Human regulator of G-protein signalling 7

Target ID:

RGS7A

Deposition Date:

Monday 4th July 2005

Families:

G-Protein Regulators

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2A72

Authors:

G.A.SCHOCH,C.JOHANSSON,C.PHILLIPS,J.DEBRECZENI,C.SMEE,J.M.ELKINS,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,F.VON DELFT,O.GILEADI,D.A.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2A72

tabs

Structure Details

Regulators of G-protein signalling (RGS) are domains that terminate the signal emanating
from G-protein coupled receptors (GPCR).
When a GPCR binds a specific ligand a signal is generated that is transmitted to the G-proteins
consisting of the Gα and Gβγ subunits. In the inactive state Gα and Gβγ
exist as a complex with the Ga GTPase in an inactive GDP bound state.
Upon activation GDP is replaced with GTP which induces Ga and Gβγ complex disassociation.
At this stage both Gα and Gβγ proteins are active.
Gα does have an intrinsic GTPase activity but this is greatly enhanced by the association with an RGS domain.
Hence, RGS proteins act as GTPase activating proteins (GAPs).
Once GTP hydrolysis has taken place the GDP-Gα protein can recombine with Gβγ
thus terminating the signal.

RGS7 is a member of the C/R7 subfamily of RGS proteins.
The proteins within this subfamily contain multiple domains besides the RGS domain.
These domains, including DEP (Disheveled, Egl-10, Pleckstrin), GGL (G-protein Gamma subunit-Like)
and R7H (R7 Homology) domains, provide extra functionality to this group of RGS containing proteins.
In the brain, RGS7 has an overlapping expression distribution with RGS17 with higher levels
in the cerebellum (Larminie et al, 2004).
This may have a bearing on their cellular function as both RGS7 and RGS17 are involved
in acute tolerance to analgesia administration
(see RGS17 description and Garson et al, 2003)
even though RGS7 and RGS17 are from different subfamilies.
However, the mechanism of mu-opoid receptor regulation is likely to differ
as the GGL domain of RGS7 is involved (Sanchez-Blazquez et al, 2003) while RGS17 lacks this domain.

Two molecules were found the crystal asymmetric unit however they were formed not from individual RGS7 domains
but by a combination of two RGS7 domains via a domain swap process.
This crystal structure suggests a possible folding pattern for RGS domains that involves first
the synthesis of the entire RGS domain before folding of the N-terminal helices around the C-terminal section.

Materials and Methods

RGS7

PDB:2A72

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RGS 7A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:Bl-21(DE3)R3 phage resistant

Construct

Prelude:
Sequence:SMKEPSQQRVKRWGFGMDEALKDPVGREQFLKFLESEFSSENLRFWLAVEDLKKRPIKEVPSRVQEIWQEFLAPGAPSAINLDSKSYDKTTHNVKEPGRYTFEDAQEHIYKLMKSDSYPRFIRSSAYQELLQAKKKSGNSMDRRTS
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:TRANSFORMATION: 2 µL of each construct DNA was added to each well of a PCR plate on ice. 90 µL of competent BL21-(DE3)R3 bacteria were added to each well, with the plate remaining on ice. The plate was left on ice for a further 20 minutes. The heat-shock procedure was carried out by transferring the plate to a 42°C water bath for 45 seconds and then returning it to sit on ice for 2 minutes. 100 µL of SOC medium (pre-warmed to 42°C) was added to each well and the plate incubated at 37°C for 60 minutes. 150 µL of each culture was plated out onto LB-agar containing 33 µg/mL kanamycin in a 5.5 cm Petri dish. The plates were incubated at 37°C overnight.

PREPARATION OF GLYCEROL STOCKS: A number of colonies were used to innoculate 1mL of 2TY with 50 µg /mL kanamycin in a 96-well block. The block was placed in a shaker overnight at 37°C and 200 rpm. 40 µL of 70% glycerol was combined with 60 µL of bacterial culture in sterile 96-well microtitre plates. The plates were placed in the -80°C freezer.

EXPRESSION: 20 mL LB + 33 µg/mL kanamycin was inoculated using the glycerol stock and grown overnight. 16 mL of the sample was used to innoculate 2 litre TB medium + 33 µg/mL kanamycin . The culture was grown at 37° C for 2.5 hours before the incubator temperature was reduced to 25°C. The cultures were induced with 1 mM IPTG one hour after the temperature shift when the OD600 of the cells was approximately 0.9.

The cells were harvested by centrifugation 4 hours after induction. All the pellets were resuspended in Lysis Buffer and stored in a -80°C freezer.

Lysis/Binding Buffer: 50 mM Hepes pH 8.0, 500 mM NaCl, 5 % glycerol, 5mM imidazole pH 8.0, 0.5 mM TCEP

Purification

Procedure
Wash Buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 5 % Glycerol, 25 mM Imidazole pH 8.0, 0.5 mM TCEP; Elute Buffer: 50 mM Hepes pH 8.0, 500 mM NaCl, 5 % Glycerol, 250 mM Imidazole pH 8.0, 0.5 mM TCEP; GF Buffer: 50 mM Tris pH 8.5, 500 mM NaCl, 0.5 mM TCEP.

Procedure: The supernatent was filtered through a 1.2 micron and then a 0.2 micron syringe filter. A new 1 mL HisTrap column was used for IMAC. After washing with water the column was equilibrated with 5 column volumes of Binding Buffer at a flow rate of 0.8 mL/min. Binding of the sample to the resin was carried out by flowing the supernatant over the resin at a rate of 0.8 mL/min. Once bound the resin was washed with a minimum of 15 mLs binding buffer followed by a minimum of 20 mLs wash buffer. The application of 5 mLs of Elution Buffer eluted the sample from the Ni2+-resin. The protein sample was immediately applied to a S200 16/60 gel filtration column at a flow rate of 1.2 mLs/min that was pre-equilibrated with GF Buffer. The main peak off the gel filtration column was RGS 7 however the sample was still only 85 % pure as judged by SDS - PAGE gel stained using Coomassie Blue.

Enzymatic treatment: HIS- TAG REMOVAL - To improve the purity of the sample the hexahistidine tag was cleaved and the cleavage products plus the contaminants rebound to a Ni2+-resin thus allowing the cleaved RGS 7 sample to run through the column. The RGS 7 fractions collected after gel filtration were pooled and cleaved overnight with 100 µL of 2.8 mg/mL Tev protease. 500 µL of 50 % Ni-sepharose (Amersham) was added and the sample inverted repeatedly on a rotator for 60 minutes at 4°C to remove the His tag with Tev and other contaminants. The protein was concentrated to a final concentration of 62 mg/mL before storing at -80°C.

Extraction

Procedure
To the cell pellet (approx. 80 mLs) was added 20 mLs of Lysis/Binding buffer and PMSF was added to a final concentration of 1.0 mM. Cell breakage: 4 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 100 mLs (estimate). PEI was added to a final concentration of 0.05 % to precipitate the DNA. Centrifuge for 40 mins at 18000 rpm and 4°C to remove cell debris. Discard pellet.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals grew from a 1:1 ratio mix of RGS 7A-to-reservoir (25 % PEG 3350 , 0.2 M ammonium acetate, 0.1 M Bis-Tris pH 5.5 ).
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Garzon, J., Lopez-Fando, A., Sanchez-Blazquez, P. (2003) The R7 subfamily of RGS proteins assists tachyphylaxis and acute tolerance at mu-opioid receptors. Neuropsychopharmacology. 28: 1983-1990.

Larminie, C., Murdock, P., Walhin, J. P., Duckworth, M., Blumer, K. J., Scheideler, M. A., Garnier, M. (2004) Selective expression of regulators of G-protein signaling (RGS) in the human central nervous system. Brain Res Mol Brain Res. 122: 24-34.

Sanchez-Blazquez, P., Rodriguez-Diaz, M., Lopez-Fando, A., Rodriguez-Munoz, M., Garzon, J. (2003) The GBeta5 subunit that associates with the R7 subfamily of RGS proteins regulates mu-opioid effects. Neuropharmacology. 45: 82-95.