Human REM2 RAS (RAD and GEM)-like GTP binding 2 in complex with GDP

Target ID:

FLJ38964A

Aliases:

FLJ38964

Deposition Date:

Friday 22nd February 2008

Families:

GTPases

Related Diseases:

MetabolismSignalling

Structure type:

protein-ligand

Download Coordinates:

3CBQ

Authors:

Nedyalkova L, Shen Y, Tong Y, Tempel W, MacKenzie F, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3CBQ

tabs

Structure Details

Description

REM2, RAS (RAD and GEM)-like GTP binding 2, belongs to the RGK branch of Ras family GTPases. RGK contains the N-terminal and C-terminal extensions beyond the core Ras domain. RGK proteins share a conserved C-terminal conserved cysteine at position -7, but lack the CAAX prenylation motif required for membrane association. RGK proteins may be localized to plasma membrane through binding to phosphorylated phosphatidylinositide (PIP) lipids (Correll et al., 2008). REM2 interacts with the beta subunit of L-type voltage-dependant Ca2+ channel (Correll et al., 2008) and negatively inhibits calcium currents, presenting a new and potentially important mechanism for regulating Ca2+-triggered exocytosis in hormone-secreting cells (Finlin et al., 2005).

We solved the crystal structure of the core Ras domain of REM2 in complex with GDP and Mg2+ at 1.82Å. While the REM2 adopts a typical G-fold, the switch I region of REM2 is placed at a position 15-20Å from its canonical position of other Ras GTPases in the GDP-bound state. This implies that REM2 uses a mechanism different from ordinary Ras GTPases in cell signaling.

Materials and Methods

REM2

PDB:3CBQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC035663
Entry Clone Source:MGC AT56-F8
SGC Clone Accession:HPC073-E12
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgQKDGIFKVMLVGESGVGKSTLAGTFGGLQGDSAHEPENPEDTYERRIMVDKEEVTLVVYDIWEQGDAGGWLRDHCLQTGDAFLIVFSVTDRRSFSKVPETLLRLRAGRPHHDLPVILVGNKSDLARSREVSLEEGRHLAGTLSCKHIETSAALHHNTRELFEGAVRQIRLRRGRNHA
Vector:pET28-mhl (GI:134105571)

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and 25 µg/mL chloramphenicol at 37 °reeC. When OD600 reached ~3.0, the temperature of the medium was lowered to 15 µC and the culutre was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 °C.

Purification

Procedure
The lysate was centrigued at 15,000 rpm for 45 minutes and the supernatants were loaded onto 5mL HiTrap HP column charged with Ni2+ ion. The column was washed steply using washing buffer containing 30, 60, and 500 mM imidazole. Fractions washed using 500 mM imidazole was collected and pooled together and further purifed by a Superdex-75 gel filtraton column pre-equilibrated with 20 mM HEPES buffer at pH 7.0, with 10 mM MgCl2 and 2 mM BME. Fractions were collected and concentrated with Amicon Ultra-15 centrifugal filter. The purify of the preparation is tested by SDS-PAGE to be around 95%.

Extraction

Procedure
Frozen cells were thawed and resuspended in 150 mL extraction buffer with freshly added 0.5% CHAPS and 2mM BME, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 3 &microL benzonase (Sigma Catalog # E1014, 250U/&microL), and lysed using microfluidizer at 15,000 PSI.
Concentration:38-40 mg/mL
Ligand
GDP, Mg2+MassSpec:Native: 21942.11, expected 21941.61
Crystallization:GDP were added to the concentrated protein to a final concentration of 5mM. Crystallization were set up using SGC-I and Red Wings screen with and without 1:100 different proteases. Crystal were seen for the following conditions:No protease: RW-G12
Chymotrypsin: SGC-B11
Trypsin: RW-B7, RW-A2
Clustered plates like crystals appear in the drop in one week.
Crystal used for structure determination were grown in Red Wings initial screen B07: 20.0% PEG3350, 0.2M KCl, no buffer in the mother liquor. Sitting drop vaporization. Cryo used: 20% PEG 3350 + 20% EG (Last updated YTONG 20080328)
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Correll,R.N., Botzet,G.J., Satin,J., Andres,D.A., and Finlin,B.S. (2008). Analysis of the Rem2 - voltage dependant calcium channel beta subunit interaction and Rem2 interaction with phosphorylated phosphatidylinositide lipids. Cell Signal. 20, 400-408.

Finlin,B.S., Mosley,A.L., Crump,S.M., Correll,R.N., Ozcan,S., Satin,J., and Andres,D.A. (2005). Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. 280, 41864-41871.

Last updated by ytong 20080402