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

Kornelius Zeth,Oleksandra Fokina,Karl Forchhammer

doi:10.1074/jbc.m113.536557

Kornelius Zeth, Oleksandra Fokina + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m113.536557

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Abstract

PII 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 PII 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 PII protein and thereby affect its structure. Here we used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of PII-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric PII, 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 PII-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 PII-ADP. In consequence, the PII-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the PII-N-acetyl-L-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of PII by its receptors allows PII to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction.

Highlights

Conserved PII signaling proteins monitor carbon/nitrogen balance and energy status
In the present study we investigated the structural transitions of effector-free and ADP-loaded PII subunits from S. elongatus and propose a mechanism of ADP sensing by PII
PII Structures Carry Flexible Modules to Adapt to Effector and Protein Interaction—Crystal structures of PII enzymes have shown a variety of conformational states, with transitions typically affecting flexible modules surrounding the structurally invariant ␤-sheet core

Summary

Background

Conserved PII signaling proteins monitor carbon/nitrogen balance and energy status. Results: Crystals of homotrimeric Synechococcus elongatus PII with ADP reveal the structural basis of anti-cooperative ADP binding. PipX and N-acetyl-L-glutamate kinase are receptors of PII 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 PII protein and thereby affect its structure. We used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. In the PII protein from the cyanobacterium Synechococcus elongatus PCC 7942, the binding of ATP and ADP to the three sites is anti-cooperative, with ATP having a higher affinity than ADP [9]. In living cells ATP and ADP are simultaneously present, and they compete for the three nucleotide-binding sites of the PII

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION