title: From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.
abstract: The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.
authors.affiliation: Interfaculty Institute for Microbiology and Infection Medicine (IMIT), Eberhard Karls University, Tübingen, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Lüddecke
authors.affiliation: Interfaculty Institute for Microbiology and Infection Medicine (IMIT), Eberhard Karls University, Tübingen, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Forchhammer
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046940:Synechococcus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D046912:Multiprotein Complexes; D051922:PII Nitrogen Regulatory Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase