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J. Biol. Chem., Vol. 282, Issue 49, 35733-35740, December 7, 2007

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

From the Department of Biological Sciences and the Alberta Ingenuity Centre for Carbohydrate Science, University of Calgary, Calgary, Alberta T2N 1N4, Canada

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 Trp²² at the start of the 1-helix, which are both adjacent to the ATP-binding site of PII, contact two β-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 substrate-binding sites. The binding of Mg²⁺·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.

Received for publication, August 24, 2007 , and in revised form, September 21, 2007.

The atomic coordinates and structure factors (code 2RD5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by Natural Sciences and Engineering Research Council of Canada Discovery grants (to K. K.-S. N. and G. B. G. M.), the Alberta Ingenuity Centre for Carbohydrate Science, a Canadian Institutes of Health Research New Investigator Award (to K. K.-S. N.), and an Alberta Heritage Foundation for Medical Research Medical Senior Scholar Award (to K. K.-S. N.). X-ray diffraction data were collected with the support of the Alberta Synchrotron Institute at beamline CMCF-1 08-ID-1 of the Canadian Light Source Inc., which is supported by the Natural Sciences and Engineering Research Council, the National Research Council of Canada, the Canadian Institutes of Health Research, and the University of Saskatchewan. The Alberta Synchrotron Institute synchrotron access program is supported by grants from the Alberta Science and Research Authority and the Alberta Heritage Foundation for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Dept. of Biological Sciences, University of Calgary, 2500 University Drive N. W., Calgary, Alberta T2N 1N4, Canada. Tel.: 403-220-4320; Fax: 403-289-9311; E-mail: ngk{at}ucalgary.ca.

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