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Dissection of the functional and structural domains of phosphorelay histidine kinase a of bacillus subtilis

Academic Article
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Overview

authors

  • Wang, L.
  • Fabret, C.
  • Kanamaru, K.
  • Stephenson, K.
  • Dartois, V.
  • Perego, Marta
  • Hoch, James

publication date

  • May 2001

journal

  • Journal of Bacteriology  Journal

abstract

  • The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.

subject areas

  • Adenosine Triphosphate
  • Amino Acid Substitution
  • Bacillus subtilis
  • Bacterial Proteins
  • Carrier Proteins
  • Catalytic Domain
  • Gene Deletion
  • Histidine
  • Maltose-Binding Proteins
  • Molecular Weight
  • Mutation
  • Phosphorylation
  • Protein Kinases
  • Protein Structure, Secondary
  • Signal Transduction
  • Spores, Bacterial
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Identity

International Standard Serial Number (ISSN)

  • 0021-9193

Digital Object Identifier (DOI)

  • 10.1128/jb.183.9.2795-2802.2001

PubMed ID

  • 11292798
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Additional Document Info

start page

  • 2795

end page

  • 2802

volume

  • 183

issue

  • 9

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