Human GTP-Binding Protein RAB14

Target ID:

RAB14

Aliases:

FBPRAB-14

Deposition Date:

Friday 22nd July 2005

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

Superceded by 4DRZ

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2AED

tabs

Structure Details

The 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 effector proteins 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 Rabs localize to distinct membrane-bound cellular compartments in mammalian cells.3

Rab14 is a close homolog of Rab2 and Rab4, which are known to localize and function in endoplasmic reticulum (ER)-Golgi and endosomal compartments, respectively.2,3 Although the function of Rab14 is unknown, studies with Rab14 from rat brain revealed that Rab14 is localized to both the biosynthetic (ER, Golgi and trans-Golgi network) as well as endosomal compartments, suggesting a role in biosynthetic as well as recycling pathways between the Golgi and endosomal compartments.4

We have solved the structure of human Rab14 bound to GDP at 2.3Ã…. The Rab14-GDP structure illustrates the nucleotide binding site and the specific interactions between Rab14 and GDP. The structure of Rab14-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 different substrates will help us understand the function and regulation of these molecular switch GTPases.

Materials and Methods

Rab14

PDB:2AED

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:19923483
Entry Clone Source:MGC
SGC Clone Accession:1E-01 (HPC006:F5)
Tag:N-terminal: His-tag with integrated thrombin protease site before the last Ser: MGSSHHHHHHSSGLVPRGS
Host:E. coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMATAPYNYSYIFKYIIIGDMGVGKSCLLHQFTEKKFMADCPHTIGVEFGTRIIEVSGQKIKLQIWDTAGQGRFRAVTRSYYRGAAGALMVYDITRRSTYNHLSSWLTDARNLTNPNTVIILIGNKADLEAQRDVTYEEAKQFAEENGLLFLEASAKTGENVEDAFLEAAKKIYQNIQ
Vector:p28a-LIC

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 37 degC 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 18 degC in the SGC LEX bubbling system.

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 2.8 mL. The protein concentration estimated based on the extinction coefficient of the protein to be 19.0 mg/mL. About 53.1 mg of protein was obtained from 3.6 L of cell culture.

Extraction

Procedure
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 4degC.
Concentration:19.0 mg/mL
Ligand
Guanidine diphosphate (GDP)MassSpec:
Crystallization:Purified RAB14 was crystallized using the hanging drop vapor diffusion method at room temperature. Crystals grew in one day when the protein (19.0 mg/ml) was mixed with the reservoir solution in a 2:2 volume ratio, and the drop was equilibrated against a reservoir solution containing 20% PEG3350, 0.2M Calcium acetate, pH7.2. 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. Junutula, J.R., De Maziere, A.M., Peden, A.A., Ervin, K.E., Advani, R.J., van Dijk, S.M., Klumperman, J. and Scheller, R.H. (2004). Rab14 is involved in membrane trafficking between the Golgi complex and endosomes. Mol. Biol. Cell 15, 2218-2229.