Human distinct subgroup of the Ras family, member 2

Target ID:

DIRAS2A

Aliases:

Di-Ras2DKFZp761C07121

Deposition Date:

Tuesday 25th October 2005

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2ERX

Authors:

E.PAPAGRIGORIOU,X.YANG,J.ELKINS,F.E.NIESEN,N.BURGESS,E.SALAH,O.FEDOROV,L.J.BALL,F.VON DELFT,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,D.DOYLE

Site:

Oxford

ICM Datapack:

ICM Datapack

2ERX

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.

DIRAS2 is the first structure of the subfamily of DIRAS small GTPases. The family members are characterised by a number of alterations that would be expected to produce a constitutively active form (Kontani et al, 2002). The most significant is the lack of a glutamine residue, the equivalent of which is highly conserved in catalytically active small GTPases and is known to be involved in the catalysis process. DIRAS family members are also likely to use a different signalling pathway from the standard Ras-Raf-1 kinase signalling pathway as a hydrophobic residue is present in place of an Asp in Ras which forms part of the Ras-Raf-1 binding site.

Crystals of DIRAS2 were grown in the presence of the GTP nucleotide as this protein was expected to be catalytically active. Surprisingly, the crystal structure clearly showed that DIRAS2 is able to catalysis the hydrolysis of GTP as both products, GDP and Pi, are present in the electron density. These products along a Ser side chain and the Thr side chain from the Switch I loop form a square planar coordination around the Mg2+ ion. Two water molecule above and below the Mg2+ ion complete the expected octahedral coordination.

Materials and Methods

DIRAS2

PDB:2ERX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:DIRAS2A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:BL21(DE3)R3 phage resistant strain

Construct

Prelude:
Sequence:SMSNDYRVAVFGAGGVGKSSLVLRFVKGTFRESYIPTVEDTYRQVISCDKSICTLQITDTTGSHQFPAMQRLSISKGHAFILVYSITSRQSLEELKPIYEQICEIKGDVESIPIMLVGNKCDESPSREVQSSEAEALARTWKCAFMETSAKLNHNVKELFQELLNLEKRRTVSLQ
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Freshly transformed E. coli cells was used to inoculate 1 litre of TB plus 50 µg/mL kanamycin. When OD600 reached ~1.0 the temperature was shifted down from 37°C to 25°C for 1 hour before induction with the addition of 1 mM IPTG. Protein expression was allowed to carry on for a further 4 hours before harvest.

Purification

Procedure
Column 1 : Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).

Buffers: Wash buffer I (WB1): 50 mM Tris pH 8.0, 150 mM NaCl, 5 % Glycerol, 10mM MgCl2, 10mM Imidazole pH 8.0. Wash Buffer II (WBII): 50 mM Tris pH 8.0, 150 mM NaCl, 5 % Glycerol, 10 mM MgCl2, 30 mM Imidazole pH 8.0. Elution buffer (EB): 50 mM Tris pH 8.0, 150 mM NaCl, 5 % Glycerol, 10 mM MgCl2, 250 mM Imidazole pH 8.0.

Procedure: Total volume of Ni-NTA added to BioRad drip column: 4 mLs (50%). Resin washed with 12.5 mL of WB1. The supernatent was applied to a column using 5 mL pipette and allowed to pass over the resin. The flow through was collected in a 50 mL falcon tube and applied once more to the column. Two wash steps followed. Wash with 12.5 mL of WBI. Wash with 12.5 mL column vols of WBII. Elute with 14 mLs of EB into 7x2 mL fractions. At this stage the purity of the protein was greater than 95 % based on SDS - PAGE analysis. The C-terminal hexahistidine tag was removed by TEV protease treatment. The TEV protease, a hexahistidine-tagged construct, was over-expressed and purified in-house to a final concentration of 2.5 mg/mL.

Extraction

Procedure
Cell Lysis - Resuspension buffer (RS): 50 mM Tris pH 8.0, 150 mM NaCl, 5 % Glycerol, 10 mM Imidazole pH 8.0, 10mM MgCl2 . 1 proteinase inhibitor tablet (EDTA free) was added to 10mL Resuspension Buffer. This was used to syspend the pellet before homogenisation. Total vol:45 mLs (estimate) Cell breakage: 5 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 50 mLs (estimate). Centrifuge for 40 mins at 16000 rpm and 4°C to remove cell debris. Discard pellet.
Concentration:
Ligand
MassSpec:
Crystallization:Column 1 : Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).

References

Kontani K, Tada M, Ogawa T, Okai T, Saito K, Araki Y, Katada T. (2002). Di-Ras, a distinct subgroup of ras family GTPases with unique biochemical properties. J. Biol. Chem. 277: 41070-41078.