Human GTP-Binding Protein RAB2B

Target ID:

RAB2B

Aliases:

FLJ14824

Deposition Date:

Thursday 30th June 2005

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2A5J

Authors:

A.DONG,J.WANG,Y.SHEN,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,A.BOCHKAREV,H.W.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2A5J

tabs

Structure Details

Rab proteins constitute the largest subfamily of the Ras superfamily.
These proteins are small-molecular-weight guanosine triphosphatases (GTPases)
that act as molecular switches between a GDP-bound inactive form and GTP-bound active form
in which they recruit downstream effectors onto membranes.
Rab proteins play key regulatory roles in vesicular trafficking between various subcellular compartments within eukaryotic cells.1 Sequence analysis of human genome indicates the presence of ~60 different Rab genes,2 and it is well established that different Rab proteins localize to distinct membrane-bound cellular compartments in mammalian cells.3

Rab2 is a resident of pre-Golgi intermediate and is required for protein transport from the endoplasmic reticulum to the Golgi complex. Two Rab2 genes (Rab2a and Rab2b) have been identified in human genome, with 82% protein sequence identity. Rab2b transcripts were found extremely high in colon tumor cells, suggesting its close relationship with colon tumors.4 This is different from Rab2a, which is associated with neoplastic diseases during immunological events.5
Here we solved the structure of human Rab2b bound to GDP at 1.5Ã….

The Rab2b-GDP structure illustrates the nucleotide binding site and the specific interactions between Rab2b and GDP. The structure of Rab2b-GDP is a valuable addition to the already known structures of Rab family. Analysis and comparison of these related structures and their conformational changes with GTP will help us understand the function and regulation of these molecular switch GTPases.

Materials and Methods

Rab2b

PDB:2A5J

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:21361884
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site before the last Ser: MGSSHHHHHHSSGLVPRGS
Host:E. coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsYLFKYIIIGDTGVGKSCLLLQFTDKRFQPVHDLTIGVEFGARMVNIDGKQIKLQIWDTAGQESFRSITRSYYRGAAGALLVYDITRRETFNHLTSWLEDARQHSSSNMVIMLIGNKSDLESRRDVKREEGEAFAREHGLIFMETSAKTACNVEEAFINTAKEIYRKIQQGLF
Vector:

Growth

Medium:
Antibiotics:
Procedure:We prepared the seeds by inoculating glycerol stock of E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After overnight growth, all of the seeds were inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin at 37ºC and grown to an OD600 about 2 to 3. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.0 mM and grown overnight at 18ºC in the SGC LEX bubbling system. Extraction method Cultures were centrifuged and the cell pellets were harvested and stored at -80 ºC before use. Cells were thawed and suspended in 100 mL the binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole) with 0.5% CHAPS (Sigma) a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride) and lysed with microfluidizer. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was used for subsequent steps of purification. All the extraction steps were carried out at 4ºC.

Purification

Procedure
The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 2 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4ºC. The Ni-NTA column was washed with 150 mL of the wash buffer (10mM Tris pH7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). Protein eluted from the Ni-NTA column was further purified by a gel filtration column superdex G75 with a buffer containing 20 mM Tris pH 8.0, 0.15 M NaCl, 10 mM DTT. Protein peak fractions were combined, and 5 molar equivalents of GDP and 5 mM MgCl2 were added to the combined fraction before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 0.5 mL. The protein concentration estimated based on the extinction coefficient of the protein to be 39.1 mg/mL. About 19.5 mg of protein was obtained from 3.6 L of cell culture.

Extraction

Procedure

Concentration:
Ligand
MassSpec:
Crystallization:Purified RAB2 was crystallized using the sitting drop vapor diffusion method at room temperature. Crystals grew in one 3 days when the protein (39.1 mg/ml) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 30% PEG5000 MME, 0.2M (NH4)2SO4, 0.1M MES, PH6.5. The crystals were flash frozen with the mother liquor with 15% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Pereira-Leal , J. B. and Seabra, M. C. (2001). Evolution of the Rab family of small GTP-binding proteins. J. Mol. Biol. 313, 889-901

2. Bock, J.B., Matern, H.T., Peden, A.A. and Scheller, R.H. (2001). A genomic perspective on membrane compartment organization. Nature 409, 839-841

3. Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers. Nat. Rev. Mol. Cell. Biol. 2, 107-117

4. Ni, X., Ma, Y., Cheng, H., Jiang, M., Guo, L., Ji, C., Gu, S., Cao, Y., Xie, Y. and Mao, Y. (2002). Molecular cloning and characterization of a novel human Rab ( Rab2B) gene. J. Hum. Genet. 47:548-551

5. Culine, S., Honore, N., Closson, V., Lang, P., Bertoglio, J., Tavitian A. and Olofsson, B. (1993). A possible role for the Ras-related Rab2 protien in the immunological events associated with hematological malignancies. Nouv Rev Fr Hematol 35:41-44