Human Ras homolog gene family, member D

Target ID:

RHODA

Aliases:

ARHDRhRHOHP1RHOM

Deposition Date:

Monday 14th August 2006

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2J1L

Authors:

A.C.W.PIKE,C.JOHANSSON,C.GILEADI,F.H.NIESEN,F.SOBOTT,G.SCHOCH,J.ELKINS,C.SMEE,F.GORREC,S.WATT,J.BRAY,A.P.TURNBULL,F.VON DELFT,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,M.SUNDSTROM,D.DOYLE

Site:

Oxford

ICM Datapack:

ICM Datapack

2J1L

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 family of Rho GTPases are involved in cell motility and proliferation through the reorganisation of actin and vesicle trafficking. Activation of RhoD causes decreased motility of endothelial cells (Murphy et al, 2001) and fibroblasts (Tsubakimoto et al, 1999);
however, it confers invasive activity in kidney and colonic epithelial cells (Nguyen et al, 2002).

Intracellularly, RhoD regulates early endosome organisation (Murphy et al, 1996). The signalling pathway involved in this process involves the proteins c-Src kinase and a splice variant of human Diaphanous with the result that activated RhoD slows down the endosome movement but also markedly blocks motility (Gasman et al, 2003).

Materials and Methods

RHOD: Human Ras homolog gene family, member D

PDB:2J1L

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC001338
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfqs*(m), TEV-cleavable (*), N-terminal his6 tag.
Host:Rosetta-R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmAGEEAP PGVRSVKVVLVGDGGCGKTSLLMVFADGA FPESYTPTVFERYMVNLQVKGKPVHLHIW DTAGQDDYDRLRPLFYPDASVLLLCFDVT SPNSFDNIFNRWYPEVNHFCKKVPIIVVG CKTDLRKDKSLVNKLRRNGLEPVTYHRGQ EMA
Vector:pNIC28-Bsa4.

Growth

Medium:
Antibiotics:
Procedure:Medium: TB + 50 µg/ml Kanamycin + 34 ug/ml chloramp .
2 x 1 liter TB in 2.5-L baffled flasks were inoculated with 2 x 10 ml overnight culture and grown at 37°C. The protein expression was induced with 1 mM IPTG at OD600 = 3.3 at 18°C over night. The cells were collected by centrifugation and frozen at -80°C.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280nm was automatically collected.

Column 2 : Hiload 16/60 Superdex 75 prep grade 120 ml (GE/Amersham Biosciences)
The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 0.80 ml/min. Eluted proteins were collected in 2 ml fractions.

Concentration: A five fold molar excess of GDP , 5 mM DTT, 20 m M MgCl 2 and 10 mM imidazole was added to the diluted protein prior to concentration in Amicon (5 K) to 20 mg/ml. The protein concentration was determined spectrophotometrically using the predicted molar extinction coefficient 28420 (M-1 cm-1).

Extraction

Procedure
Frozen cell pellets from 2 liter were thawed at 37 o C and resuspended in a total volume of 100 ml lysis buffer. The cells were disrupted by high pressure (20 kpsi) and nucleic acids and cell debris removed by adding 0.15 % PEI , followed by centrifugation for 30 minutes at 40000 x g. The supernatant was further clarified by filtration (0.20 m m).
Concentration:
Ligand
MassSpec:The expected mass for RHODAp003 was 23981 Da. Two peaks of 24012.0 Da (+31 Da) and 23832 Da (-149 Da) were detected .
Crystallization:Crystals were grown by vapor diffusion at 20°C. A sitting drop consisting of 200 nl protein (20.0 mg/ml) and 200 nl well solution was equilibrated against well solution containing 0.4 M NH4Cl, 15 % ethylene-glycol, 19 % PEG6000 and 1 M MES pH 6. 15% ethylene-glycol was used as cryoprotectant for low temperature mounting.
NMR Spectroscopy:
Data Collection:Resolution: 2.5 Å; X-ray source: Synchrotron SLS -X10, single wavelength.
Data Processing:

References

Gasman S, Kalaidzidis Y, Zerial M. (2003). RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase. Nat. Cell Biol. 5: 195-204. Erratum in: Nat. Cell Biol. (2003) 5:680.
Murphy C, Saffrich R, Grummt M, Gournier H, Rybin V, Rubino M, Auvinen P, Lutcke A, Parton RG, Zerial M. (1996). Endosome dynamics regulated by a Rho protein. Nature. 384: 427-432.
Murphy C, Saffrich R, Olivo-Marin JC, Giner A, Ansorge W, Fotsis T, Zerial M. (2001). Dual function of rhoD in vesicular movement and cell motility. Eur. J. Cell. Biol. 80: 391-398.
Nguyen QD, Faivre S, Bruyneel E, Rivat C, Seto M, Endo T, Mareel M, Emami S, Gespach C. (2002). RhoA- and RhoD-dependent regulatory switch of Galpha subunit signaling by PAR-1 receptors in cellular invasion. FASEB J. 16: 565-576.
Tsubakimoto K, Matsumoto K, Abe H, Ishii J, Amano M, Kaibuchi K, Endo T. (1999). Small GTPase RhoD suppresses cell migration and cytokinesis. Oncogene. 18: 2431-2440.