Sensation and signaling of alpha-ketoglutarate and adenylylate energy charge by the Escherichia coli PII signal transduction protein require cooperation of the three ligand-binding sites within the PII trimer.
PII proteins are sensors of alpha-ketoglutarate and adenylylate energy charge that regulate signal transduction proteins, metabolic enzymes, and permeases involved in nitrogen assimilation. Here, purified Escherichia coli PII and two of its receptors, ATase and NRII, were used to study the mechanisms of sensation by PII. We assembled heterotrimeric forms of PII from wild-type and mutant subunits, which allowed us to assess the role of the three binding sites for alpha-ketoglutarate and adenylylate nucleotide in the PII trimer. Signaling of alpha-ketoglutarate and adenylylate energy charge by these heterotrimeric PII proteins required multiple binding sites for these effectors, and the ligand-binding sites on different subunits could influence the function of a single subunit interacting with a receptor, implying communication between PII subunits. Wild-type and heterotrimeric forms of PII were also used to examine the effects of alpha-ketoglutarate and ADP on PII activation of the adenylyltransferase (AT) activity of ATase. Previous work showed that when ATP was the sole adenylylate nucleotide, alpha-ketoglutarate controlled the extent of PII activation but did not alter the PII activation constant (K(act)). We show that ADP affected both the PII K(act) and the extent of activation by PII. When ATP was present, ADP dramatically reduced the K(act) for wild-type PII, and this effect was antagonized by alpha-ketoglutarate. Consequently, when ATP was present, the antagonism between ADP and alpha-ketoglutarate allowed each of these effectors to influence the PII K(act) for activation of ATase. A study of heterotrimeric forms of PII suggested that the major part of the ability of ADP to improve the binding of PII to ATase required multiple nucleotide binding sites and intersubunit communication. We also used nondenaturing gel electrophoresis to investigate the effect of ADP and alpha-ketoglutarate on the binding of PII to ATase and NRII. These studies showed that ATase and NRII differ in their requirements for interaction with PII, and that under the appropriate conditions, the antagonism between alpha-ketoglutarate and ADP allowed each of these effectors to influence the binding of PII to receptors.
Jiang
Peng
P
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA. pejiang@umich.edu
Ninfa
Alexander J
AJ
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004586:Electrophoresis; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008024:Ligands; D009713:Nucleotidyltransferases / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction
D000227:Adenine Nucleotides; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D008024:Ligands; D051922:PII Nitrogen Regulatory Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D009713:Nucleotidyltransferases; D010749:Phosphoprotein Phosphatases