Specificity and regulation of interaction between the P_II and AmtB₁ proteins in Rhodospirillum rubrum

The nitrogen regulatory protein P_II, and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P_II homolog, GlnJ, has higher affinity for an AmtB₁-containing membrane than the other two P_II homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB₁ complex in the presence of ATP and 2-KG, supporting a role for P_II as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K_d of 2-KG for GlnJ bound to AmtB₁ is 340 μM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 μM. This supports a model where multiple 2-KG and ATP molecules must bind a P_II trimer to stimulate release of P _II from AmtB₁, in contrast to the lower 2-KG requirement for productive uridylylation of P_II by GlnD.