Andrea E Rawlings

Publications (2)6.94 Total impact

• Article: Structure of the phosphatase domain of the cell fate determinant SpoIIE from Bacillus subtilis.

  Vladimir M Levdikov, Elena V Blagova, Andrea E Rawlings, Katie Jameson, James Tunaley, Darren J Hart, Imrich Barak, Anthony J Wilkinson
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  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 σ(F) 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.
  Journal of Molecular Biology 11/2011; 415(2):343-58. · 4.00 Impact Factor
• Source

  Available from: Ludmila Vavrova

  Article: Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen.

  Andrea E Rawlings, Vladimir M Levdikov, Elena Blagova, Vicki L Colledge, Philippe J Mas, James Tunaley, Ludmila Vavrova, Keith S Wilson, Imrich Barak, Darren J Hart, Anthony J Wilkinson
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  ABSTRACT: SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.
  Protein Engineering Design and Selection 11/2010; 23(11):817-25. · 2.94 Impact Factor