PMID:11063581
| Citation |
Pioszak, AA, Jiang, P and Ninfa, AJ (2000) 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 39:13450-61 |
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| 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. |
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| Keywords |
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 |
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