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.
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.
Pioszak
A A
AA
Department of Biological Chemistry and Human Genetics Training Program, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Jiang
P
P
Ninfa
A J
AJ
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005464:Fluorobenzenes; D008301:Maleimides; D016297:Mutagenesis, Site-Directed; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012694:Serine / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005464:Fluorobenzenes; D008301:Maleimides; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments; C073711:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; D012694:Serine; C404143:PIID regulatory protein, Bacteria; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D010749:Phosphoprotein Phosphatases; D003545:Cysteine