Structural Basis for the Regulation of N-Acetylglutamate Kinase by PII in Arabidopsis thaliana

Abstract

PII is a highly conserved regulatory protein found in organisms across the three domains of life. In cyanobacteria and plants, PII relieves the feedback inhibition of the rate-limiting step in arginine biosynthesis catalyzed by N-acetylglutamate kinase (NAGK). To understand the molecular structural basis of enzyme regulation by PII, we have determined a 2.5-Å resolution crystal structure of a complex formed between two homotrimers of PII and a single hexamer of NAGK from Arabidopsis thaliana bound to the metabolites N-acetylglutamate, ADP, ATP, and arginine. In PII, the T-loop and Trp 22 at the start of the ␣1-helix, which are both adjacent to the ATP-binding site of PII, contact two ␤-strands as well as the ends of two central helices (␣E and ␣G) in NAGK, the opposing ends of which form major portions of the ATP and N-acetylglutamate substratebinding sites. The binding of Mg 2؉ ⅐ATP to PII stabilizes a conformation of the T-loop that favors interactions with both open and closed conformations of NAGK. Interactions between PII and NAGK appear to limit the degree of opening and closing of the active-site cleft in opposition to a domain-separating inhibitory effect exerted by arginine, thus explaining the stimulatory effect of PII on the kinetics of arginine-inhibited NAGK.PII (GlnB) is now recognized as one of the most ancient and conserved signal transduction proteins known, with orthologs spread across the three domains of life (1, 2). Originally discovered as a factor necessary for the inactivation of Escherichia coli glutamine synthetase, PII is now known to play roles in the regulation of gene transcription, enzyme activity, and membrane channel function, all in response to cellular carbon, nitrogen, and energy status (3-6). PII interprets the metabolic status of the cell by directly binding to ATP and 2-ketoglutarate, and in certain bacteria, nitrogen status is sensed via covalent modification of PII. PII then directly interacts with a variety of proteins to regulate their function in response to the detected metabolic conditions.A eukaryotic PII protein has been discovered in several algae and higher plants. Arabidopsis thaliana PII is Ͼ50% identical to proteobacterial and cyanobacterial PII (7), but unlike these orthologs, it does not appear to be regulated by phosphorylation or uridylylation in response to nitrogen metabolites (8). Plant PII has a conserved chloroplast transit peptide that is cleaved upon entry into the chloroplast, where PII performs its function. PII knock-out plants display altered carbon and nitrogen metabolite levels as well as increased sensitivity to nitrite (9).To date, the sole interacting protein discovered for plant PII is the chloroplast enzyme N-acetylglutamate kinase (NAGK), 2 which catalyzes the second and rate-limiting step in the pathway of arginine biosynthesis (10). We (11) and others (12) have performed enzyme kinetics to define the role of PII in NAGK activity: although PII activates NAGK slightly, the primary purpose of binding appears to be relief of feedback inhi...

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

“…It has been shown that 2-OG controls P II target interactions that involve the T loop, with X-ray structural information available for the S. elongatus and A. thaliana P II -NAGK complex (22,23), the S. elongatus P II -PipX complex (27), and the E. coli GlnK-AmtB complex (28,29). The mechanism of 2-OGmediated P II -target control was clarified here with the cognate P II -NAGK complex.…”

“…The ATP molecule observed in the S2 site is significantly distorted relative to that in S1 and S3. ADP could involve additional residues, possibly from its prolonged C-terminal segment, which contacts the effector moleculebinding site (22). The fact that all residues revealed here to be involved in 2-OG binding are highly conserved among P II proteins (see also 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).…”

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

“…The C-terminal residues extend out from the b4 strand to form a C-loop (Asp97 to Leu112) and has a 3 10 helix embedded within it between Gly108 and Ala111, consistent with other PII like protein structures. 16,24,[30][31][32][33][34][35] Structure of the ATP bound Mtb PII protein…”