Human RAB25 - member of RAS oncogene family

Target ID:

RAB25

Aliases:

CATX-8

Deposition Date:

Thursday 11th January 2007

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2OIL

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2OIL

tabs

Structure Details

Rab proteins constitute the largest branch of the Ras GTPase superfamily. Rabs use the guanine nucleotide-dependent switch mechanism common to the superfamily to regulate each of the four major steps in membrane traffic: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment. These different tasks are carried out by a diverse collection of effector molecules that bind to specific Rabs in their GTP-bound state.

Rabs are a ubiquitously expressed family of small (20â€“29 kDa) monomeric Ras-like GTPases. To date, 11 Rabs have been identified in yeast (including Sec4p and the Ypt proteins) and >60 in mammalian cells. Rab proteins distinguish themselves from other members of the Ras GTPase family with five so-called Rab family sequences F1 â€“ F5 (F1: IGVDF; F2: KLQIW (β3); F3: RFrsiT (loop 4); F4: YYRGA (α2 â€“ loop 5); F5: LVYDIT (β4 â€“ loop 6)). These sequences are locating in and around switch I and switch II regions, the regions that change conformation upon GDP or GTP binding.

Rab proteins have switch conformations between GTP-Rab proteins (active form) and GDP-Rab proteins (inactive form). Effector proteins interact with the active GTP-Rab proteins. Conformational differences within the switch regions (switch I and II) of different Rab proteins lead to the specific binding of various effector proteins to the targeted Rab proteins, although effectors are also found to interact with non-conserved regions of Rab proteins, which further increasing binding specificity. In some cases, the variability in Rab-GDP switch domain structures is influenced by the presence and nature of the bound metal ion. Rab proteins share some conserved residues, these residues point their side chains at different angles in the relation to the β-strands of the core. These angle shifts create very distinct surfaces of related GTPases that are surely important for their ability to interact specifically with distinct effector proteins. Thus, Rab proteins have a related overall shape, but they are very different in subtle ways that will be recognized by binding partners.

Rab 25 belongs to the Rab11 family of small GTPases. It has been reported Rab25 determines aggressiveness of ovarian and breast cancers. A better understanding of the structural features of Rab25 will open new opportunities for therapeutic intervention and better outcomes.

We solved the structure of Rab25 at 2.3Å. The overall structure of this protein share common structure with other Rab proteins with relatively subtle variations. The crystal structure of Rab25 consisting of a core formed by a six stranded β-sheet surrounded by five α-helices. The conserved phosphate-binding loop (P-loop) motif in this structure is 19GESGVGKT26, with bound Mg (II) and GDP. The structure of Rab25 with bound GDP is very similar to the structure of Rab11 with bound GDP (PDB code: 1OIX) [ ] except in two loop regions (residues 39 â€“ 47 and residues 67 â€“ 77). Although the sequence identity between Rab 25 and Rab 11 is high (> 72%), even in conserved secondary structure regions, exposed surfaces between two proteins are different due to the different orientations of side chains. The switch regions and the some distinct surface area may play crucial roles for the potential specific binding partner recognition.

Materials and Methods

Rab25- Ras-related GTP-binding protein 25 with bound GDP

PDB:2OIL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_020387
Entry Clone Source:Codon Devices
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:E.coli. BL21 (DE3) codon(+) RIL

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsEDYNFVFKVVLIGESGVGKTNLLSRFTRNEFSHDSRTTIGVEFSTRTVMLGTAAVKAQIWDTAGLERYRAITSAYYRGAVGALLVFDLTKHQTYAVVERWLKELYDHAEATIVVMLVGNKSDLSQAREVPTEEARMFAENNGLLFLETSALDSTNVELAFETVLKEIFAKVSKQ
Vector:p28a-thrombin-lic

Growth

Medium:
Antibiotics:
Procedure:The target protein was expressed in E.coli. BL21 (DE3) coden (+) RIL containing the plasmid. The 100 mL overnight culture in Luria-Bertani medium was inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin and 50 µg/mL chloramphenicol at 37 ºC and grown to an OD600 ~ 3.0 in the SGC LEX bubbling system. The culture was induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.0 mM and grown overnight at 18 ºC. The culture was harvested with centrifugation. Pellets were flash frozen and stored at -80 ºC.

Purification

Procedure
Column 1: Ni-NTA beads
Procedure: 1 ml of Ni-NTA suspension solution was added into 100 ml cell lysis supernatant solution. The mixture was shaken for 1 hour at 4 ºC. Beads were collected by centrifugation at 2500 rpm for 5 minutes. Beads were washed with 75 ml washing buffer, then collected by centrifugation. Protein was eluted with 15 ml elution buffer. Thrombin (1 unit/mg protein) was added into the eluted protein solution, and the solution was shaken at 4 ºC overnight to remove the His tag.

Column 2: Size exclusion chromatography (Superdex 75 26/60)
The fractions eluted off the Ni-affinity column were applied to a Superdex 75 column equilibrated in SEC buffer at a flow rate of 2.0 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.

Protein concentration: Amicon ultra centrifugal filter with a 5 kDa cut off in SEC-buffer

Extraction

Procedure
Cells were thawed and re-suspended in 100 mL binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5 mM imidazole) with 0.5% CHAPS (Sigma) and 1 mM phenylmethyl sulfonyl fluoride (PMSF), 0.5% (v/v) protease inhibitor cocktail (Sigma), 1 mM Benzamidine, 1600 units Benzonase (Sigma), and lysed with a microfluidizer. The lysate was centrifuged at 16000 rpm for 45 min and the supernatant was used for subsequent steps of purification. All the extraction steps were carried out at 4 ºC.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method. The protein solution (27 mg/ml) contained 5 fold molar of GDP and MgCl₂. 0.5 µl of the concentrated protein was mixed with 0.5 µl of a well solution containing 20% PEG3350, 0.2 M NH4Cl. Crystals appeared after two days at 18°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using a mixture of PEG3350 and PEG400, and flash frozen in liquid nitrogen. Diffraction data were collected at Rigaku FR-E with Rigaku R-AXIS IV++ detector to 2.3 Å.
Data Processing:

References

Grosshans BL, Ortiz D, Novick P., Rabs and their effectors: achieving specificity in membrane traffic., Proc Natl Acad Sci U S A. 2006, 103, 11821-7.
Chavrier, P. & Goud, B. Curr. Opin. Cell. Biol. 1999, 11, 466â€“475.
Schultz, J., Doerks, T., Ponting, C. P., Copley, R. R. & Bork, P. Nat. Genet. 2000, 25, 201â€“204.
Pereira-Leal, J. B., and Seabra, M. C. The Mammalian Rab Family of Small GTPases:
Definition of Family and Subfamily Sequence Motifs Suggests a Mechanism for Functional Specificity in the Ras Superfamily, J. Mol. Biol., 2000, 301, 1077â€“87.
Grosshans BL, Ortiz D, Novick P., Rabs and their effectors: achieving specificity in membrane traffic., Proc Natl Acad Sci U S A. 2006, 103, 11821-7.
Pfeffer, SR., Structural Clues to Rab GTPase Functional Diversity, J. Biol. Chem., 2005, 280, 15485-88.
Cheng, KW., et. al., The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers., Nat Med. 2004, 1251-6.
Pasqualato, S., and Cherfils J., Crystallographic Evidence for Substrate-Assisted GTP Hydrolysis by a Small GTP Binding Protein, Structure, 2005, 13, 533-540.