Human regulator of G-protein signalling 2

Target ID:

RGS2A

Aliases:

G0S8

Deposition Date:

Monday 25th July 2005

Families:

G-Protein Regulators

Related Diseases:

CancerSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2AF0

Authors:

E.PAPAGRIGORIOU,C.JOHANNSON,C.PHILLIPS,C.SMEE,J.M.ELKINS,J.WEIGELT,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,F.VON DELFT,D.A.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2AF0

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
that consist of the Gα and Gβγ subunits.
In the inactive state Gα and Gβγ exist as a complex
with the Gα GTPase in an inactive GDP bound state.
Upon activation GDP is replaced with GTP which induces Gα 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.

RGS2 is a member of the B/R4 subfamily of RGS proteins and was originally identified
as a protein that was up regulated in blood mononuclear cells 2 hours
after the addition of lectin and cycloheximide (Siderovski et al, 1990, Siderovski et al, 1994) .
All of these subfamily members have an N-terminal amphipathic helix which enhances the localisation
of the protein to the membrane surface (Bernstein et al, 2000; Chen et al, 1999)
where it is ideally placed to interact with the G-proteins.

RGS2 has been shown to be directly involved in control of blood pressure
making it an exciting new pharmaceutical target for hypertension
(Heximer et al, 2003; Tang et al, 2003; Sun et al, 2005).

Materials and Methods

RGS2

PDB:2AF0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RGS2A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:BL-21(DE3)R3 phage resistant

Construct

Prelude:The valine marked in red in the sequence has been modelled as Alanine in the deposited PDB strutcure.
Sequence:MHHHHHHSSGVDLGTENLYFQSMKPSPEEAQLWSEAFDELLASKYGLAAFRAFLKSEFCEENIEFWLACEDFKKTKSPQKLSSKARKIYTDFIEKEAPKEINIDFQTKTLIAQNIQEATSGCFTTAQKRVYSLMENNSYPRFLESEFYQDLCKKPQITTEPHAT
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:10 microL of RGS 2A-c230 DNA was pipetted into a well of a 96-well plate. Added 50 µl competent BL-21(DE3)R3 cells before incubating on ice for 30 minutes. Next the cells were heat shocked for 42 seconds at 42°C in a water bath before returning onto ice for a further 2 minutes. 100 µl of SOC medium was added and the plate incubated for 1 hour at 37°C. A total of 135 µl of the cell suspension was plated onto an LB agar plate that contained 50 µg/ml kanamycin.

Glycerol stocks: Colonies from the transformation were used to inoculate 1 ml TB containing 50 µg/ ml kanamycin. The cells were then grown at 700 RPM , 37° C in a GlasCol shaker for approximately 12 hrs. Next, 250 µ l of 60% glycerol was added to the culture, mixed and 50 µl pipetted into a well of a 96-well plate before storing at -80°C.
Using the glycerol stock, 10 ml of TB + 50 µg/ml kanamycin was used inoculated and grown overnight. This was used as a starter culture for a 1 litre culture of TB + 50 µg/ml kanamycin. The culture was grown at 37°C, transferred to 25°C when the OD600 reached a value of 2. The culture was grown at this temperature until OD600 reached approximately 3 when protein production was induced with the addition of 1 mM IPTG. The cells were then grown overnight at 25°C. The next day the cells were collected by centrifugation and frozen at -80°C.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)
Procedure: The cell extract was loaded on the column at 0.8 ml/min on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of Lysis buffer, 10 column volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gel filtration, Hiload 16/60 Superdex 200 prep grade 120 ml, Code no. 17-1069-01 Amersham Biosciences
Procedure: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions.

Concentration : Using a 2 ml Vivaspin 3 K cutoff concentrator the RGS 2A-p001 was concentrated to 30 mg/ml. Concentration was determined from the absorbance at 280 nm.

Extraction

Procedure
Frozen cell pellets were thawed on ice over night and resuspended in a total volume of 50 ml lysis buffer. The cells were disrupted by four passes at 20,000 psi through a high pressure homogeniser followed by sonication. Nucleic acids and cell debris were removed by adding 0.15 % PEI , stirring for 15 minutes, then centrifugation for 20 minutes at 40,000xg. The supernatant was then further clarified by filtration (0.45 µm).
Concentration:
Ligand
MassSpec:Calculated mass of the full length construct is 19040 Da and the determined mass was 19039 Daltons .
Crystallization:Crystals grew from a 2:1 ratio mix of RGS 2A-to-reservoir ( 2.0 M ammonium sulphate, 0.2M NaCl, 0.1 M cacodylate pH 6.5 ).
NMR Spectroscopy:
Data Collection:Resolution: 2.3Å, X-ray source: Synchrotron SLS -X10, single wavelength.
Data Processing:

References

Bernstein, L. S., Grillo, A. A., Loranger, S. S., Linder, M. E. (2000).
RGS4 binds to membranes through an amphipathic alpha -helix. J. Biol. Chem. 275: 18520-18526.

Chen, C., Seow, K. T., Guo, K., Yaw, L. P., Lin, S. C. (1999). The membrane association domain of RGS16 contains unique amphipathic features that are conserved in RGS4 and RGS5. J. Biol. Chem. 274:19799-19806.

Heximer, S. P., Knutsen, R. H., Sun, X., Kaltenbronn, K. M., Rhee, M. H., Peng, N., Oliveira-dos-Santos, A., Penninger, J. M., Muslin, A. J., Steinberg, T. H., Wyss, J. M., Mecham, R. P., Blumer, K. J. (2003). Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. J Clin Invest. 111:445-452.

Siderovski, D. P., Blum, S., Forsdyke, R. E., Forsdyke, D. R. (1990). A set of human putative lymphocyte G0/G1 switch genes includes genes homologous to rodent cytokine and zinc finger protein-encoding genes. DNA Cell Biol. 9: 579-587.

Siderovski, D. P., Heximer, S. P., Forsdyke, D. R. (1994). A human gene encoding a putative basic helix-loop-helix phosphoprotein whose mRNA increases rapidly in cycloheximide-treated blood mononuclear cells. DNA Cell Biol. 13: 125-147.

Sun, X., Kaltenbronn, K. M., Steinberg, T. H., Blumer, K. J. (2005). RGS2 is a mediator of nitric oxide action on blood pressure and vasoconstrictor signaling. Mol. Pharmacol. 67: 631-639.

Tang, M., Wang, G.; Lu, P., Karas, R. H., Aronovitz, M., Heximer, S. P., Kaltenbronn, K. M., Blumer, K. J., Siderovski, D. P., Zhu, Y., Mendelsohn, M. E. (2003). Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. Nature Med. 9: 1506-1512.