Crystallization of SpoIISA toxin and SpoIISB antitoxin from different Bacilli species
Patrik Florek1, Jana Melničáková1, Anthony J. Wilkinson2, S. Rešetárová1 and Imrich Barák1
of Molecular Biology,
2Department of Chemistry, University of York, York, UK
SpoIISA-SpoIISB killer-antikiller system was originally found in B. subtilis and its homologues were identified also in B. cereus and several other relative Bacilli. In absence of SpoIISB protein, activity of SpoIISA toxin blocks B. subtilis sporulation process in early stages and its forced expression during vegetative growth leads to cell lysis both in B. subtilis and E. coli .
SpoIISA protein (248 aa) was proposed to comprise two domains – N-terminal, which is formed by three membrane spanning helices and globular C-terminal part, which contitutes negatively charged cytosolic domain (C-SpoIISA). SpoIISB (56 aa), on the other hand, is basic, hydrophilic protein, which was shown to bind SpoIISA through its predicted cytosolic domain .
In our previous work we prepared crystals and solved molecular structure of C-SpoIISA–SpoIISB complex, where we observed binding of two SpoIISB monomers to opposite sites of C-SpoIISA dimer [unpublished data].
Here, to further understand mechanism of regulation of SpoIISA toxicity, we purified and crystallized cytosolic domain of SpoIISA protein without its cognate antitoxin partner. For now, these crystals were successfully tested for diffraction, but the further optimization of the crystallization conditions and search for better cryoprotectants is still required. We also determined expression level of SpoIISA protein during life cycle of B. subtilis. Moreover, we focused on SpoIISA-SpoIISB killer-antikiller system in B. cereus and analyzed SpoIISA toxic activity in E. coli. We purified protein complex of cytosolic domain SpoIISA and SpoIISB for further crystallization experiments.
1. E. Adler,
2. P. Florek, K. Muchová, P. Pavelčíková, I. Barák. FEMS Microbiol. Lett. 278 (2008) 177-184.
This work was supported by the grant APVT-51-027804, No. ESF-EC-0106, LPP-0218-06 and VEGA grant 2/7007/27 from the Slovak Academy of Sciences and The Wellcome Trust Grant 082829/Z/07/Z.