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.
Highlights
PII proteins represent one of the most widespread and most ancient signal transduction protein families in nature
To its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - N-acetyl-Lglutamate kinase (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
The possibility of using PII for FRET approaches opens a wide field of possible sensor applications as PII proteins have many different interaction partners whose interactions depend on different metabolic conditions
Summary
PII proteins represent one of the most widespread and most ancient signal transduction protein families in nature. They are found in bacteria, archea and in chloroplasts of green algae and plants. PII proteins act as signal processing units by integrating the cellular energy and nitrogen supply through binding of the central metabolites 2-oxoglutarate (2-OG), ATP and ADP in an interdependent manner [1,2,3,4]. The conformational changes induced by binding of these molecules enable PII proteins to interact in a regulated manner with a wide range of partners including enzymes, transcription factors and membrane proteins. The three effector binding sites are positioned in the three intersubunit clefts, contacting the proximal part of the respective
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