Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor σ F in Bacillus subtilis

Abstract

The activity of the developmental transcription factor σ F in Bacillus subtilis is governed by a switch involving the dual function protein SpoIIAB. SpoIIAB is an antisigma factor that forms complexes with σ F 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 σ F. 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, σ F. 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-σ F 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.