Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis

Abstract

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor σF from an inhibitory complex with the anti-sigma factor SpoIIAB. SpoIIE therefore couples morphological development to differential gene expression. Here, we determined the crystal structure of the phosphatase domain of SpoIIE to 2.6 Å spacing, revealing a domain-swapped dimer. SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) analysis however suggested a monomer as the principal form in solution. A model for the monomer was derived from the domain-swapped dimer in which 2 five-stranded β-sheets are packed against one another and flanked by α-helices in an αββα arrangement reminiscent of other PP2C-type phosphatases. A flap region that controls access of substrates to the active site in other PP2C phosphatases is diminished in SpoIIE, and this observation correlates with the presence of a single manganese ion in the active site of SpoIIE in contrast to the two or three metal ions present in other PP2C enzymes. Mapping of a catalogue of mutational data onto the structure shows a clustering of sites whose point mutation interferes with the proper coupling of asymmetric septum formation to sigma factor activation and identifies a surface involved in intramolecular signaling.

Stoichiometry and Mobility Switching of a Morphogenetic Protein in Live Differentiating Cells

“…Crystallographic and biophysical studies reveal that SpoIIE(590-827), comprising the phosphatase domain, is a monomer while SpoIIE(457-827), comprising the phosphatase plus part of the upstream regulatory domain, is dimeric ( Fig. 6A) (47,48). Comparison of these structures and mapping of mutational data onto them led to the proposal that PP2C domains in SpoIIE(590-827) and SpoIIE(457-827) crystals represent inactive and active states respectively.…”

A conserved regulatory switch controls phosphatase activity and specificity

“…SpoIIAA and SpoIIAB were expressed in BL21(DE3) Rosetta2 pLysS cells as 6H-sumo fusions in pET23a (made by ligation of the coding sequence in pET23a 6H-sumo digested with NotI/AgeI as reported (Bradshaw and Losick, 2015)). SpoIIAA-P was produced in BL21(DE3) cells containing pET-YSBLIC 6H-3C-spoIIAA-spoIIAB as reported (Levdikov et al, 2012).…”

Regulation of σ factors by conserved partner switches controlled by divergent signalling systems

“…When a signal is launched, the PP2C-type phosphatase SpoIIE, which is a membrane-anchored protein that perceives the signal dephosphorylates specifically SpoIIAA. The anti-r antagonist becomes thus efficient to interact with SpoIIAB, leading to the release of r F (Diederich et al, 1994;Duncan et al, 1996;Magnin et al, 1997;Campbell et al, 2002;Masuda et al, 2004;Levdikov et al, 2012).…”