Functional and biochemical analysis of
truncated SpoIISA toxin in Bacillus subtilis
J. Makroczyová1, P. Florek1, S. Rešetárová1, A. J. Wilkinson2 and I. Barák1
1Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia
2Department of Chemistry, University of York, Heslington, York YO1 5OD, UK
Toxin-antitoxin were found to be encoded on vast number of bacterial
plasmids and genomes and are thought to be associated with the maintenance of extrachromosomal DNA, bacteriostasis
and possibly altruistic cell death. Chromosomally encoded SpoIISA-SpoIISB
system appears to define a new family of toxin-antitoxin modules that is
conserved among Bacilli.
SpoIISA proteic toxin from B. subtilis
was shown to affect cell mebrane of either mother
cell during sporulation process or, when artificially
over-expressed in vegetatively growing cells. This
248-amino residue protein was predicted to be composed of two domains – a
membrane spanning domain consisting of three helices and a negatively charged cytosolic domain (CSpoIISA),
which binds small, positively charged SpoIISB protein
[1].
Recently solved
crystal structure of CSpoIISA2:SpoIISB2 heterotetrameric complex [2] revealed that the toxin
portion of the complex constitutes so called GAF (cGMP
phospho- diesterase, adenylate cyclase, FhlA) domain, a fold which is widespread in proteins
of diverse function including cell signalling proteins, transcription factors
and proteins involved in light detection in bacteria, fungi and plants [3]. The
GAF domain has three tiers, a basal layer of two or more α-helices, a
middle tier of four or more strands that form a mixed β-pleated sheet and
a distal layer of more variable structure made up of segments connecting the
strands of the β-sheet. Many GAF domains are dimers
formed by the packing of α-helices from the basal layer to form a four
helix bundle. In case of SpoIISA, the two protomers of its cytosolic domain
form extensive hydrophobic interactions through an intermolecular 4-helix bundle
formed by the first and the last CSpoIISA alpha
helices [2].
Since alternative
prediction of the SpoIISA topology suggested that the
last CSpoIISA α-helix might swap from
intermolecular interface and become incorporated into cytoplasmic
membrane, in this work we assessed the toxicity of the C-terminally truncated SpoIISA lacking the last alpha helix and the ability of SpoIISB antitoxin to neutralize the eventual lethal
activity in B. subtilis cells. Moreover, we
examined the effect of this C-terminal SpoIISA
deletion on the CSpoIISA dimerization
and the CSpoIISA-SpoIISB complex formation in
vitro.
1. E. Adler, I. Barák, P. Stragier, J. Bacteriol., 183, (2001), 3574.
2. P. Florek, V. M. Levdikov, E. Blagova, A. A. Lebedev, R. Škrabana, S. Rešetárová, P. Pavelčíková, I. Barák, A. J. Wilkinson, (2010), doi:10.1074/jbc.M110.172429.
3. L. Aravind, C. P. Ponting,,
Trends Biochem. Sci. 22, (1997), 458.
This work was supported by Grant 2/0016/10 from Slovak Academy of Sciences, by grants from the Slovak Research and Development Agency under the contract No. APVT-51-0278 and No. LPP-0218-06, by Grant from European Science Foundation ESF-EC-0106, and by Grant 082829/Z/07/Z from The Wellcome Trust.