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

Abstract

The phosphorelay signal transduction pathway controls sporulation initiation in Bacillus subtilis. Transfer of a phosphoryl group from multiple kinases (KinA and KinB) through a single domain response regulator homologue (Spo0F), a phosphotransferase (Spo0B), and ultimately to a transcriptional regulator, (Spo0A) activates sporulation. Counteracting this response are phosphatases (RapA and RapB), which can short-circuit this phosphorelay via dephosphorylation of Spo0F. In vitro assays of RapB activity on phosphorylated Spo0F alanine-scanning mutants have been used to identify Spo0F residues critical 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 largest number of residues in which an alanine substitution leads to resistance or decreased sensitivity to RapB phosphatase activity. Other mutations desensitizing 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 but not the same as the surface identified for KinA and Spo0B interactions with Spo0F. Divalent metal ions were shown to be required for RapB activity, and this activity was insensitive to vanadate, suggesting that Rap phosphatases 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 similarities between the mechanisms for RapB catalyzed Spo0F similar to P hydrolysis and GAP (GTPase activating protein)-assisted GTP hydrolysis of Ras are discussed.