The crystal structure of the complex of P _II and acetylglutamate kinase reveals how P _II controls the storage of nitrogen as arginine

Abstract

Photosynthetic organisms can store nitrogen by synthesizing arginine, and, therefore, feedback inhibition of arginine synthesis must be relieved in these organisms when nitrogen is abundant. This relief is accomplished by the binding of the P II signal transduction protein to acetylglutamate kinase (NAGK), the controlling enzyme of arginine synthesis. Here, we describe the crystal structure of the complex between NAGK and P II of Synechococcus elongatus, at 2.75-Å resolution. We prove the physiological relevance of the observed interactions by site-directed mutagenesis and functional studies. The complex consists of two polar P II trimers sandwiching one ring-like hexameric NAGK (a trimer of dimers) with the threefold axes of these molecules aligned. The binding of P II favors a narrow ring conformation of the NAGK hexamer that is associated with arginine sites having low affinity for this inhibitor. Each PII subunit contacts one NAGK subunit only. The contacts map in the inner circumference of the NAGK ring and involve two surfaces of the P II subunit. One surface is on the PII body and interacts with the C-domain of the NAGK subunit, helping widen the arginine site found on the other side of this domain. The other surface is at the distal region of a protruding large loop (T-loop) that presents a novel compact shape. This loop is inserted in the interdomain crevice of the NAGK subunit, contacting mainly the N-domain, and playing key roles in anchoring P II on NAGK, in activating NAGK, and in complex formation regulation by MgATP, ADP, 2-oxoglutarate, and by phosphorylation of serine-49.arginine synthesis ͉ regulation ͉ x-ray structure ͉ signaling ͉ cyanobacteria I n photosynthetic organisms nitrogen can be stored by synthesizing arginine (1, 2) and, therefore, feedback inhibition of arginine synthesis must be relieved when nitrogen is abundant. The enzyme of arginine biosynthesis that is the target of arginine inhibition, N-acetyl-L-glutamate (NAG) kinase (NAGK) (1, 3-5), was found in cyanobacteria and plants (2, 4-8) to be a target of the carbon/ nitrogen P II signaling protein (9, 10), forming with it a complex in which arginine inhibition is alleviated (6, 7). P II signaling proteins are homotrimers of a 12-to 13-kDa subunit that interact with enzymes, transcription factors, and ammonia channels, regulating their activity (9, 10) and carbon/ nitrogen homeostasis. Numerous structures of P II proteins, including those for cyanobacteria and plants (9-12), are known, but it was unclear how P II proteins carry out their functions. The body of the P II trimer is roughly hemispheric. Its subunits have ␤␣␤␤␣␤ topology, with ␣ helices looking outward and the ␤ sheet inward and providing the intersubunit interactions. Each subunit has three loops: the B-and C-loops and the larger flexible T-loop. The T-loop residues Y51 and S49 are, respectively, the sites of the regulatory uridylylation and phosphorylation in enterobacterial and cyanobacterial P II proteins (9, 10), with S49 phosphorylation abolishing interaction wi...

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

“…Moreover, both P II variants were impaired in NAGK binding, confirming that K58 is indeed pivotal for folding the T loop in the tightly bent structure. The R9 side chain is near the contact surface to NAGK and appears to stabilize the B-loop-T-loop interface (23) (Fig. S2).…”

“…In Fig. 1, this structure is compared to the structures of ligand-free P II (14) and P II in complex with NAGK (23). In the ligand-free structure, the T loop, and the C terminus adopt highly extended conformations away from the ATP-binding site (see Fig.…”

“…By contrast, a recently published structure of a P II homologue form Azospirillum brasiliense in complex with Mg-ATP and 2-OG revealed the 2-OG-binding site close to the base of the T loop and near the ATP-binding site (21). However, neither of these two structures was proved by biochemical studies nor could they explain the anticooperative binding of 2-OG.The structures of complexes of P II with its regulatory target NAGK from Synechococcus elongatus and A. thaliana are highly similar (22,23), and the mode of interaction and regulation is apparently conserved in cyanobacteria and plants (24). The P II -NAGK complex involves one hexameric (trimer of dimers) NAGK toroid sandwiched between two P II trimers with the threefold axis aligned (23).…”

“…The structures of complexes of P II with its regulatory target NAGK from Synechococcus elongatus and A. thaliana are highly similar (22,23), and the mode of interaction and regulation is apparently conserved in cyanobacteria and plants (24). The P II -NAGK complex involves one hexameric (trimer of dimers) NAGK toroid sandwiched between two P II trimers with the threefold axis aligned (23).…”

“…The P II -NAGK complex involves one hexameric (trimer of dimers) NAGK toroid sandwiched between two P II trimers with the threefold axis aligned (23). Each P II subunit engages two contact surfaces in NAGK binding: A smaller surface involves the B loop and a larger is formed by the T loop, which adopts a tightly bent conformation that fits into the interdomain crevice of NAGK.…”