Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB

The phosphorelay signal transduction pathway controls sporulation initiatio n in Bacillus subtilis. Transfer of a phosphoryl group from multiple kinase s (KinA and KinB) through a single domain response regulator homologue (Spo 0F), a phosphotransferase (Spo0B), and ultimately to a transcriptional regu lator, (Spo0A) activates sporulation. Counteracting this response are phosp hatases (RapA and RapB), which can short-circuit this phosphorelay via deph osphorylation of Spo0F. In vitro assays of RapB activity on phosphorylated Spo0F alanine-scanning mutants have been used to identify Spo0F residues cr itical for interactions between these proteins. The Spo0F surface comprised of the beta 1-alpha 1 loop and N-terminal half of helix ctl has the larges t number of residues in which an alanine substitution leads to resistance o r decreased sensitivity to RapB phosphatase activity. Other mutations desen sitizing Spo0F to RapB are also located near the site of phosphorylation on the beta 3-alpha 3 and beta 4-alpha 4 loops. This surface is similar to bu t not the same as the surface identified for KinA and Spo0B interactions wi th Spo0F. Divalent metal ions were shown to be required for RapB activity, and this activity was insensitive to vanadate, suggesting that Rap phosphat ases catalyze acyl phosphate hydrolysis by inducing conformational changes in phosphorylated Spo0F, which results in increased autodephosphorylation. Arginine 16 of Spo0F is propos ed to pray a role in catalysis, and similari ties between the mechanisms for RapB catalyzed Spo0F similar to P hydrolysi s and GAP (GTPase activating protein)-assisted GTP hydrolysis of Ras are di scussed.