Human Glutamine Synthetase in complex with ADP and Methionine Sulfoximine Phosphate

Target ID:

GLULA

Aliases:

GLNSGLULGSPIG43PIG59

Deposition Date:

Tuesday 19th June 2007

Families:

AA metabolic enzymes

Related Diseases:

MetabolismNeurobiology

Structure type:

protein-ligand

Download Coordinates:

2QC8

Site:

Stockholm

2QC8

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, Parkinson's, Alzheimer's and Huntington'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 (PDB code 2OJW). 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 (PDB code
2QC8). 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

GLUL

PDB:2QC8

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:MHHHHHHSSGVDLGTENLYFQSMLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETG
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:30 µl BL21(DE3) gold pRARE2 cells were transformed with 2 µl plasmid. The mix was kept on ice for 30 min followed by a heatshock at 42°C for 45 sec. SOC, 125 µl, was added to the cellsuspension which then was incubated for 1 hour at 37°C and plated on LA-plates containing kanamycin (50 µg/ml) and chloramphenicol (34 µg/ml). 20 ml TB (supplemented with 8 g/l glycerol, 100 µg kanamycin/ml and 34 µg/ml chloramphenicol) was inoculated with 5-10 colonies and grown overnight at 30°C. The 20 ml of the inoculation culture was added to 1.5 l TB (supplemented 8g glycerol/L and 50 µg kanamycin/ml) in 2 l bottles. The flask was incubated in the LEX system-water bath at 37 ºC until OD600 reached ~2. At this time the flask was transferred to an 18°C 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 18 hours. Cells were harvested by centrifugation 5500 x g for 10 minutes (WCW 35.1 g).

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 40 mg/ml.

Extraction

Procedure
Pellets were resuspended in ~60 ml 50 mM Na-phosphate pH 7.5, 500 mM NaCl, 10 % glycerol, 10mM imidazole, 0.5 mM TCEP and Complete EDTA-free protease inhibitor (Roche Biosciences). Resuspended cells were stored at -80°C until further use. Before lyses, 8 µl of 250U/µl Benzonase (Novagen) was added to the thawed cells and sonicated (Sonics VibraCell) at 80% amplitude for 3 min (pulse: 4 s on and 4 s off).The sample was spun for 30 min at 49000 x g and the soluble fraction was decanted and filtered through a 0.45 µm syringe filter.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals of the GS complex were grown using vapor diffusion at 4°C by mixing equal amounts of protein solution at 20 mg/ml including 2mM added Methionine sulfoximine and reservoir solution containing 1.1M Sodium-Malonate, 0.5% Jeffamine ED-2001, 100 mM HEPES pH 7.0. Crystals appeared after three days.
NMR Spectroscopy:
Data Collection:Data to 2.6 Å resolution were collected from a single crystal at ESRF (ID29), Grenoble, France. Crystal belonged to C2 space group with cell parameters of a=181.2 Å, b=126.1 Å, c=188.2 Å, α= 90°, β=92.1°, γ= 90°.
Data Processing:The structure was solved by molecular replacement using the previously solved human Glutamine synthetase as a search model (PDB entry: 2OJW) with the program MolRep. The asymmetric unit contains a decamer. The space group was C2 with cell dimensions a=181.2Å, b=126.1Å, c=188.2Å and β=92.1°. Refmac5 was used for refinement and Coot for model building. NCS and 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 39.9-2.60Å resolution was used and at the end of the refinement the values for R= 16.8% and Rfree= 21.7%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 2QC8.

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.
PubMed 12135980
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.
PubMed 15820321
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.
PubMed 16341942
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.
PubMed 16267323
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.
PubMed 16829528