Human regulator of G-protein signalling 1 in complex with the activated state of Galphai1

Target ID:

XX01RGS1A

Deposition Date:

Friday 28th April 2006

Families:

G-Protein Regulators

Related Diseases:

MetabolismNeurobiologySignallingCancer

Structure type:

protein-protein complex

Download Coordinates:

2GTP

Authors:

M.SOUNDARARAJAN,A.P.TURNBULL,E.UGOCHUKWU,F.GORREC,F.VONDELFT,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2GTP

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 Ga and Gbg subunits. In the inactive state Ga and Gbg 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 Gbg complex disassociation. At this stage both Ga and Gbg proteins are active. Ga 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-Ga protein can recombine with Gbg thus terminating the signal.

RGS1 is a member of the B/R4 subfamily of RGS proteins. 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.

Stimulation of the immune system results in a complex, coordinated signalling process that prepares the body to respond to an infection and elliminated the infection. A major element of this process is the activation of the B-cells which produce the antibodies that target the pathogen for destruction. Within the B-cells several genes are up-regulated upon activation. RGS1 is one that is immediately activated (Newton et al 1993). The GAP activity of RGS1 is directed against Gia and Gqa G-proteins with the result that the direction of B-cell migration is altered (Moratz et al, 2000). RGS1 even alters the movement of B-cells within lymph nodes (Han et al, 2005). Demonstration that RGS1 is also upregulated by bacterial lipopolysaccharides (Panetta et al, 1999) enhances the suggestion that RGS1 plays a key role in the signalling response of the immune system.

Here we present the crystal structure of RGS1 in complex with the activated state of G alpha I

Materials and Methods

RGS1 + G alpha I: Human regulator of G-protein signalling 1 in complex with the activated state of G alpha I

PDB:2GTP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Prof D Siderovski
SGC Clone Accession:
Tag:
Host:

Construct

Prelude:
Sequence:MREVKLLLLGAGESGKSTIVKQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLFDSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAAAYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDVIIKNNLKDCGLF
Vector:pProEXHT

Growth

Medium:
Antibiotics:
Procedure:Transformation: 2 microL of the construct was added and mixed to 90 microLof phage-resistant BL21 (DE3) with the Rosetta plasmid and left on ice for 20 minutes.The heat-shock procedure was done by transferring the plate to a 42degC water bathfor 45 seconds and then returning it to ice for a further 2 minutes.100 microL of LB medium (pre-warmed to 42degC) was added to the well and the plate incubated at 37degC for 40 minutes.

Large scale expression:The RGS 1A-c004 fresh transformants were used to innoculate 20 mL LB media with 100 microG/mL Ampicillinwhich was placed in a 37degC shaker overnight.The next day this starter culture was used to innoculate 4 x 1 litre of TB medium which contained 100 microG/mLAmpicillin.Protein induction was carried out with the addition of 0.5 mM IPTG after the cellsreached an OD600 of 0.7 and the incubation temperature decreased to 25degC.After 4 hours the cells were harvested by centrifugation. The cell pellet was frozen in the -80degC freezer.

Purification

Buffers
Wash Buffer I (WBI): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % glycerol, 10 mM imidazoleWash Buffer II: 50 mM Tris pH 8.0, 300 mM NaCl, 5 % glycerol , 30 mM imidazoleElute Buffer: 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % glycerol, 250 mM imidazole
Procedure
Total volume of Ni-NTA added to BioRad drip column: 4 ml (50 %). Resin washed with 12.5 mL of WBI.The supernatent was applied to a column using 5 ml pipette and allowed to pass over the resin.The flow through was collected in a 50 mL falcon tube and applied once more to the column.Two wash steps followed. Wash with 12.5 mL of WBI.Wash with 12.5 ml column vols of WBII. Elute with 14 ml of EB into 7x2 ml fractions.

The fractions from gel filtration that contained G protein a1 were pooled and concentratedbefore loading on to S200 16/60 gel filtration column.The fractions containing protein were identified on a coomasie blue stained gel.

Enzymatic treatment : His-Tag removal. The eluate was treated with Tev protease. The sample was rebound to Ni sepharose to remove Tev and other contaminants. The His tag cleaved protein was concentrated to 300 ÂµM.

Common steps:Purification of G protein a 1 and RGS 1A complex:Equimolar concentrations of G protein and RGS 1A was mixed and incubated at 4 degrees for 15 minutes.The sample was passed through S200 gelfiltration column which was prequilibratedwith 20 mM Hepes pH 7.5, 100 mM NaCl, 5 % glycerol, 2 mM DTT, 30 m M AlCl3, 100 mM GDP and 20 mM NaF.The proteins eluted as a complex was analysed using PAGE and the fractions were pooled and concentrated to 24 mg/mLand used for crystallisation set up.

Extraction

Buffers
Lysis Buffer: 50 mM Hepes pH 7.5, 300 mM NaCl, 5 % Glycerol, 10 Mm imidazole
Procedure
1 tablet protein inhibitor in 10 mL Lysis Buffer was added to the 1L growth pellet.Total vol: 45 mls (estimate).Cell breakage: 3 passes through the Emulsiflex C5 high pressure homogeniser.Centrifuge for 45 mins at 16000 rpm and 4degC to remove cell debris.
Concentration:
Ligand
MassSpec:The expected mass of N terminally 30 aminoacids truncated GNAI1A-C001 without histidine tag is 37113.The experimentally determined mass was 37111.
Crystallization:Crystals grew from a 1:1 ratio mix of G protein a1 , RGS 1A and precipitant containing 0.2M NaNO 3 ; 0.1M BT Prop pH 6.5, 20 % PEG 3350; 10 % EtGly.
NMR Spectroscopy:
Data Collection:Resolution: 2.55Å; 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.

Han, S. B., Moratz, C., Huang, N. N., Kelsall, B., Cho, H., Sh,i C. S., Schwartz, O., Kehrl, J. H. (2005). Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles. Immunity. 22: 343-354.

Moratz, C., Kang, V. H., Druey, K. M., Shi, C. S., Scheschonka, A., Murphy, P. M., Kozasa, T., Kehrl, J. H. (2000). Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes. J Immunol. 164: 1829-1838.

Newton, J. S., Deed, R. W., Mitchell ,E. L., Murphy, J. J., Norton, J. D. (1993). A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an alpha helical basic phosphoprotein.
Biochim Biophys Acta. 1216: 314-316.

Panetta, R., Guo, Y., Magder, S., Greenwood, M. T. (1999). Regulators of G-protein signaling (RGS) 1 and 16 are induced in response to bacterial lipopolysaccharide and stimulate c-fos promoter expression.
Biochem. Biophys. Res. Commun. 259: 550-556.