Structural basis for the regulation of N-acetylglutamate kinase by PII in Arabidopsis thaliana.
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-A 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 alpha1-helix, which are both adjacent to the ATP-binding site of PII, contact two beta-strands as well as the ends of two central helices (alphaE and alphaG) in NAGK, the opposing ends of which form major portions of the ATP and N-acetylglutamate substrate-binding sites. The binding of Mg(2+).ATP 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.
Mizuno
Yutaka
Y
Department of Biological Sciences and the Alberta Ingenuity Centre for Carbohydrate Science, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Moorhead
Greg B G
GB
Ng
Kenneth K-S
KK
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001120:Arginine / Q000096:biosynthesis / Q000737:chemistry; D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000737:chemistry* / Q000378:metabolism; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D013329:Structure-Activity Relationship
D029681:Arabidopsis Proteins; D046912:Multiprotein Complexes; D051922:PII Nitrogen Regulatory Proteins; C473074:PII protein, Arabidopsis; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase; D008274:Magnesium