Abstract
DivIB(FtsQ), FtsL, and DivIC(FtsB) are enigmatic membrane proteins that are central to the process of bacterial cell division. DivIB(FtsQ) is dispensable in specific conditions in some species, and appears to be absent in other bacterial species. The presence of FtsL and DivIC(FtsB) appears to be conserved despite very low sequence conservation. The three proteins form a complex at the division site, FtsL and DivIC(FtsB) being associated through their extracellular coiled-coil region. We report here structural investigations by NMR, small-angle neutron and x-ray scattering, and interaction studies by surface plasmon resonance, of the complex of DivIB, FtsL, and DivIC from Streptococcus pneumoniae, using soluble truncated forms of the proteins. We found that one side of the "bean"-shaped central beta-domain of DivIB interacts with the C-terminal regions of the dimer of FtsL and DivIC. This finding is corroborated by sequence comparisons across bacterial genomes. Indeed, DivIB is absent from species with shorter FtsL and DivIC proteins that have an extracellular domain consisting only of the coiled-coil segment without C-terminal conserved regions (Campylobacterales). We propose that the main role of the interaction of DivIB with FtsL and DivIC is to help the formation, or to stabilize, the coiled-coil of the latter proteins. The coiled-coil of FtsL and DivIC, itself or with transmembrane regions, could be free to interact with other partners.
Highlights
OCTOBER 2, 2009 VOLUME 284 NUMBER 40 nomenclature applies to Gram-negative organisms, whereas Div nomenclature applies to Gram-positive bacteria
We decided to produce proteins with a truncated N terminus with respect to our proteolysis results to exclude the fully conserved Glu-222, which is absent from the -domain from G. stearothermophilus, and appears to be part of the linker between the ␣- and -domains from E. coli and Y. enterocolitica
Our NMR and proteolysis results show that the POTRA domain from S. pneumoniae is not folded in the recombinant soluble form of the protein, as was observed with DivIB from G. stearothermophilus (26)
Summary
Plasmid Constructions—Proteins were produced in E. coli from expression plasmids inducible with isopropyl -D-1-thio-galactopyranoside. The protein EC with a Strep-tag was purified from the cell lysate in 20 mM Tris, pH 8, 150 mM NaCl by affinity chromatography on a streptactin resin (IBA). The urea was removed by dialysis three times against 40 volumes of 20 mM Tris, pH 8, 150 mM NaCl. The (KL/EC) complex, where EC has a Strep tag and KL a poly-His tag, was prepared from a lysate of cells co-expressing both proteins. Following concentration with an Amicon Ultra device, the complex was further purified by size exclusion chromatography on a Superdex S200 column (16 ϫ 600 mm) equilibrated with 20 mM Tris, pH 8, 150 mM NaCl. For the transverse relation optimized spectroscopy NMR experiment, the buffer was exchanged by desalting on a PD10 column (GE Healthcare) against 100 mM ammonium acetate, pH 7, prior to lyophilization.
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