Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus P _II signal transduction protein

Abstract

P II proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, 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 2þ , 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 2þ 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.metabolic signaling | nitrogen regulation | cyanobacteria | chloroplasts T he P II proteins constitute one of the largest and most widely distributed family of signal transduction proteins present in archaea, bacteria, and plants. They control key processes of nitrogen metabolism in response to central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance (1-4). These effectors bind to P II in an interdependent manner (see below), thereby transmitting metabolic information into structural states of this sensor protein (3, 5). Furthermore, P II proteins may be posttranslationally modified (1, 6). Depending on the signal input states, P II proteins bind and thereby regulate the activity of key metabolic and regulatory enzymes, transcription factors, or transport proteins (1-3). In cyanobacteria and plants, the controlling enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase (NAGK), is a major P II target (7-9). Moreover, P II affects gene expression in cyanobacteria through binding to the transcriptional coactivator of NtcA, PipX (10). In plants, acetyl-CoA carboxylase was recently shown to be regulated by P II , providing an additional link between carbon and nitrogen regulation (11). Although these P II targets share no common structural element, interaction with P II is inhibited by 2-OG.P II proteins are homotrimers of 12-to 13-kDa subunits, built of a double ferredoxin-like fold-containing core (βαβ-βαβ), with a characteristic and highly cons...

From cyanobacteria to plants: conservation of PII functions during plastid evolution

“…2a): from one subunit, Arg38 and Gln39 residues (belonging to the basal part of the T-loop) contact the c-phosphate of ATP, whereas Lys90 and Gly89 contact the b-and a-phosphate; from the opposite subunit, two Arg residues (101 and 103) of the C-loop contact c-and b-phosphates, whereas Thr29 and Val64 coordinate the adenosine moiety. The mode of 2-oxoglutarate binding to PII has been clarified recently by solving the crystal structures of PII proteins with Mg-ATP and 2-OG from the archaeon Archaeoglobus fulgidus (Maier et al 2011), the proteobacterium Azospirillum brasiliense (Truan et al 2010), and the cyanobacterium S. elongatus (Fokina et al 2010a). In the latter case, a snapshot of the sequential anticooperative binding of 2-OG to the three sites could be obtained.…”

Crystal structure of the GlnZ-DraG complex reveals a different form of PII-target interaction

“…The nucleotide binding sites are mainly formed by the B-and C-loops from opposing subunits; ADP and ATP can occupy these sites competitively (4,6). The recent structures of Azospirillum brasilense GlnZ (7) and of Synechococcus elongatus P II (8) revealed that the P II 2-OG binding site is also located in the intersubunit cleft adjacent to the nucleotide binding pocket.…”

PipX, the coactivator of NtcA, is a global regulator in cyanobacteria

“…P II contains three binding sites (one per subunit) for 2-OG and ATP (6,7), and it regulates the activity of N-acetyl-glutamate-kinase (NAGK), a key enzyme for biosynthesis of arginine, by direct protein-protein interactions (3,8,9). The 2-OG stimulates binding of NtcA to target sites (10), transcription activation in vitro (11), and complex formation between NtcA and PipX (12).…”