Characterization of interactions between a two-component response regulator, Spo0F, and its phosphatase, RapB.
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 beta1-alpha1 loop and N-terminal half of helix alpha1 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 beta3-alpha3 and beta4-alpha4 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 proposed to play a role in catalysis, and similarities between the mechanisms for RapB catalyzed Spo0F approximately P hydrolysis and GAP (GTPase activating protein)-assisted GTP hydrolysis of Ras are discussed.
Tzeng
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YL
Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Feher
V A
VA
Cavanagh
J
J
Perego
M
M
Hoch
J A
JA
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D004352:Drug Resistance, Microbial; D008239:Lysine / Q000235:genetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D013171:Spores, Bacterial / Q000201:enzymology / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D001426:Bacterial Proteins; D002413:Cations, Divalent; D008670:Metals; D010446:Peptide Fragments; C080951:Spo0F protein, Bacillus subtilis; D014443:Tyrosine; C091155:rapB protein, Bacillus subtilis; D010749:Phosphoprotein Phosphatases; D008239:Lysine; D000409:Alanine