The Escherichia coli PII signal transduction protein regulates the activities of the two-component system transmitter protein NRII by direct interaction with the kinase domain of the transmitter module

Biochemistry. 2000 Nov 7;39(44):13450-61. doi: 10.1021/bi000795m.

Abstract

The PII signal transduction protein regulates the transcription of nitrogen-regulated genes by controlling the kinase and phosphatase activities of NRII. We used a cross-linking approach to study the interaction of the T-loop of the PII protein with NRII. Cross-linking of PII to NRII required ATP and 2-ketoglutarate, allosteric effectors known to control PII activity, and was not affected by the presence of excess nonspecific proteins such as bovine serum albumin. The purified cross-linked species appeared to consist mainly of PII trimers in which one of the three subunits was cross-linked to a single subunit of the NRII dimer; this complex had the phosphatase activity characteristic of the un-cross-linked PII-NRII complex, and had significant phosphatase activity in the absence of 2-ketoglutarate, suggesting that once PII was tethered to NRII the active conformation was stabilized. Studies with truncated forms of NRII indicated that the purified N-terminal "sensory" domain of NRII was not cross-linked to PII, nor was a polypeptide consisting of NRII residues 1-189. In contrast, polypeptides containing the kinase domain of the transmitter module of NRII (residues 190-349) were cross-linked to PII in an ATP- and 2-ketoglutarate-dependent reaction. These results indicate that PII controls NRII by interaction with the conserved kinase domain of the transmitter module.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Bacterial Proteins / physiology*
  • Chromatography, Gel
  • Cross-Linking Reagents
  • Cysteine / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation / genetics
  • Escherichia coli / enzymology*
  • Escherichia coli / metabolism
  • Fluorobenzenes
  • Maleimides
  • Mutagenesis, Site-Directed
  • PII Nitrogen Regulatory Proteins
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Peptide Mapping
  • Phosphoprotein Phosphatases / isolation & purification
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Protein Structure, Tertiary / genetics
  • Serine / genetics
  • Signal Transduction* / genetics

Substances

  • Bacterial Proteins
  • Cross-Linking Reagents
  • Fluorobenzenes
  • Maleimides
  • PII Nitrogen Regulatory Proteins
  • Peptide Fragments
  • N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide
  • Serine
  • PIID regulatory protein, Bacteria
  • Protein Kinases
  • protein kinase-phosphatase NTRB
  • Phosphoprotein Phosphatases
  • Cysteine