Structural Basis and Target-specific Modulation of ADP Sensing by the Synechococcus elongatus PII Signaling Protein

Background: Highly conserved P-II signaling proteins monitor carbon/nitrogen balance and energy status. Results: Crystals of homotrimeric Synechococcus elongatus P-II with ADP reveal the structural basis of anti-cooperative ADP binding. Interaction with PipX receptor changes the ATP/ADP preference of P-II. Conclusion: P-II signaling is modulated by conformational states imposed by the receptor protein. Significance: Molecular information is processed through protein structure plasticity. P-II signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of P-II signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric P-II protein and thereby affect its structure. Here we used the P-II protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of P-II-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric P-II, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the P-II-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of P-II-ADP. In consequence, the P-II-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the P-II-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of P-II by its receptors allows P-II to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction.