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

J Mol Biol. 1997 Sep 19;272(2):200-12. doi: 10.1006/jmbi.1997.1226.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine
  • Amino Acid Sequence
  • Bacillus subtilis / genetics
  • Bacillus subtilis / physiology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphoproteins
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Protein Structure, Secondary
  • Sigma Factor*
  • Signal Transduction / physiology*
  • Spores, Bacterial
  • Transcription Factors*


  • Bacterial Proteins
  • Phosphoproteins
  • Sigma Factor
  • Spo0F protein, Bacillus subtilis
  • Transcription Factors
  • kinA protein, Bacillus subtilis
  • spoIIR protein, Bacillus subtilis
  • spore-specific proteins, Bacillus
  • Protein Kinases
  • Alanine