Human Glutamine Synthetase

Target ID:

GLULA

Aliases:

GLNSGLULGSPIG43PIG59

Deposition Date:

Monday 15th January 2007

Families:

AA metabolic enzymes

Related Diseases:

NeurobiologyMetabolism

Structure type:

apo

Download Coordinates:

2OJW

Authors:

T.KARLBERG,J.UPPENBERG,C.ARROWSMITH,H.BERGLUND,R.D.BUSAM,R.COLLINS,A.EDWARDS,S.FLODIN,A.FLORES,S.GRASLUND,B.M.HALLBERG,M.HAMMARSTROM,M.HOGBOM,I.JOHANSSON,T.KOTENYOVA,M.MOCHE,M.E.NILSSON,P.NORDLUND,T.NYMAN,D.OGG,C.PERSSON,J.SAGEMARK,P.STENMARK,M.SUNDSTROM,A.G.THORSELL,S.VAN DENBERG,K.WALLDEN,J.WEIGELT,L.HOLMBERG-SCHIAVONE,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Stockholm

ICM Datapack:

ICM Datapack

2OJW

tabs

Structure Details

Human Glutamine synthetase (EC 6.3.1.2) (GS or Glutamate-ammonia ligase) regenerates L-glutamine from the neurotransmitter glutamate and ammonia. The enzyme plays a major role in ammonia detoxification and interorgan nitrogen flux and activity and expressed levels of GS are hypothesized to be related to neurodegenerative diseases such as schizophrenia, Parkinsonâ€™s, Alzheimerâ€™s and Huntingtonâ€™s (Behrens et al., 2002, Bruneau et al., 2005; Burbaeva et al., 2005). Recently rare recessive inherited mutations in the glutamine synthetase gene were shown to result in lethal glutamine deficiency (Haberle et al., 2005).

We have solved the structure of human GS in the presence of ATP and MnCl2 at 2.05 Å resolution. The overall structure is composed of two ring-like pentamers forming an active decamer with five two-fold axes perpendicular to a central five-fold axis. There is a high resemblance to the recently determined plant GS structure (Unno et al., 2006), however significant differences are seen when comparing with known bacterial GS forming active dodecamers. The active sites lie in between the individual subunits resulting in a total of ten active sites per decamer. Interestingly, clear electron density is observed for one ADP, one phosphate and four manganese ions per active site.

We have also solved the structure of human GS in the presence of ATP, MnCl2 and Methionine sulfoximine to 2.6 Å resolution. Clear electron density is observed for one ADP, one phosphorylated Methionine sulfoximine (MSO-P) and three manganese ions per active site. MSO is an inihibitor that binds to the glutamate binding site. The structures reveal new insights into the mechanism of the enzyme and for designing novel drugs by structural comparison with GS from pathogenic bacteria.

Materials and Methods

GS

PDB:2OJW

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC011852
Entry Clone Source:MGC
SGC Clone Accession:GLULA-s002
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavagesite: mhhhhhhssgvdlgtenlyfq*sm
Host:BL21 (DE3) Gold pRare2

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETG
Vector:pNIC-Bsa4

Growth

Medium:TB
Antibiotics:
Procedure:30 microL BL21(DE3) gold pRARE2 cells were transformed with 2 microL plasmid miniprep for 30 min on ice followed by heatshock at 42degC for 45 sec. SOC, 125 microL, was added to the cellsuspension which was then incubated for 1 hour at 37degC and plated on LB-plates containing kanamycin (50 microG/mL). 30 mL TB (supplemented with 8 g/L glycerole, 100 µg kanamycin/ml and 3.4 microG/ml chloramphenicol) was inoculated with cells and grown overnight (ON) at 30degC. 20 ml of the inoculation culture was added to 1.5 L TB (supplemented 8g glycerole/L and 50 microG kanamycin/mL) in 2 L bottles. The flask was incubated in the LEX system-water bath at 37degC until OD600 reached 2. At this time the flask was transferred to an 18degC water bath in the LEX-system. Expression of protein was induced after approximately 1 hour by addition of 0.5 mM IPTG and continued for approximately 20 hours. Cells were harvested by centrifugation in a SLC-6000 rotor for 10 minutes at 5000 rpm (WCW 28.8 g). Pellets were suspended in 140 mL 50 mM Naphosphate pH 7.5, 10 % glycerol, 0.5 mM TCEP and 500 mM NaCl, 10mM imidazole and Complete EDTA-free protease inhibitor (Roche Biosciences). Suspended cells were stored at -80degC until further use.

Purification

Procedure
The cleared lysate was loaded onto a HiTrap IMAC column (Amersham Biosciences) using an ÄktaExpress system. Eluted protein was run through a Superdex 200 16/60 gel filtration column. Fractions containing protein were pooled and the TCEP-concentration adjusted to 2 mM. Purified GS only remained stable in the presence of ATP and MnCl2 and thus 10 mM ATP and MnCl2 were added to the protein before concentration to 20 mg/ml.

Extraction

Procedure
Before lyses, 8 microL of 250U/microL benzonase (Novagen) was added to the suspended cells and the sample was sonicated (Sonics VibraCell) at 80% amplitude for 3 min (pulse: 4 sec on and 4 sec off).The sample was spun for 30 min at 20500 rpm in a Sorvall SA-800 rotor. The soluble fraction was decanted and filtered through a 0.45 microM syringe filter
Concentration:
Ligand
MassSpec:
Crystallization:Crystals of GS were grown using vapor diffusion at 4degC by mixing equal amounts of protein solution at 20 mg/ml and reservoir solution containing 10% Isopropanol, 200 mM Sodium Chloride, 100 mM HEPES pH 7.5. Plate-like crystals appeared after one to three days.
NMR Spectroscopy:
Data Collection:Data to 2.05 Å resolution were collected from a single crystal at ESRF (ID14-2), Grenoble, France. Crystal belonged to C2 space group with cell parameters of a=177.6 Å, b=122.6 Å, c=126.6 Å, α= 90°, β=130.6°, γ= 90°.
Data Processing:The structure was solved by molecular replacement using Glutamine synthetase from maize as a search model (PDB entry: 2D3A) with the program MolRep. The asymmetric unit contains a pentamer. The space group was C2 with cell dimensions a=177.6Å, b=122.6Å, c=126.6Å and beta=130.6. Refmac5 was used for refinement and Coot for model building. TLS restrained refinement was used in the refinement process. The TLS groups were selected using the tlsmd server http://skuld.bmsc.washington.edu/~tlsmd/. Data in the interval 39.25-2.05Å resolution was used and at the end of the refinement the values for R= 16.2% and Rfree= 21.2%. A few residues are disordered and not visible in the electron density map. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 2OJW.

References

Behrens PF, Franz P, Woodman B, Lindenberg KS and Landwehrmeyer GB (2002). Impaired glutamate transport and glutamate-glutamine cycling: downstream effects of the Huntington mutation. Brain 125:1908-22.
Bruneau EG, McCullumsmith RE, Haroutunian V, Davis KL and Meador-Woodruff JH (2005). Increased expression of glutaminase and glutamine synthetase mRNA in the thalamus in schizophrenia. Schizophr. Res.75, 27-34.
Burbaeva GSh, Boksha IS, Tereshkina EB, Savushkina OK, Starodubtseva LI, Turishcheva MS (2005). Glutamate metabolizing enzymes in prefrontal cortex of Alzheimer's disease patients. Neurochem Res. 30:1443-51.
Haberle J, Gorg B, Rutsch F, Schmidt E, Toutain A, Benoist JF, Gelot A, Suc AL, Hohne W, Schliess F, Haussinger D and Koch HG (2005). Congenital glutamine deficiency with glutamine synthetase mutations. 353, 1926-1933.
Unno H, Uchida T, Sugawara H, Kurisu G, Sugiyama T, Yamaya T, Sakakibara H, Hase T and Kusunoki M (2006). Atomic structure of plant glutamine synthetase: a key enzyme for plant productivity. J. Biol. Chem. 281, 29287-29296.