title: Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.
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.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
authors.forename: P
authors.initials: P
authors.lastname: Jiang
authors.forename: P
authors.initials: P
authors.lastname: Zucker
authors.forename: M R
authors.initials: MR
authors.lastname: Atkinson
authors.forename: E S
authors.initials: ES
authors.lastname: Kamberov
authors.forename: W
authors.initials: W
authors.lastname: Tirasophon
authors.forename: P
authors.initials: P
authors.lastname: Chandran
authors.forename: B R
authors.initials: BR
authors.lastname: Schefke
authors.forename: A J
authors.initials: AJ
authors.lastname: Ninfa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D008024:Ligands; D051922:PII Nitrogen Regulatory Proteins; C404143:PIID regulatory protein, Bacteria; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D009713:Nucleotidyltransferases; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; C020658:glutamine-synthetase adenylyltransferase; D010749:Phosphoprotein Phosphatases