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-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 α1-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 (αE and αG) 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. 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 α1-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 (αE and αG) 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. 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 (1Ninfa A.J. Atkinson M.R. Trends Microbiol. 2000; 8: 172-179Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 2Arcondeguy T. Jack R. Merrick M. Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (352) Google Scholar). 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 (3Moorhead G.B.G. Smith C.S. Plant Physiol. 2003; 133: 492-498Crossref PubMed Scopus (41) Google Scholar, 4Forchhammer K. FEMS Microbiol. Rev. 2004; 28: 319-333Crossref PubMed Scopus (205) Google Scholar, 5Ninfa A.J. Jiang P. Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (211) Google Scholar, 6Osanai T. Tanaka K. Plant Cell Physiol. 2007; 48: 908-914Crossref PubMed Scopus (39) Google Scholar). 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 >50% identical to proteobacterial and cyanobacterial PII (7Hsieh M.H. Lam H.M. van de Loo F.J. Coruzzi G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13965-13970Crossref PubMed Scopus (198) Google Scholar), but unlike these orthologs, it does not appear to be regulated by phosphorylation or uridylylation in response to nitrogen metabolites (8Smith C.S. Morrice N.A. Moorhead G.B.G. Biochim. Biophys. Acta. 2004; 1699: 145-154Crossref PubMed Scopus (36) Google Scholar). 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 (9Ferrario-Mery S. Bouvet M. Leleu O. Savino G. Hodges M. Meyer C. Planta. 2005; 223: 28-39Crossref PubMed Scopus (55) Google Scholar). To date, the sole interacting protein discovered for plant PII is the chloroplast enzyme N-acetylglutamate kinase (NAGK), 2The abbreviations used are: NAGK, N-acetylglutamate kinase; SASA, solvent-accessible surface area. which catalyzes the second and rate-limiting step in the pathway of arginine biosynthesis (10Sugiyama K. Hayakawa T. Kudo T. Ito T. Yamaya T. Plant Cell Physiol. 2004; 45: 1768-1778Crossref PubMed Scopus (72) Google Scholar). We (11Chen Y.M. Ferrar T.S. Lohmeier-Vogel E. Morrice N. Mizuno Y. Berenger B. Ng K.K.-S. Muench D.G. Moorhead G.B.G. J. Biol. Chem. 2006; 281: 5726-5733Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and others (12Ferrario-Mery S. Besin E. Pichon O. Meyer C. Hodges M. FEBS Lett. 2006; 580: 2015-2020Crossref PubMed Scopus (67) Google Scholar) 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 inhibition by the downstream product arginine. Quite elegantly, the binding of PII changes the kinetics of NAGK from sigmoidal to hyperbolic in the presence of arginine. A similar form of regulation is also found in cyanobacteria (13Burillo S. Luque I. Fuentes I. Contreras A. J. Bacteriol. 2004; 186: 3346-3354Crossref PubMed Scopus (103) Google Scholar, 14Maheswaran M. Urbanke C. Forchhammer K. J. Biol. Chem. 2004; 279: 55202-55210Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 15Heinrich A. Maheswaran M. Ruppert U. Forchhammer K. Mol. Microbiol. 2004; 52: 1303-1314Crossref PubMed Scopus (110) Google Scholar). Recently, we solved the crystal structure of the first eukaryotic PII protein from A. thaliana (16Mizuno Y. Berenger B. Moorhead G.B.G. Ng K.K.-S. Biochemistry. 2007; 46: 1477-1483Crossref PubMed Scopus (25) Google Scholar). As in bacteria, plant PII forms a trimer with three protruding T-loops on one surface as well as a plant-specific N-terminal extension on the opposing surface that may be involved in protein-protein interactions and signaling. We have now determined the structure of A. thaliana PII bound to NAGK. This structure reveals for the first time the molecular mechanism underlying the regulation of an enzyme by PII. Protein Expression and Purification—PII was expressed and purified as described previously (16Mizuno Y. Berenger B. Moorhead G.B.G. Ng K.K.-S. Biochemistry. 2007; 46: 1477-1483Crossref PubMed Scopus (25) Google Scholar). The NAGK gene from A. thaliana was amplified by PCR using primers 5′-GGGAATTCCATATGACCGTATCAACACCACCT-3′ and 5′-GAAGATCTTTATCCAGTAATCATAGTTCCAGC-3′, Vent polymerase (New England Biolabs), and the His-tagged NAGK expression clone in the pRSET-A plasmid (Invitrogen) as a template (11Chen Y.M. Ferrar T.S. Lohmeier-Vogel E. Morrice N. Mizuno Y. Berenger B. Ng K.K.-S. Muench D.G. Moorhead G.B.G. J. Biol. Chem. 2006; 281: 5726-5733Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The amplified product was digested with NdeI and BamHI and then ligated to pET-3a (Novagen) digested with the same enzymes. The expression plasmid was transformed into Rosetta-gami pLysS cells (Novagen). Four 1-liter cultures of LB medium supplemented with ampicillin and chloramphenicol were induced with 0.2 mm isopropyl β-d-thiogalactopyranoside and grown for 20 h at 25 °C. Cells were centrifuged, resuspended in 160 ml of Buffer A (20 mm Tris-Cl (pH 8.0), 150 mm NaCl, 0.5 mm EDTA, 0.5 mg of DNase, and 0.25 mm phenylmethylsulfonyl fluoride), and lysed by sonication. The extract was clarified by centrifugation, diluted 1:5 (v/v) with Buffer B (20 mm NaHEPES (pH 7.0), 1 mm NaEDTA, 2 mm dithiothreitol, and 10% glycerol), and loaded at 1 ml/min onto a blue dye column (5 × 2.5 cm; Bio-Rad) equilibrated with Buffer B. The column was washed with 80 ml of Buffer B containing 250 mm NaCl, and 8-ml fractions were eluted using Buffer B containing 1.2 m NaCl. Peak fractions were pooled and precipitated with ammonium sulfate between 40 and 55% saturation. The precipitated protein was collected by centrifugation, dissolved with Buffer C (20 mm Tris-Cl (pH 8.0), 0.2 mm NaEDTA, 2 mm dithiothreitol, and 10% glycerol), dialyzed overnight against Buffer C containing 30 mm NaCl, and loaded at 1 ml/min onto a 5-ml HiTrap Q HP anion exchange column (Amersham Biosciences) equilibrated with Buffer C containing 20 mm NaCl. NAGK was eluted at ∼0.15 m NaCl using a 0.02-1 m NaCl linear gradient and loaded onto a Superdex 200 10/300 GL gel filtration column (Amersham Biosciences) equilibrated with 100 mm NaCl, 10 mm Tris-Cl (pH 8.0), 1 mm NaEDTA, 2 mm dithiothreitol, and 5% glycerol. The purity of NAGK was assessed by SDS-PAGE, and NAGK was concentrated to 9 mg/ml using Amicon Ultrafree-15 centrifugal concentrators. Structure Determination of the PII·NAGK Complex—NAGK and PII were independently concentrated and mixed at a 2:1 mass ratio to produce a mixture containing 40 mm l-arginine, 20 mm MgCl2, 10 mm ADP, 10 mm N-acetylglutamate, 4 mg/ml NAGK, and 2 mg/ml PII. Protein concentrations were determined using the Bradford dye binding assay (Bio-Rad) (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Crystals were grown using the hanging drop vapor diffusion method at room temperature by mixing 2 μl of the protein mixture with an equal volume of reservoir solution containing 8.5% (w/v) polyethylene glycol 8000, 8.5% (w/v) polyethylene glycol 1000, 0.1 m NaHEPES (pH 7.0), 0.4 m trimethylamine N-oxide, 50 mm l-arginine, 2 mm dithiothreitol, and 12% (w/v) glycerol. This mixture was seeded with small crystals from a previous crystallization trial under the same conditions and equilibrated against 1 ml of reservoir solution. The crystallizations were dehydrated by adding 50 μl of 80% (w/v) glycerol to the well solution on days 7 and 15 after the initial setup. On day 17, 100 μl of 80% (w/v) glycerol was added. On day 18, a single crystal (cubic shape with edges of ∼0.15 mm) was mounted on a polyimide loop (MiTeGen) and flash-cooled at 100 K with a nitrogen gas stream (Oxford Cryostream). The crystal was stored in liquid nitrogen and transferred to Canadian Light Source beamline CMCF-1 08-ID-1 for data collection using a MarCCD detector. Data were measured on 331 images with 1-s exposures and 0.5° of and that the most was with cell a and Data were and using PubMed Scopus Google Scholar) and from PubMed Scopus Google Scholar). are in The may be in to the of data K. Biol. PubMed Scopus Google collection cell cell resolution from the resolution are in from the resolution are in from the resolution are in from the resolution are in where is the of a and is the of all of from the resolution are in for the of data used in for the 5% of data from of and from temperature in by PubMed Scopus Google from the resolution are in where is the of a and is the of all of for the of data used in for the 5% of data from in a The structure of the PII·NAGK complex was determined with the molecular method using the structure of PII from A. thaliana Protein Data (16Mizuno Y. Berenger B. Moorhead G.B.G. Ng K.K.-S. Biochemistry. 2007; 46: 1477-1483Crossref PubMed Scopus (25) Google Scholar) and the structure of NAGK from Protein Data S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar) as The of the crystal was to be two PII and two NAGK are in the PubMed Scopus Google Scholar). were using Biol. 2001; PubMed Scopus Google Scholar), a solution for the of two of the NAGK and the of these two NAGK a solution for the of two of the PII was for the of for and Biol. PubMed Scopus Google Scholar) was used for and J. Biol. PubMed Scopus Google Scholar) was used to into PubMed Scopus Google Scholar) was used to the in the was the of in both of NAGK. This may the presence of a covalent for which we were to a Structure of the PII·NAGK understand the molecular of enzyme regulation by PII, we determined the structure of a complex formed between two homotrimers of PII and a single hexamer of NAGK. The structure of the PII·NAGK complex was solved by molecular using the of PII from A. thaliana Data (16Mizuno Y. Berenger B. Moorhead G.B.G. Ng K.K.-S. Biochemistry. 2007; 46: 1477-1483Crossref PubMed Scopus (25) Google Scholar) and NAGK from P. Data S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar) as crystal forms were under a of well crystals were in the presence of ADP, N-acetylglutamate, and arginine. of these are bound to NAGK and are well in ATP and not is bound to PII. ATP was or the ATP was bound to PII the protein was from E. The of the PII·NAGK complex crystal two of NAGK and two of PII. A with the molecular of the complex to the complex that forms in solution The of the complex are × × with the the from the N-terminal of one PII trimer to the N-terminal of the second PII PII trimer forms interactions with opposing of the NAGK with PII a single NAGK two of PII contact Trp22 at the of the and in the T-loop of PII contact on helices and as well as and on the of NAGK the active-site A small of solvent-accessible surface for PII is upon complex and PII trimer appears to independently on of the NAGK This of the for PII and 5% of the for NAGK the interactions between in PII trimer and NAGK the of upon binding of a single PII trimer to the NAGK hexamer of the for the PII trimer and for the NAGK PII trimer independently on of the NAGK The of the two PII in the complex appear to be similar to the structure of PII, with the of the T-loop and the The T-loop is in PII, but it is well and forms with NAGK in the of the interacting between NAGK and PII are conserved in all plants and the of the binding interactions in the A. thaliana complex and The of and in the T-loop of PII form with the and of and in NAGK In interactions are formed by the of and with the of and of NAGK. The of also forms a with the of from the the of the plant-specific Trp22 at the of the first of PII forms a with the of of NAGK as well as van with and The PII·NAGK complex also reveals that the NAGK hexamer of a as previously in the and NAGK from P. and S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). NAGK an of the kinase of an N-terminal that N-acetylglutamate and a that the ATP S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar, S. A. I. Structure Full Text Full Text PDF PubMed Scopus Google Scholar). is to adjacent at two A and B. A is formed by interactions between the of the central an adjacent and two B is formed between the N-terminal helices of adjacent A appears to be found in all and NAGK but B and the N-terminal to be found in organisms in which NAGK as a hexamer and arginine an S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar), as in plants (11Chen Y.M. Ferrar T.S. Lohmeier-Vogel E. Morrice N. Mizuno Y. Berenger B. Ng K.K.-S. Muench D.G. Moorhead G.B.G. J. Biol. Chem. 2006; 281: 5726-5733Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). As previously in the NAGK from P. and T. arginine the of the N-terminal and may inhibition by an open conformation of NAGK as described for the of NAGK by structure of the PII·NAGK complex reveals for the first time the molecular basis for the regulation of an enzyme by PII. In bacteria, and plants, PII metabolic by on the carbon, and nitrogen status appears to be in PII proteins the binding of The PII·NAGK complex reveals the molecular mechanism which ATP binding the regulation of an The binding of ATP to PII favors a conformation of the T-loop that interacts with NAGK. the of ATP changes on the structure of PII and its interactions with NAGK. The in a that is by in the of ATP of from the ATP-binding the of the from an by the T-loop in the PII·NAGK As is conserved in plants and in a found in plant PII (16Mizuno Y. Berenger B. Moorhead G.B.G. Ng K.K.-S. Biochemistry. 2007; 46: 1477-1483Crossref PubMed Scopus (25) Google Scholar). The changes in the conformations of the and the T-loop may be of an found in plants. In PII, the binding of the may a in a similar that is also involved with signaling. The of ATP also a role in a conformation of the T-loop that favors interactions with NAGK. The in with the of at the of the T-loop to the conformation of the T-loop for interactions with NAGK is conserved in PII from bacteria, and all A previous in E. coli has also that the of the with PII interactions with as well as the binding of and ATP P. P. Atkinson M.R. P. A.J. J. Bacteriol. PubMed Scopus (85) Google Scholar). The binding of the of ATP in A. thaliana PII from the of ATP-binding in from E. coli Y. E. van J. Mol. Biol. 1998; PubMed Scopus Google Scholar, Y. T. J. Biochem. 2001; PubMed Scopus Google Scholar) and C. T. Y. T. S. M. S. J. Biol. 2005; PubMed Scopus Google Scholar) as well as from E. coli A. Merrick M. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). the of ATP binding is similar to the binding in PII from O. C. S. J. 2007; PubMed Scopus (55) Google Scholar). The of the as well as in both A. thaliana and M. the of the of in A. thaliana In M. the peptide is in the and the does not with structural that the binding of to the of ATP may to the of ATP binding on the structure of the surface of PII. As the binding of ATP to PII also to with PII, the carbon signal after of energy status A.J. Jiang P. Curr. Opin. Microbiol. 2005; 8: 168-173Crossref PubMed Scopus (211) Google Scholar, C.S. Moorhead G.B.G. Plant J. 2003; PubMed Scopus Google Scholar). As it is that ATP and not is bound to PII. A reveals that the ATP is well bound to PII of with the of the in the of ATP was not or the ATP was bound to PII the protein was from E. and of NAGK of the PII·NAGK complex is that the two of NAGK found in the which is found in the the complex is as in an conformation with B. the two conformations are the N-terminal of A is by from the 4 is as by the S. J. Mol. PubMed Scopus Google Scholar), which between the two and The closed conformation of NAGK the two N-acetylglutamate and ATP, the of was as a to the of a mixture of and ATP were The closed conformation the of of the of In the open the between these two by In by the of and the of to the appear to be formed in the closed conformation of the substrate-binding site for NAGK. determined of NAGK from and also that a mixture of with of be found in the same crystal as well as in from crystals S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). This that of may be an of NAGK structure that is to function. that the closed conformation the of the as has been in the NAGK from and S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar, S. I. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). In the open conformations the entry of and the of with E. coli structure of the PII·NAGK complex reveals a of to and from with the determined of bound to the ammonium A. Merrick M. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). the complex is the structure of a complex containing a In both the T-loop of PII the of and the structure of PII the structure of the binding the the of interactions between the T-loop and NAGK from the interactions with The T-loop of E. coli in the complex is the of the In the T-loop of A. thaliana PII forms a as it from the surface of the PII to the of the trimer The in T-loop conformation in the two that the of the T-loop may be in to with a of and of by the of is the product of the pathway in which NAGK as the second but rate-limiting is a feedback and to NAGK between the N-terminal and the at a site B of adjacent This binding site is formed by a in the of NAGK that is similar to the binding site previously in the NAGK enzyme from T. S. A. I. J. Mol. Biol. 2006; PubMed Scopus Google Scholar). of the of NAGK from P. and NAGK from T. that the binding of arginine may a open conformation of NAGK by interactions between the and the N-terminal The degree of in and structure between the plant and NAGK that the binding of arginine may also a open conformation in plant NAGK. arginine binding or stabilizes the open conformation of NAGK, then arginine inhibition may from the closed conformation that is necessary for The binding of PII to NAGK in plants (11Chen Y.M. Ferrar T.S. Lohmeier-Vogel E. Morrice N. Mizuno Y. Berenger B. Ng K.K.-S. Muench D.G. Moorhead G.B.G. J. Biol. Chem. 2006; 281: 5726-5733Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, S. Besin E. Pichon O. Meyer C. Hodges M. FEBS Lett. 2006; 580: 2015-2020Crossref PubMed Scopus (67) Google Scholar) and cyanobacteria (13Burillo S. Luque I. Fuentes I. Contreras A. J. Bacteriol. 2004; 186: 3346-3354Crossref PubMed Scopus (103) Google Scholar, 14Maheswaran M. Urbanke C. Forchhammer K. J. Biol. Chem. 2004; 279: 55202-55210Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 15Heinrich A. Maheswaran M. Ruppert U. Forchhammer K. Mol. Microbiol. 2004; 52: 1303-1314Crossref PubMed Scopus (110) Google Scholar) has been to the inhibition by arginine. The structure of the PII·NAGK complex a mechanism by which PII may PII with both open and closed conformations of NAGK. The of the site is found on the of NAGK the opening of the active-site cleft and on the of for the two domains This PII to with a of NAGK conformations in a similar small changes in of the PII binding may not the open or the closed conformation of NAGK, both of which are necessary for enzyme function. PII with both open and closed conformations of NAGK, the PII·NAGK structure that PII may NAGK from The arginine-inhibited structure of NAGK from T. reveals a degree of between the and domains with the open conformation of NAGK in the plant PII·NAGK complex as well as an opening of a in A. thaliana and in T. of these changes appear to to a of the adjacent and these the of and the of and which form interactions with PII these interactions may limit of and of the of NAGK. these changes are that may be by the binding of arginine, then the by PII binding may effect and the kinetics of NAGK and PII interactions in the NAGK complex and the of A. thaliana NAGK in the presence of arginine the of We for of and and for and the of We and for with data collection at the Canadian Light Source

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