Functional Dissection of the Dimerization and Enzymatic Activities of Escherichia coli Nitrogen Regulator II and Their Regulation by the PII Protein

Abstract

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.

The Phosphoryl Transfer Domain of UhpB Interacts with the Response Regulator UhpA

“…It remains to be shown whether the dominant-negative effect of GST-Zc or GST-ZBc on porin expression results from sequestration of OmpR (34), as proposed for the effect by GST-Bc on UhpA, or from its action as P-OmpR phosphatase (15,51). Nonetheless, the portion of UhpB which appears to be involved in the binding of UhpA overlaps the homologous region of a VanSderived peptide which binds VanR (46), and portions of NtrB and EnvZ which are necessary for phosphatase activity on NtrC and OmpR, respectively (19,23).…”

Direct Interaction of the NifL Regulatory Protein with the GlnK Signal Transducer Enables the Azotobacter vinelandiiNifL-NifA Regulatory System to Respond to Conditions Replete for Nitrogen

“…Because adenosine nucleotides bind to the kinase-like domain to stimulate the NifL-NifA interaction, it is possible that this domain of NifL represents either an ancient precursor of the histidine protein kinases or has evolved from the "classical" kinases with loss of catalytic function (nucleoside triphosphate hydrolysis), so that nucleotide binding is utilized to drive conformational changes that promote interaction with NifA (3,4). The homology between the kinase-like domains of NifL and NtrB (NRII) is of particular interest in view of the recent finding that this domain of NtrB (residues 190 -339) interacts with Ec PII (35,38). The C-terminal region of NifL is implicated in the interaction with Av GlnK because the N-terminal region (residues 1-284) did not interact, whereas the central plus the C-terminal regions were competent to do so.…”

“…Although the kinase-like domain of NifL binds adenosine nucleotides (2), no autokinase or phosphotransferase activities have been detected in vitro (28). Nevertheless, the similarity between NifL and the histidine protein kinase family is of interest, in light of the recent demonstration that the Cterminal kinase domain of NtrB (NRII) interacts with enteric PII (35,38,39).…”

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…The helix in the middle domain close to the N-terminal domain includes histidine 139, the site of autophosphorylation (173). The C-terminal domain includes the putative nucleotide binding site (174)(175)(176). After dimerization, the two helices of each monomer may together form a four-helix bundle (176).…”