The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis

Patrik Florek; Vladimir M. Levdikov; Elena Blagova; Andrey A. Lebedev; Rostislav Skrabana; Stanislava Resetarova; Pamela Pavelcikova; Imrich Barak; Anthony J. Wilkinson

doi:10.1074/jbc.M110.172429

The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis

Patrik Florek, Vladimir M. Levdikov, Elena Blagova, Andrey A. Lebedev, Rostislav Skrabana, Stanislava Resetarova, Pamela Pavelcikova, Imrich Barak, Anthony J. Wilkinson

Chemistry

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

Abstract

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase sigma factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, sigma(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 angstrom spacing, revealing a CSpoIISA(2) . SpoIISB(2) heterotetramer. CSpoIISA has a single domain alpha/beta structure resembling a GAF domain with an extended alpha-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA . SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

Original language	English
Pages (from-to)	6808-6819
Number of pages	12
Journal	Journal of Biological Chemistry
Volume	286
Issue number	8
DOIs	https://doi.org/10.1074/jbc.M110.172429
Publication status	Published - 25 Feb 2011

Keywords

GRAM-POSITIVE BACTERIA
ESCHERICHIA-COLI
CRYSTAL-STRUCTURE
MOLECULAR-GRAPHICS
STATIONARY-PHASE
DIFFRACTION DATA
GAF DOMAIN
EXPRESSION
COMPLEX
PROTEIN

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10.1074/jbc.M110.172429

http://www.jbc.org/content/286/8/6808

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title = "The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis",

abstract = "Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase sigma factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, sigma(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 angstrom spacing, revealing a CSpoIISA(2) . SpoIISB(2) heterotetramer. CSpoIISA has a single domain alpha/beta structure resembling a GAF domain with an extended alpha-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA . SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.",

keywords = "GRAM-POSITIVE BACTERIA, ESCHERICHIA-COLI, CRYSTAL-STRUCTURE, MOLECULAR-GRAPHICS, STATIONARY-PHASE, DIFFRACTION DATA, GAF DOMAIN, EXPRESSION, COMPLEX, PROTEIN",

author = "Patrik Florek and Levdikov, {Vladimir M.} and Elena Blagova and Lebedev, {Andrey A.} and Rostislav Skrabana and Stanislava Resetarova and Pamela Pavelcikova and Imrich Barak and Wilkinson, {Anthony J.}",

year = "2011",

month = feb,

day = "25",

doi = "10.1074/jbc.M110.172429",

language = "English",

volume = "286",

pages = "6808--6819",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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T1 - The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis

AU - Florek, Patrik

AU - Levdikov, Vladimir M.

AU - Blagova, Elena

AU - Lebedev, Andrey A.

AU - Skrabana, Rostislav

AU - Resetarova, Stanislava

AU - Pavelcikova, Pamela

AU - Barak, Imrich

AU - Wilkinson, Anthony J.

PY - 2011/2/25

Y1 - 2011/2/25

N2 - Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase sigma factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, sigma(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 angstrom spacing, revealing a CSpoIISA(2) . SpoIISB(2) heterotetramer. CSpoIISA has a single domain alpha/beta structure resembling a GAF domain with an extended alpha-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA . SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

AB - Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase sigma factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, sigma(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 angstrom spacing, revealing a CSpoIISA(2) . SpoIISB(2) heterotetramer. CSpoIISA has a single domain alpha/beta structure resembling a GAF domain with an extended alpha-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA . SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

KW - GRAM-POSITIVE BACTERIA

KW - ESCHERICHIA-COLI

KW - CRYSTAL-STRUCTURE

KW - MOLECULAR-GRAPHICS

KW - STATIONARY-PHASE

KW - DIFFRACTION DATA

KW - GAF DOMAIN

KW - EXPRESSION

KW - COMPLEX

KW - PROTEIN

UR - http://www.scopus.com/inward/record.url?scp=79953230878&partnerID=8YFLogxK

U2 - 10.1074/jbc.M110.172429

DO - 10.1074/jbc.M110.172429

M3 - Article

SN - 0021-9258

VL - 286

SP - 6808

EP - 6819

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 8

ER -

The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis

Abstract

Keywords

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