Human regulator of G-protein signalling 8 in complex with Galphai3

Target ID:

XX02RGS8A

Deposition Date:

Friday 22nd December 2006

Families:

G-Protein Regulators

Related Diseases:

NeurobiologySignallingCancerMetabolism

Structure type:

protein-protein complex

Download Coordinates:

2ODE

Authors:

C.GILEADI,M.SOUNDARARAJAN,A.P.TURNBULL,J.M.ELKINS,E.PAPAGRIGORIOU,A.C.W.PIKE,G.BUNKOCZI,F.GORREC,C.UMEANO,F.VON DELFT,J.WEIGELT,A.EDWARDS,C.H.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2ODE

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.

RGS8 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.

RGS8 modulates the activity of the G-protein coupled inward rectifying K+ channel Kir3.1/Kir3.2 (GIRK1/2; Doupnik et al, 1997; Saitoh et al, 1997). It is the Gbg subunits that bind directly to GIRK, activating the channel (Logothetis et al, 1987). As described above, RGS8 does have a GAP activity through its RGS domain, resulting in deactivation of the GIRK channel however RGS8 also enhances channel activity which has been attributed to its N-terminal region (Jeong & Ikeda, 2001). Explanations for this behaviour have been accounted for through the generation of macromolecular assemblies involving ion channels, G-proteins, GPCR and RGS proteins (Benians et al, 2005; Lavine et al, 2002; Riven et al, 2006).

Here we present the crystal structure of part of the macromolecular assembly that of RGS8 in complex with the Galphai3 subunit.

Materials and Methods

XX02RGS8A: Human regulator of G-protein signalling 8 in complex with Galphai3

PDB:2ODE

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RGS8A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:BL-21(DE3)R3 (phage resistant)

Construct

Prelude:
Sequence:SMLSTEEATRWADSFDVLLSHKYGVAAFR AFLKTEFSEENLEFWLACEEFKKTRSTAK LVSKAHRIFEEFVDVQAPREVNIDFQTRE ATRKNLQEPSLTCFDQAQGKVHSLMEKDS YPRFLRSKMYLDLLSQSQRRLS
Vector:pLIC- SGC1

Growth

Medium:
Antibiotics:
Procedure:

Purification

Procedure
Column 1: Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).
Procedure: Total volume of Ni-NTA added to BioRad drip column: 4 mls (50 %). Resin washed with 12.5 ml of WB1. 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 mls of EB into 7x2 ml fractions.

Column 2 : Size exclusion using a S200 16/60 column
Procedure: The column was pre-equilibrated with two column volumes of GF buffer (flow rate 1 ml/min). The fractions from gel filtration that contained RGS8A were pooled and concentrated before 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 µM.

Extraction

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 4°C to remove cell debris. Discard pellet.
Concentration:
Ligand
MassSpec:The expected mass of RGS8A-C007 without histidine tag is 16195.2. The experimentally determined mass was 16195.17.
Crystallization:
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Benians A, Nobles M, Hosny S, Tinker A. (2005). Regulators of G-protein signaling form a quaternary complex with the agonist, receptor, and G-protein. A novel explanation for the acceleration of signaling activation kinetics. J. Biol. Chem. 280: 13383-13394.
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.
Doupnik CA, Davidson N, Lester HA, Kofuji P. (1997). RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels. Proc. Natl. Acad. Sci. U S A. 94: 10461-10466.
Jeong SW, Ikeda SR. (2001).Differential regulation of G protein-gated inwardly rectifying K(+) channel kinetics by distinct domains of RGS8. J. Physiol. 535: 335-347.
Lavine N, Ethier N, Oak JN, Pei L, Liu F, Trieu P, Rebois RV, Bouvier M, Hebert TE, Van Tol HH. (2002). G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase. J. Biol. Chem. 277: 46010-46019.
Logothetis DE, Kurachi Y, Galper J, Neer EJ, Clapham DE. (1987). The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 325: 321-326.
Riven I, Iwanir S, Reuveny E. (2006). GIRK Channel Activation Involves a Local Rearrangement of a Preformed G Protein Channel Complex. Neuron. 51: 561-573.
Saitoh O, Kubo Y, Miyatani Y, Asano T, Nakata H. (1997). RGS8 accelerates G-protein-mediated modulation of K+ currents. Nature. 390: 525-529.