Functional dissection of the dimerization and enzymatic activities of Escherichia coli nitrogen regulator II and their regulation by the PII protein.
The dimeric two-component system transmitter protein NRII (NtrB) of Escherichia coli, product of glnL (ntrB), controls transcription of nitrogen-regulated genes by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI (NtrC). Previous studies showed that the PII signal transduction protein inhibits the kinase activity of NRII and activates its phosphatase activity. We observed that PII greatly stimulated the NRII phosphatase activity under conditions where the cleavage of ATP was prevented, indicating that the phosphatase activity did not result simply from prevention of the antagonistic NRII kinase activity by PII. Rather, PII was an activator of the phosphatase activity. To study this regulation, we examined the dimerization and enzymatic activities of NRII and various polypeptides derived from NRII, and their regulation by PII. Our results were consistent with the hypothesis that NRII consists of three domains: an N-terminal domain found only in NRII proteins and two domains formed by the conserved transmitter module of NRII, the phosphotransferase/phosphatase/dimerization (central) domain and the kinase domain. All three domains were involved in regulating the kinase and phosphatase activities of NRII. The N-terminal domain was involved in intramolecular signal transduction, and controlled access to the NRII active site for the isolated dimeric central domain added in trans. The central domain was responsible for dimerization and the phosphotransferase and phosphatase activities of NRII, but the latter activity was weak in the isolated domain and was not regulated by PII. The C-terminal kinase domain was responsible for the kinase activity. The PII protein appeared to interact with the isolated transmitter module of NRII, and not with the N-terminal domain as previously thought, since PII dramatically increased the stoichiometry of autophosphorylation of the isolated transmitter module. However, the phosphatase activity of the transmitter module of NRII was low even in the presence of PII, suggesting that the N-terminal domain was necessary for the central domain to assume the conformation necessary for potent phosphatase activity. Also, PII significantly reduced the rate of transphosphorylation of the isolated central domain by the isolated kinase domain, suggesting that PII interacts directly with the kinase domain. We hypothesize that the binding of PII to the kinase domain of NRII results in an altered conformation that is transmitted to the central and N-terminal domains; this causes the central domain to assume the conformation with potent phosphatase activity.
Jiang
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P
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Atkinson
M R
MR
Srisawat
C
C
Sun
Q
Q
Ninfa
A J
AJ
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D051922:PII Nitrogen Regulatory Proteins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; C461599:maltose transport system, E coli; C404143:PIID regulatory protein, Bacteria; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D010749:Phosphoprotein Phosphatases