Solution structure of SpoIIAA, a phosphorylatable component of the system that regulates transcription factor sigmaF of Bacillus subtilis

Abstract

The establishment of differential gene expression in sporulating Bacillus subtilis involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded beta-sheet and four alpha-helices. Knowledge of the structure helps to account for the phenotype of several strains of B. subtilis that carry known spoIIAA mutations and should facilitate investigations of the conformational consequences of phosphorylation.

Figure 1

Summary of the ³J_NHα coupling constants and sequential- and medium-range NOE connectivities for backbone atoms and secondary-structure elements. •, ³J_NHα coupling constant >8 Hz; ○, ³J_NHα <6 HZ; ×, 6 Hz < ³J_NHα < 8 Hz. The plot distinguishes between weak and strong (thin and thick bar) only for sequential d_αN, d_NN, and d_βN distance constraints, the cutoff of the strong constraint being 3.0, 3.6, and 3.6 Å, respectively. The normalized deviations of the chemical shift of the ¹³C^α atoms from their random coil values (24) are shown on the bottom line. Light shading, β-strand; dark shading, α-helical region.

Figure 2

Stereo view of the superposition of the backbone atoms N, C^α, and C′ to the mean in the structured regions (residues 3–72 and 77–113) of the 24 best conformers. The α-helices are shown in red, the β-strands are in yellow, and coils are in cyan.

Figure 3

(a) Global fold of the nonphosphorylated SpoIIAA. The position of the phosphorylatable Ser-58 is highlighted by a green sphere. The figure is drawn by using molscript (25), povray (http:/www.povray.org/), and povscript (http:/www.rose.brandeis.edu/users/peisach/rayscript/). (b) Mutational sites in SpoIIAA. Gly-20, Gly-60, Gly-62, Gly-76, and Gly-95 are denoted with cyan spheres. The figure is drawn by using molscript, povray, and povscript.

Figure 4

Multiple sequence alignment of SpoIIAA, obtained through a search in the EMBL/GenBank DNA data base with fasta version 3.0.7 (31) using the program default parameters. The amino acids are denoted by single-letter code, dashes indicate gaps, and amino acid position is indicated on the left. Consensus residues that are present in at least four of seven sequences are boxed. Conserved changes are indicated by stippled boxes. The location of the β-strands is marked with strands and the α-helical regions are marked by cylinders. The conserved phosphorylation site is marked with a star, and the sites of mutation (30) shown in Fig. 3b and discussed in the text are marked with vertical arrows. Accession numbers from the GenBank/EMBL and SwissProt data bases are as follows: SpoIIAA-B.sub, P10727; SpoIIAA-B.lic, P26777; SpoIIAA-B.meg, P35147; SpoIIAA-B.coa, Z54161; SpoIIAA-B.ste, L47358; SpoIIAA-P.pol, L47359; SpoIIAA-B.sph, L47360.