bibliographical note

Keywords

TRANSLOCATION DOMAIN
CRYSTAL-STRUCTURE
NUCLEASE DOMAIN
PORE FORMATION
ENDONUCLEASE
TOLA
IDENTIFICATION
IMMUNITY
BTUB
SITE

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10.1128/JB.186.14.4520-4527.2004

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Cite this

@article{73f6fe8f668845ceae70b7f85d47d12e,

title = "Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells",

abstract = "The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil Mull receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.",

keywords = "TRANSLOCATION DOMAIN, CRYSTAL-STRUCTURE, NUCLEASE DOMAIN, PORE FORMATION, ENDONUCLEASE, TOLA, IDENTIFICATION, IMMUNITY, BTUB, SITE",

author = "Penfold, {Christopher N.} and Bryan Healy and Housden, {Nicholas G.} and Ruth Boetzel and Mireille Vankemmelbeke and Colin Kleanthous and Moore, {Geoffrey R.} and Richard James",

note = "Copyright {\textcopyright} 2004, American Society for Microbiology.",

year = "2004",

month = jul,

doi = "10.1128/JB.186.14.4520-4527.2004",

language = "English",

volume = "186",

pages = "4520--4527",

journal = "Journal of Bacteriology",

issn = "0021-9193",

publisher = "American Society for Microbiology",

number = "14",

}

TY - JOUR

T1 - Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

AU - Penfold, Christopher N.

AU - Healy, Bryan

AU - Housden, Nicholas G.

AU - Boetzel, Ruth

AU - Vankemmelbeke, Mireille

AU - Kleanthous, Colin

AU - Moore, Geoffrey R.

AU - James, Richard

PY - 2004/7

Y1 - 2004/7

N2 - The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil Mull receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.

AB - The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil Mull receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.

KW - TRANSLOCATION DOMAIN

KW - CRYSTAL-STRUCTURE

KW - NUCLEASE DOMAIN

KW - PORE FORMATION

KW - ENDONUCLEASE

KW - TOLA

KW - IDENTIFICATION

KW - IMMUNITY

KW - BTUB

KW - SITE

U2 - 10.1128/JB.186.14.4520-4527.2004

DO - 10.1128/JB.186.14.4520-4527.2004

M3 - Article

SN - 0021-9193

VL - 186

SP - 4520

EP - 4527

JO - Journal of Bacteriology

JF - Journal of Bacteriology

IS - 14

ER -