Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio

Abstract

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated in vitro by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/ nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in R. rubrum, a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

Control of AmtB-GlnK Complex Formation by Intracellular Levels of ATP, ADP, and 2-Oxoglutarate

“…However, for regulation of AmtB-GlnK complex formation, the antagonistic effect of ADP on 2-OG binding also appears to be critical. Many authors have proposed that P II proteins can sense changes in adenylate charge (16,21,42,43 (40,44), and our data are consistent with this. Consequently, it remains to be established whether there is a common underlying physiological link between the ATP/ADP ratio and regulation of P II interactions with specific targets.…”

“…At high 2-OG and in the absence of AmtB, formation of the GlnK-ATP complex was favored compared with GlnK-ADP by 2.7 kcal/ mol, whereas at low 2-OG and in the presence of AmtB, the reciprocal situation pertained, with formation of the AmtBGlnK-ADP complex favored by 2.4 kcal/mol (Table 1). These data for E. coli GlnK are largely consistent with similar studies of ligand binding to E. coli GlnB, which was assessed using an alternative technique (16). GlnB also shows negative cooperativity of ATP binding and synergistic binding of ATP and 2-OG.…”

“…In Vivo Pools of 2-OG, ATP, and ADP in Pre-and Post-ammonium-shocked Cells-A number of studies have suggested that in vivo formation of an AmtB-P II complex is influenced by the intracellular pools of MgATP, ADP, and 2-OG metabolites (15)(16)(17)23). To establish the relationship between these metabolites and the cellular nitrogen status, we determined the intracellular pools of ATP, ADP, and 2-OG in E. coli over a period of 15 min following a 200 M ammonium shock.…”

P II signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate

“…A number of authors also have proposed that in addition to sensing the cellular nitrogen-status, P II proteins may also act as sensors of cellular adenylate energy charge; that is, as monitors of the ATP:ADP ratio in the cell (13)(14)(15)(16)(17)(18). The majority of those studies report either the differential binding of ATP and ADP to P II proteins in vitro or the in vitro effects of ATP and ADP on interactions of P II proteins with a number of their targets.…”

“…It was long considered that ATP could not play a regulatory role because its intracellular concentration is typically 1-5 mM, whereas the affinity of P II proteins for the nucleotide is in the micromole range (K d ∼50 μM) (11,12). However, the subsequent recognition that ADP is also a physiological effector (10) led to a reevaluation of the role of nucleotides, and a number of studies concluded that P II proteins might also act as sensors of cellular energy status, as reflected by fluctuations in the ATP/ADP ratio (13)(14)(15)(16)(17)(18).…”