title: 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.
abstract: The homotrimeric PII signal transduction protein of Escherichia coli interacts with two small-molecule effectors, 2-ketoglutarate and ATP, regulates two protein receptors, the kinase/phosphatase nitrogen regulator II (NRII) and the glutamine synthetase (GS) adenylyltransferase (ATase), and is subject to reversible uridylylation, catalyzed by the uridylyltransferase/uridylyl-removing enzyme (UTase/UR). The site of PII uridylylation, Y51, is located at the apex of the solvent-exposed T-loop (E. Cheah, P. D. Carr, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Structure 2:981-990, 1994), and an internally truncated PII lacking residues 47 to 53 formed trimers that bound the small-molecule effectors but were unable to be uridylylated or activate NRII and ATase (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179:4342-4353, 1997). We investigated the ability of heterotrimers containing delta47-53 and wild-type subunits to become uridylylated and activate NRII and ATase. Heterotrimers were formed by denaturation and renaturation of protein mixtures; when such mixtures contained a fivefold excess of A47-53 subunits, the wild-type subunits were mostly redistributed into trimers containing one wild-type subunit and two mutant subunits. The resulting population of trimers was uridylylated and deuridylylated by UTase/UR, stimulated the phosphatase activity of NRII, and stimulated adenylylation of GS by ATase. In all except the ATase interaction, the activity of the hybrid trimers was greater than expected based on the number of wild-type subunits present. These results indicate that a single T-loop region within a trimer is sufficient for the productive interaction of PII with its protein receptors. We also formed heterotrimers containing wild-type subunits and subunits containing the G89A alteration (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). The G89A mutant form of PII does not bind the small-molecule effectors, does not interact with UTase or with NRII, and interacts poorly with ATase. Heterotrimers formed with a 10/1 starting ratio of G89A to wild-type subunits interacted with UTase/UR and ATase to a lesser extent than expected based on the number of wild-type subunits present but activated NRII slightly better than expected based on the number of wild-type subunits present. Thus, intersubunit interactions within the PII trimer can adversely affect the activity of wild-type subunits and may affect the interactions with the different receptors in a variable way. Finally, we formed heterotrimers containing delta47-53 and G89A mutant subunits. These heterotrimers were not uridylylated, did not interact with NRII, and interacted with the ATase only to the extent expected based on the number of G89A subunits present. Thus, the G89A subunits, which contain an intact T-loop region, were not "repaired" by inclusion in heterotrimers along with delta47-53 subunits.
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: A J
authors.initials: AJ
authors.lastname: Ninfa
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D051922:PII Nitrogen Regulatory Proteins; C404143:PIID regulatory protein, Bacteria; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D009713:Nucleotidyltransferases; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; C020658:glutamine-synthetase adenylyltransferase; D010749:Phosphoprotein Phosphatases