Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus P_(II) signal transduction protein

摘要

P_(II) proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADR and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P_(II) and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P_(II) target, whose activity responds to 2-OG via P_(II). Here we show structures of the Synechococcus elongatus P_(II) protein in complex with ATP, Mg~(2+), and 2-OG, which clarify how 2-OG affects P_(II)-NAGK interaction. P_(II) trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P_(II) trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg~(2+) ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B- and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P_(II) target. Structures of P_(II) trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P_(II) senses effectors in a wide range of concentrations.