Human ral guanine nucleotide dissociation stimulator, RAS-associating domain

Target ID:

RALGDS

Deposition Date:

Friday 30th October 2009

Families:

G-Protein Regulators

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

3KH0

Authors:

Shen Y, Tempel W, Wang H, Tong Y, Guan X, Crombet L, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3KH0

tabs

Structure Details

RALGDS, as one kind of effector molecules of Ras proteins, is found to specifically stimulate the dissociation of guanine nucleotides from the Ras family member Ral, and also identified as a binding partner for Ras in yeast two-hybrid screening (Albright et al., 1993). RalGDS contains a central RasGEF (Guanine nucleotide exchange factor for Ras-like small GTPases) domain, an N-terminal REM (Ras-exchange motif) domain and a C-terminal RA (Ras-associating) domain. Whereas the catalytic domain of the RALGDS is specific for Ral, RALGDS binds to other members of the Ras family including R-Ras, H-Ras, K-Ras and Rap (Hofer et al., 1994 and Spaargaren et al., 1994). This Ras-binding of RalGDS is through its C-terminal, non-catalytic segment, RA domain, and the interaction between Ras and RalGDS is dependent on activation of Ras by GTP.

We crystallized the Ras-associationg (RA) domain of RALGDS in the presence of Endoproteinase Glu-C and solved the crystal structure at 2.1 Å. The RA domain of RALGDS adopted a ubiquitin-like conformation characterized by a ββαββαβ-type tertiary structure, and the C-terminal residues of one monomer forms parallel β-sheet interaction with the β-strand of another molecule.

Materials and Methods

RALGDS

PDB:3KH0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:MGC:AT65-H11, BC059362.1
Entry Clone Source:MGC
SGC Clone Accession:HPC09Q-F03
Tag:N-terminal tag: mhhhhhhssgrenlyfq*g
Host:BL21-V2R-pRARE2

Construct

Prelude:RALGDS:N793-F914
Tag was not removed.
Sequence:mhhhhhhssgrenlyfqgNQQVGDCCIIRVSLDVDNGNMYKSILVTSQDKAPAVIRKAMDKHNLEEEEPEDYELLQILSDDRKLKIPENANVFYAMNSTANYDFVLKKRTFTKGVKVKHGASSTLPRMKQKGLKIAKGIF
Vector:pET28-mhl (GI:134105571)

Growth

Medium:
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 2 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and 25 µg/mL chloramphenicol at 37 degC. When OD600 reached ~3.0, the temperature of the medium was lowered to 15 °C and the culutre was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 degC.

Purification

Procedure
The lysate was centrifuged at 16,000 rpm for 1 hour and the supernatants were mixed with 12 mL 50% flurry of Ni-NTA beads and incubated at 4 degC on rotary shaker for 0.7 hour. The mixture was then centrifuged at 2300 rpm for 5 min and the supernant discarded. The beads were then washed with washing buffers containing 5 mM and 10 mM Imidazole, and finally the bound protein was eluted with the elution buffer. The flow-trough was collected and further purified by a Superdex-75 gel filtration column pre-equilibrated with gel filtration buffer. Fractions containing the protein were collected and concentrated with Amicon Ultra-15 centrifugal filter (cut-off 5 kDa). The purity of the preparation is tested by SDS-PAGE to be greater than 95%.

The His6-tag was not removed for crystallization.

Extraction

Procedure
Frozen cells from 4L TB culture were thawed and resuspended in 500 mL Binding buffer with freshly added 0.5% CHAPS, and supplemented with 1.7 mL protease inhibitor cocktail (SIGMA Catalog # P8849), and 10 microL benzonase (Sigma Catalog # E1014, 250U/microL), and lysed using sonication at 120W for 8 minutes.
Concentration:23.0 mg/mL
Ligand
MassSpec:Native expected 15998.3 Da, measured 15999.6 Da.
Crystallization:Crystallization was setup using in situ proteolysis method in sitting drops with Red Wings and SGC-I screens. Diffracting crystals were found from initial screen drops.

Crystal used for structure determination was grown in 2.0 M (NH4)2SO4, 2.0% PEG400, 0.1M Na HEPES buffer pH 7.5, with 1:100 Endoproteinase Glu-C (w/w) in sitting drop setup. 0.3 uL protein solution plus 0.3 uL well solution was used. Crystals grow to a mountable size in 3 days.
Paratone was used as cryoprotectant.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Albright, C. F., Giddings, B. W., Liu, J., Vito, M. and Weinberg, R. A. (1993)
Characterizaton of a guanine nucleotide dissociation stimulator for a ras-related GTPase.
EMBO J. 12, 339-347
Hofer, F., Fields, S., Schneider, C. and Martin, G. S. (1994) Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator Proc. Natl. Acad. Sci. U.S.A. 91,
11089-11093
Spaargaren, M. and Bischoff, J. R. (1994) Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras and
Rap. Proc. Natl. Acad. Sci. U.S.A. 91, 12609-12613