Interactions between the PII protein and its receptors revealed by NanoBiT technology

Abstract

The PII proteins are notable members of a vast and ancient protein family involved in signal transduction. These molecules are found in all living organisms and are primarily recognized for their ability to sense metabolites such as ATP, ADP, and 2-oxoglutarate (2-OG). When the effector molecules are non-covalently bound by PII, they cause several structural changes in PII proteins, particularly in their flexible T-loops, which serve as dynamic modules for protein-protein interactions. The interpretation of metabolic data sent by PII is dependent on the binding state of metabolites and the resulting conformation of PII receptors. To thoroughly investigate the complex interactions between PII and target proteins, analytical methods that maintain the natural cellular milieu are needed. In light of the limitations inherent in alternative methodologies such as immobilization on sensor surfaces in Surface-Plasmon-Resonance (SPR) and Biolayer Interferometry (BLI), as well as the reliance on sizable fluorescence proteins in Förster Resonance Energy Transfer (FRET), our research endeavors focused on the development of an innovative NanoBiT sensor. The focus of this sensor is on the interaction of the PII protein derived from Synechocystis sp. PCC6803 with the PII-interacting protein X (PipX), N-acetyl-L glutamate kinase (NAGK) and the PII-interacting regulator of arginine synthesis (PirA). Using the NanoBiT technology, we have attained an advanced comprehension, enabling the calculation of KD values for the PII-NAGK and PII-PipX complexes, which have not been previously reported. The test also demonstrated an increased level of sensitivity, allowing for the detection of low-affinity interactions, such as the one seen between the PII-S49E variant and NAGK. The study also highlights astounding proof indicating that the development of the PII-NAGK complex is impacted by the presence of ADP, which reduces the complex affinity. Additional analysis by the NanoBiT method and enzymatic assays provided further evidence that the PII-NAGK complex exhibits specific feed forward activation in response to increasing concentrations of NAG. These two sensors were also applied to investigate the real time metabolic fluctuations in response to nitrogen upshift or nitrogen depletion treatments. Furthermore, our exploration extended to a small protein encoded by the ssr0692 gene in Synechocystis sp. PCC 6803. The protein regulates the flux into the ornithine-ammonia cycle (OAC), a pivotal mechanism for the accumulation and redistribution of nitrogen in cyanobacteria. The regulation described in this context arises from the connection between the PII protein and the OAC cycle. PII traditionally regulates the key enzyme NAGK, which catalyzes arginine production. The Ssr0692 protein competes with NAGK for PII binding, resulting in the inhibition of NAGK activation and a consequent reduction in arginine synthesis. In light of its function, we have identified it as the PII Interacting Regulator of Arginine Synthesis (PirA). The interaction between PirA and PII depends on the presence of ADP and is hindered by mutations in PII that affect the structure of the T-loop. Therefore, we propose that PirA serves as a crucial mediator, directing flux into nitrogen storage compounds by considering both the availability of nitrogen and the energy level of the cell.

Dissertation ist gesperrt bis 31. Juli 2025 !