Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

P II signalling proteins constitute one of the largest families of signalling proteins in nature. An even larger superfamily of trimeric sensory proteins with the same architectural principle as P II proteins appears in protein structure databases. Large surface-exposed flexible loops protrude from the intersubunit faces, where effector molecules are bound that tune the conformation of the loops. Via this mechanism, P II proteins control target proteins in response to cellular ATP/ADP levels and the 2-oxoglutarate status, thereby coordinating the cellular carbon/nitrogen balance. The antagonistic (ATP versus ADP) and synergistic (2-oxoglutarate and ATP) mode of effector molecule binding is further affected by P II -receptor interaction, leading to a highly sophisticated signalling network organized by P II . Altogether, it appears that P II is a multitasking information processor that, depending on its interaction environment, differentially transmits information on the energy status and the cellular 2-oxoglutarate level. In addition to the basic mode of P II function, several bacterial P II proteins may transmit a signal of the cellular glutamine status via covalent modification. Remarkably, during the evolution of plant chloroplasts, glutamine signalling by P II proteins was re-established by acquisition of a short sequence extension at the C-terminus. This plant-specific C-terminus makes the interaction of plant P II proteins with one of its targets, the arginine biosynthetic enzyme N-acetyl-glutamate kinase, glutamine-dependent. General properties of P II proteinsThe designation 'P II protein' refers to proteins belonging to a class of small trimeric signal-transducing proteins characterized by special structural and functional features (Fig. 1A). P II proteins have been studied ever since the late 1960s and, subsequently, they have been reviewed extensively [1][2][3][4][5][6][7][8][9][10]. In general, they are encoded by glnB, glnK or nifI genes (in some cases, glnK paralogues are termed glnZ or glnJ) and, in almost all cases, these P II proteins are engaged in coordination of central anabolic metabolism; in particular, the regulation of anabolic reactions related to nitrogen metabolism. Members of the P II family occur in almost all free-living bacteria, in nitrogen-fixing archaea and in the chloroplasts of red algae and green plants. As such, they constitute one of the largest signalling protein families in nature. The basic mechanism of signal perception by P II proteins is highly conserved via binding of adenylnucleotides and 2-oxoglutarate (2-OG). P II proteins process the information via conformational changes imposed by effector molecule binding and they transduce the resulting state to their targets via protein interactions. In the course of evolution, P II proteins have been recruited to coordinate a variety of different cellular processes revolving around nitrogen assimilation. In many bacteria, P II proteins of the GlnB subfamily are involved in the regulation of different steps of nitrogen assim...

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.

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