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.
