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Human Ras-related GTP-binding protein 4B

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PDB Code 2O52 Target Class GTPases

Target RAB4B
Alias FLJ78649, MGC52123
Disease Area/Function signalling
Date Deposited 2006-12-05
Authors H.ZHU, W.TEMPEL, J.WANG, Y.SHEN, R.LANDRY, C.H.ARROWSMITH, A.M.EDWARDS, M.SUNDSTROM, J.WEIGELT, A.BOCHKAREV, H.PARK, STRUCTURAL GENOMICS CONSORTIUM (SGC)

Struc Details 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 (20–29 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 – F5 (F1: IGVDF; F2: KLQIW (β3); F3: RFrsiT (loop 4); F4: YYRGA (α2 – loop 5); F5: LVYDIT (β4 – 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 [6].

Rab4 was found to be associated with early endosomes playing a role in their sorting and recycling. Rab4 is also implicated in the regulation of the recycling of internalized receptors back to the plasma membranes. Furthermore, Rab4 seems to have a specialized role in receptor-mediated antigen processing in B-lymphocytes, in calcium-induced a-granule secretion in platelets, and in a-amylase exocytosis in exocrine pancreatic cells. In addition, Rab4 appears to control the subcellular distribution of the glucose transporter isoform Glut4, specifically expressed in the insulin-sensitive adipose and muscle tissues. Therefore, it is assumed that Rab4 is implicated in early endosome trafficking by controlling steps necessary for sorting, recycling and degradation [reviewed in 7].

A number of proteins that specifically bind to the GTP-bound form of Rab4 have been identified, such as Rabaptin-5, Rabaptin-5b, Rabaptin-4/5a, Rabenosyn-5, Rabip4, Rabip40, Rab coupling protein_(RCP), Dynein light intermediate chain-1, syntaxin 4, and CD2AP/CMS. Most of the identified Rab effectors are divalent, interacting with more than one Rab protein. These divalent effector proteins might function as membrane-tethering molecules [reviewed in 7].

We solved the structure of Rab4B at 2.2 Å. The overall structure of this protein share common structure with other Rab proteins with relatively subtle variations. The crystal structure of Rab4B 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 17GXXXXGKS24, with bound Mg (II) and GDP. Superimposing this structure with crystal structure of Rab4A (2BMD), the largest deviations were observed in the switch II region (T66 – T76), the switch I loops in both protein structures are disordered. Although the sequence identity between Rab 4A and Rab 4B is high (> 80%), 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.

References

  1. 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.
  2. Chavrier, P. & Goud, B. Curr. Opin. Cell. Biol. 1999, 11, 466–475.
  3. Schultz, J., Doerks, T., Ponting, C. P., Copley, R. R. & Bork, P. Nat. Genet. 2000, 25, 201–204.
  4. 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, 1077–87.
  5. 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.
  6. Pfeffer, SR., Structural Clues to Rab GTPase Functional Diversity, J. Biol. Chem., 2005, 280, 15485-88.
  7. Huber, S., Scheidig, A., FEBS Lett. 2005, 579, 2821-9.
Materials & Methods
StructureRAB4B-GDP
PDB Code2O52
Entry clone accession NM_016154
Entry clone source Codon Devices
Tag N-terminal hexahistidine tag
Construct sequencemgsshhhhhhssglvprgsIWSDFLFKFLVIGSAGTGKSCLLHQFIENKFKQDSNHTIGVEFGSRVVNVGGKTVKLQIWD
TAGQERFRSVTRSYYRGAAGALLVYDITSRETYNSLAAWLTDARTLASPNIVVILCGNKKDLDPEREVTFLEASRFAQEN
ELMFLETSALTGENVEEAFLKCARTILNKIDSGELDPERM
Vector p28a-thrombin-lic
Expression host E.coli. BL21 (DE3) codon(+) RIL
Growth medium Terrific Broth
Growth method 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.
Extraction procedure Cells were thawed and resuspended 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.
Purification buffersWash buffer: 50 mM Tris pH 8.0, 0.5 M NaCl, 30mM Imidazole, 1 mM TCEP.
Elution buffer: 50 mM Tris, pH 8.0, 0.5 M NaCl, 250 mM Imidazole, 1 mM TCEP, 2 mM MgCl2.
Size exclusion chromatography (SEC) buffer: 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM TCEP.
Purification procedure Column 1: Ni-NTA beads
Procedure: 2.5 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.

Column 2 : Size exclusion chromatography (Superdex 75 26/60)
The fractions eluted off the Ni-affinity column were applied to a Superdex 75 column (26/60) 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 5kDa cut off in SEC-buffer

Crystallization Crystals were obtained using the vapor diffusion method. The protein solution (17 mg/mL) contained a 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 18% PEG8000, 0.2 M Mg(OAc)2, 0.1 M NaCaco, pH 6.50. Crystals appeared after three days at 18 ºC.
Data collection Crystals were cryo-protected using a mixture of 50% mineral oil and 50% parotone, and flash frozen in liquid nitrogen. Diffraction data were collected at Rigaku FR-E with Rigaku R-AXIS IV++ detector to 2.2 Å.
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