Human GTP-binding mitogen-induced T-cell protein

Target ID:

GEMA

Aliases:

KIRMGC26294

Deposition Date:

Tuesday 21st February 2006

Families:

GTPases

Related Diseases:

NeurobiologyCancer

Structure type:

apo

Download Coordinates:

2G3Y

Authors:

E.UGOCHUKWU,M.SOUNDARARAJAN,J.ELKINS,C.GILEADI,G.SCHOCH,F.SOBOTT,O.FEDOROV,J.BRAY,N.PANTIC,G.BERRIDGE,N.BURGESS,W.H.LEE,A.TURNBULL,M.SUNDSTROM,C.ARROWSMITH,J.WEIGELT,A.EDWARDS,F.VON DELFT,D.DOYLE,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2G3Y

tabs

Structure Details

Cells respond in many ways to environmental changes including altering gene expression, physically moving locations, changes to cell-cell interactions, differentiation and even altering their life spans.
This is achieved through a signalling network that consists of several signalling pathways. The network receives multiple signals, interprets the signals, amplified and then transmits the signals. One of the best characterised signalling pathways involves the ras GTPase family. The importance of this family of small GTPases to human health was first recognised through the identification of the human H-Ras, N-Ras and K-Ras genes as being oncogenic.

The RGK subfamily of small GTPases are characterised by sequence changes in the functionally important switch I loop and the G3 (DXXG) motif. These changes result in significant decrease in enzymatic turnover effectively making these proteins GTP binding proteins rather than catalytic enzymes. The significance from a functional point of view is that these proteins are likely to be always in the active state.

The family also has N- and C-terminal extensions that contain Ser/Thr residues that after phosphorylation form a complex with 14-3-3 proteins with a subsequent increase in GEM protein half life (Ward et al, 2004). Calmodulin, a protein important in calcium regulation, is also able to bind to GEM (Fisher et al, 1996). This interaction is linked to one of GEM's cellular functions which is the regulation of calcium channel activity through the beta subunit interaction (Beguin et al, 2006; Finlin et al, 2003).

Individuals with type II diabetes were found to have higher levels of GEM in skeletal muscle (Maguire et al, 1992).

Materials and Methods

GEM

PDB:2G3Y

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GEMA-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*), N-terminal his6 tag.
Host:BL-21(DE3)R3 phage resistant

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmSTDSVI SSESGNTYYRVVLIGEQGVGKSTLANIFA GVHDSMDSDCEVLGEDTYERTLMVDGESA TIILLDMWENKGENEWLHDHCMQVGDAYL IVYSITDRASFEKASELRIQLRRARQTED IPIILVGNKSDLVRCREVSVSEGRACAVV FDCKFIETSAAVQHNVKELFEGIVRQVRL RRDSKEKN
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformed 50 µl competent BL-21 (DE3) phage resistant cells with 10 µl of the plasmid DNA and plated out onto LB plate plus 50 µg/ml kanamycin. The next day colonies were picked out into fresh deep well blocks containing 1 ml TB + 50 µg/ml kanamycin. These were grown overnight and glycerol stocks prepared by adding 333 µl of 60 % glycerol to 1 ml of cell suspension, mixing and then storing in a -80°C freezer. The glycerol stock was used to innoculate 10 mls of TB + 50 µg/ml kanamycin which was grown overnight at 37°C as a starter culture for a 1 litre growth. The large scale growth was grown at 37°C until approximately 45 mins before induction when the temperature was lowered to 25°C. Protein production was induced with the addition of 1mM IPTG. The next day cells were harvested by centrifugation at 4000 rpm for 15 minutes. The pellet was resuspended in 50 mM K phosphate, pH 8, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, and then stored in the -80°C freezer.

Purification

Procedure
Column 1: Ni Sepharose Affinity column.
The Supernatant containing the protein was loaded on to manually packed Ni sepharose beads column and washed with 30 ml of wash buffer and eluted in 6 fractions of 2 ml each using the high imidazole elute buffer. The eluate was pooled and concentrated using 10 KDa cutoff millipore filters and loaded on to Gelfiltration column.

Column 2 : Gel filtration, Hiload 16/60, S75 16/60 - 120 ml
Eluted proteins were collected in 1.75 ml fractions. Using a Centricon 10 K cutoff concentrator the buffer was exchange to 10 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, 2 mM MgCl2 and 5 mM DTT. The volume was then brought to a total of 2 ml before adding 5 mM GDP and concentrated to 19.06 mg/ml. Concentration was determined from the absorbance at 280 nm.

Extraction

Procedure
After thawing, the resuspended cells were lysed by passing through Emulsiflex C5 high pressure homogeniser. PEI (stock 5 %) was added to the homogenate to a final concentration of 0.15%. The cell debris, nuclei and DNA were spun down at 16000 rpm for 45 min. The supernatant was collected.
Concentration:
Ligand
MassSpec:Calculated mass of the full length construct is 23822.5 Da and the determined mass was 23822.7 Daltons.
Crystallization:Crystals grew from a 1:1 ratio mix of GEMA (+ GDP )-to-reservoir ( 14- 30 % PEG 3350 and 0.025 - 0.3 M Ammonium citrate pH 5.0 ).
NMR Spectroscopy:
Data Collection:Resolution: 2.25 Å; X-ray source: Synchrotron ALS -8.2.1, single wavelength.
Data Processing:

References

Beguin P, Mahalakshmi RN, Nagashima K, Cher DH, Ikeda H, Yamada Y, Seino Y, Hunziker W. (2006). Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation. J. Mol. Biol. 355: 34-46.

Finlin BS, Crump SM, Satin J, Andres DA. (2003). Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases. Proc. Natl. Acad. Sci. U S A. 100: 14469-14474.

Fischer R, Wei Y, Anagli J, Berchtold MW. (1996). Calmodulin binds to and inhibits GTP binding of the ras-like GTPase Kir/Gem. J. Biol. Chem. 271: 25067-25070.

Maguire J, Santoro T, Jensen P, Siebenlist U, Yewdell J, Kelly K. (1994). Gem: an induced, immediate early protein belonging to the Ras family. Science. 265: 241-244.

Ward Y, Spinelli B, Quon MJ, Chen H, Ikeda SR, Kelly K. (2004). Phosphorylation of critical serine residues in Gem separates cytoskeletal reorganization from down-regulation of calcium channel activity. Mol. Cell. Biol. 24: 651-661.