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RAB31: Human RAB31, member of RAS oncogene family

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PDB Code 2FG5 Target Class GTPases

Target RAB31
Alias Rab22B
Disease Area/Function signalling
Date Deposited 2005-12-21
Authors W.TEMPEL, J.WANG, S.ISMAIL, C.ARROWSMITH, A.EDWARDS, M.SUNDSTROM, J.WEIGELT, A.BOCHKAREV, H.PARK, STRUCTURAL GENOMICS CONSORTIUM(SGC)

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Structure Details
Rab proteins are small-molecular-weight guanosine triphosphatases (GTPases) that control a number of intracellular events by alternating between GDP and GTP conformations. There are more than 60 different Rab genes in the human genome. Each Rab protein interacts with its specific partners, localizes to distinct membrane-bound cellular compartments, and carry out distinct functions.1

Human Rab31 was first identified and cloned from human platelets using RT-PCR (2). Here we present the structure of human Rab31 bound to a non-hydrolyzable GTP analog, guanosine-5’-(â, ã)-imidotriphosphate (GppNHp) at 2.8 Å. Like other members of the GTPase superfamily, Rab31 possesses a characteristic nucleotide binding fold that consists of six-stranded â-sheet core surrounded by five á helices. The RAB31-GppNHp structure illustrates the nucleotide binding site and the specific interactions between them. The Rab31 structure is a valuable addition to the already known structures of the Rab family members. Comparison of these related structures complexed with different substrates will help us understand the function and regulation of these molecular switch GTPases.

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. Bao, X., Faris, A., Jang, E. and Haslam, R. (2002). Molecular cloning, bacterial expression and properties of Rab31 and Rab32. Eur. J. Biochem. 269, 259-271

Materials & Methods
StructureRab31
PDB Code2FG5
Entry clone accession GI:33589861
Entry clone source MGC
Tag His-tag with integrated thrombin protease site before the last Ser: MGSSHHHHHHSSGLVPRGS
Construct sequencemgsshhhhhhssglvprgsAIRELKVCLLGDTGVGKSSIVCRFVQDHFDHNISPTIGASFMTKTVPCGNELHKFLIWDTA
GQERFHSLAPMYYRGSAAAVIVYDITKQDSFYTLKKWVKELKEHGPENIVMAIAGNKCDLSDIREVPLKDAKEYAESIGA
IVVETSAKNAINIEELFQGISRQIPPLDPHEN
Vector p28a-LIC
Expression host E. coli BL21-CodonPlus (DE-3)-RIL
Growth method We prepared the seeds by inoculating glycerol stock of E. coli cells BL21-CodonPlus (DE-3)-RIL into 100 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 and 50 µg/mL chloramphenicol at 37ºC and grown to an OD600 between 3-5. 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 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 mM imidazole) with 0.5% CHAPS (Sigma) and 1 mM phenylmethyl sulfonyl fluoride (PMSF) 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 5 mL HisTrap HP (Amersham) equilibrated with the same binding buffer at 4ºC. The HisTrap HP column was steply washed with 25 mL of binding buffer, 25 mL of binding buffer with 30 mM imidazole, and 25 mL of binding buffer with 50 mM imidazole. The His-tagged protein was eluted by linear gradient of imidazole from 50 mM to 500 mM in 50 mL. The eluted protein peak fractions detected by UV280 nm were combined and further purified by gel filtration column superdex 75 with a buffer containing 20 mM HEPES pH 8.0, 500m M NaCl, 1 mM DTT. Protein peak fractions were combined, GppNHp (Sigma) 5 times of the Rab31 in molarity, and MgCl2 to the final concentration of 5 mM were added before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 0.5 mL. The protein concentration estimated by Bradford to be 75.2 mg/mL. About 140 mg of protein was obtained from 1.8 L of cell culture.
Crystallization Purified RAB31 was crystallized using the sitting drop vapor diffusion method at room temperature. Crystals grew in 2 days when the protein (75.2 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 20% (v/v) isopropanol, 16% PEG4K and 0.1M BisTris pH6.5. The crystals were flash frozen with the mother liquor.
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