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PMID: 9209053 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.

Journal of bacteriology ·Vol. 179 ·No. 13 ·1997-07-00 ·Pages 4342-53

Jiang P, Zucker P, Atkinson MR, Kamberov ES, Tirasophon W, Chandran P, Schefke BR, Ninfa AJ

Abstract

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.

MeSH Terms
Adenosine Triphosphate/metabolism Amino Acid Sequence Bacterial Proteins/chemistry,genetics,metabolism Escherichia coli/metabolism Ketoglutaric Acids/metabolism Ligands Molecular Sequence Data Molecular Structure Mutagenesis Nucleotidyltransferases/metabolism PII Nitrogen Regulatory Proteins Phosphoprotein Phosphatases/metabolism Protein Binding Protein Kinases/metabolism Signal Transduction Structure-Activity Relationship UDPglucose-Hexose-1-Phosphate Uridylyltransferase/metabolism
Chemicals
Bacterial Proteins Ketoglutaric Acids Ligands PII Nitrogen Regulatory Proteins PIID regulatory protein, Bacteria Adenosine Triphosphate Protein Kinases protein kinase-phosphatase NTRB Nucleotidyltransferases UDPglucose-Hexose-1-Phosphate Uridylyltransferase glutamine-synthetase adenylyltransferase Phosphoprotein Phosphatases
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Jiang P
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Zucker P
Atkinson M R
Kamberov E S
Tirasophon W
Chandran P
Schefke B R
Ninfa A J
References (45)
45 references, click to expand
  1. Dideoxy sequencing method using denatured plasmid templates.
    Anal Biochem. 1986 Feb 1;152(2):232-8 PMID: 3516005
  2. Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes.
    Proc Natl Acad Sci U S A. 1980 May;77(5):2636-40 PMID: 6104810
  3. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region.
    Gene. 1987;52(2-3):279-83 PMID: 3038690
  4. The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation.
    Mol Gen Genet. 1989 Jun;217(2-3):474-80 PMID: 2570349
  5. Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.
    Arch Biochem Biophys. 1978 Dec;191(2):590-601 PMID: 33596
  6. Sensory components controlling bacterial nitrogen assimilation.
    Cell Mol Biol Res. 1994;40(3):175-91 PMID: 7874194
  7. The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli.
    Mol Microbiol. 1993 Aug;9(3):443-57 PMID: 8412694
  8. Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase NRII.
    J Biol Chem. 1991 Apr 15;266(11):6888-93 PMID: 2016302
  9. Probing interactions of the homotrimeric PII signal transduction protein with its receptors by use of PII heterotrimers formed in vitro from wild-type and mutant subunits.
    J Bacteriol. 1997 Jul;179(13):4354-60 PMID: 9209054
  10. Glutamine synthetase from Escherichia coli.
    Methods Enzymol. 1985;113:213-41 PMID: 2868385
  11. Modulation of glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the P II -regulatory protein.
    Proc Natl Acad Sci U S A. 1971 Dec;68(12):2949-53 PMID: 4399832
  12. Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli.
    J Bacteriol. 1982 Jun;150(3):1302-13 PMID: 6122676
  13. The glnB region of the Escherichia coli chromosome.
    J Bacteriol. 1993 Nov;175(22):7441-9 PMID: 8226691
  14. Regulation of glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deuridylylation of the PII regulatory protein.
    Arch Biochem Biophys. 1973 Oct;158(2):514-25 PMID: 4150122
  15. Structure of the Escherichia coli signal transducing protein PII.
    Structure. 1994 Oct 15;2(10):981-90 PMID: 7866749
  16. An additional PII in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade.
    FEMS Microbiol Lett. 1995 Oct 1;132(1-2):153-7 PMID: 7590157
  17. Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme.
    J Biol Chem. 1975 Aug 25;250(16):6264-72 PMID: 239942
  18. Characterization of Escherichia coli glnL mutations affecting nitrogen regulation.
    J Bacteriol. 1992 Jul;174(14):4538-48 PMID: 1352516
  19. Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli.
    J Bacteriol. 1985 Nov;164(2):816-22 PMID: 2865248
  20. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli.
    Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909-13 PMID: 2874557
  21. The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP.
    J Biol Chem. 1995 Jul 28;270(30):17797-807 PMID: 7629080
  22. X-ray structure of the signal transduction protein from Escherichia coli at 1.9 A.
    Acta Crystallogr D Biol Crystallogr. 1996 Jan 1;52(Pt 1):93-104 PMID: 15299730
  23. Regulation of Escherichia coli glutamine synthetase.
    Adv Enzymol Relat Areas Mol Biol. 1989;62:37-92 PMID: 2567108
  24. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC.
    Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976-80 PMID: 2839825
  25. Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.
    J Bacteriol. 1975 Aug;123(2):407-18 PMID: 238954
  26. Cloning and organization of the abc and mdl genes of Escherichia coli: relationship to eukaryotic multidrug resistance.
    Gene. 1993 Dec 22;136(1-2):231-6 PMID: 7904973
  27. Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene.
    J Biol Chem. 1987 Jun 25;262(18):8690-5 PMID: 2885322
  28. Mutational analysis of the bacterial signal-transducing protein kinase/phosphatase nitrogen regulator II (NRII or NtrB).
    J Bacteriol. 1993 Nov;175(21):7016-23 PMID: 7901195
  29. Expression of glnA in Escherichia coli is regulated at tandem promoters.
    Proc Natl Acad Sci U S A. 1985 Apr;82(7):1979-83 PMID: 2858855
  30. The products of glnL and glnG are bifunctional regulatory proteins.
    Mol Gen Genet. 1982;188(2):325-33 PMID: 6130464
  31. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli.
    J Bacteriol. 1992 Oct;174(19):6061-70 PMID: 1356964
  32. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.
    Biotechniques. 1991 Nov;11(5):620-7 PMID: 1804254
  33. Feedback inhibition of fully unadenylylated glutamine synthetase from Salmonella typhimurium by glycine, alanine, and serine.
    Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4996-5000 PMID: 8099447
  34. Isolation of the nitrogen assimilation regulator NR(I), the product of the glnG gene of Escherichia coli.
    Proc Natl Acad Sci U S A. 1983 Sep;80(18):5554-8 PMID: 16593366
  35. Protein measurement with the Folin phenol reagent.
    J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
  36. Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits.
    J Bacteriol. 1993 Nov;175(21):7024-32 PMID: 8226644
  37. Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade.
    Arch Biochem Biophys. 1978 Dec;191(2):602-12 PMID: 33597
  38. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli.
    Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919-23 PMID: 2848245
  39. Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.
    J Bacteriol. 1996 Jul;178(14):4143-9 PMID: 8763942
  40. Effect of mutations in Escherichia coli glnL (ntrB), encoding nitrogen regulator II (NRII or NtrB), on the phosphatase activity involved in bacterial nitrogen regulation.
    J Biol Chem. 1994 Nov 11;269(45):28294-9 PMID: 7961767
  41. Activation of the dephosphorylation of nitrogen regulator I-phosphate of Escherichia coli.
    J Bacteriol. 1995 Feb;177(4):926-31 PMID: 7860602
  42. DNA polymerase beta belongs to an ancient nucleotidyltransferase superfamily.
    Trends Biochem Sci. 1995 Sep;20(9):345-7 PMID: 7482698
  43. An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli.
    Mol Microbiol. 1996 Jul;21(1):133-46 PMID: 8843440
  44. The role of adenylyltransferase and uridylyltransferase in the regulation of glutamine synthetase in Escherichia coli.
    Curr Top Cell Regul. 1985;27:221-32 PMID: 2868842
  45. Reversible uridylylation of the Escherichia coli PII signal transduction protein regulates its ability to stimulate the dephosphorylation of the transcription factor nitrogen regulator I (NRI or NtrC).
    J Biol Chem. 1994 Nov 11;269(45):28288-93 PMID: 7961766
Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.179.13.4342-4353.1997
Published
1997-07-00
Pages
4342-53
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC179259
Subset
IM
Grants
NIGMS NIH HHS · GM47460 · United States
External Links
PubMed source DOI: 10.1128/jb.179.13.4342-4353.1997 PMC Full Text View journal details
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