Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis.

The phosphorelay, a signal transduction pathway composed of two-component regulatory proteins, mediates the initiation of sporulation in Bacillus subtilis. Environmental and physiological signals activate the autophosphorylation of histidine kinases, KinA and KinB, which transfer the phosphoryl group to Spo0F, a single domain homolog of the two-component response regulator. Phosphorylated Spo0F passes the phosphate to the final transcriptional regulator, Spo0A, through a phosphotransferase, Spo0B. Spo0F shares significant homology with other members of the response regulator family. It displays a (beta/alpha)5-barrel scaffold with the active site situated at the carboxyl end of the beta strands. The molecular recognition of Spo0F with its cognate proteins was investigated using a comprehensive strategy termed alanine-scanning mutagenesis. Of the total 124 residues, 79 in the region of helices and loops were individually changed to alanine using site-directed mutagenesis. The mutants with notable in vivo sporulation phenotypes were further examined in vitro to identify the corresponding effect in each protein-protein interaction. This study revealed that most, if not all, protein-protein interactions involve the residues in the vicinity of the active site. The surface-exposed residues critical for the interactions with KinA or Spo0B were identified. Surprisingly, although these interaction proteins are very different, they recognize subsets of residues comprising a common surface of Spo0F.

Tzeng YL ，Hoch JA 《JOURNAL OF MOLECULAR BIOLOGY》

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

Tzeng YL ，Feher VA ，Cavanagh J ，Perego M ，Hoch JA ... -  《BIOCHEMISTRY》

A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site.

Zapf J ，Madhusudan M ，Grimshaw CE ，Hoch JA ，Varughese KI ，Whiteley JM ... -  《BIOCHEMISTRY》

A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates.

Zapf JW ，Hoch JA ，Whiteley JM 《BIOCHEMISTRY》

Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for in vitro metalloregulation in the initiation of sporulation.

Kojetin DJ ，Thompson RJ ，Benson LM ，Naylor S ，Waterman J ，Davies KG ，Opperman CH ，Stephenson K ，Hoch JA ，Cavanagh J ... -  《BIOMETALS》