Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis.
The activity of the developmental transcription factor sigmaF in Bacillus subtilis is governed by a switch involving the dual function protein SpoIIAB. SpoIIAB is an antisigma factor that forms complexes with sigmaF and with an alternative partner protein SpoIIAA. SpoIIAB is also a protein kinase that can inactivate SpoIIAA by phosphorylating it on a serine residue. We sought to identify amino acids in SpoIIAB that are involved in the formation of the SpoIIAB-SpoIIAA complex by screening for mutants that were defective in the activation of sigmaF. This genetic screen, in combination with biochemical analysis and the construction of loss-of-side-chain (alanine substitution) mutants, led to the identification of amino acid side-chains in the N-terminal region of SpoIIAB that could contact SpoIIAA. Unexpectedly, the same amino acid side-chains (R20 and N50) that appear to touch SpoIIAA are required for binding to, and may represent sites of contact with, sigmaF. We propose that the N-terminal region of SpoIIAB forms a binding surface that is responsible for the formation of both the SpoIIAB-SpoIIAA and the SpoIIAB-sigmaF complexes, and that in some cases the same amino acid side-chains contact both partner proteins. N50 is also the defining residue of a region of amino acid sequence homology known as the N-box that is shared by SpoIIAB and related serine protein kinases, as well as by members of a mechanistically dissimilar family of protein kinases that undergo autophosphorylation at a histidine residue. We discuss the implications of this finding for the mechanism of histidine autophosphorylation.
Garsin
D A
DA
The Biological Laboratories, Harvard University, 16 Diversity Avenue, Cambridge, MA, 02138, USA.
Paskowitz
D M
DM
Duncan
L
L
Losick
R
R
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D016297:Mutagenesis, Site-Directed; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; C064971:FliA protein, Bacteria; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D000409:Alanine