title: Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein.
abstract: The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system.
authors: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland.
authors: Shoji
authors: S
authors: Maeda
authors: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland.
authors: Dawei
authors: D
authors: Sun
authors: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland.
authors: Ankita
authors: A
authors: Singhal
authors: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors: Marcello
authors: M
authors: Foggetta
authors: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors: Georg
authors: G
authors: Schmid
authors: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland.
authors: Joerg
authors: J
authors: Standfuss
authors: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors: Michael
authors: M
authors: Hennig
authors: pRED Pharma Research and Early Development, Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors: Roger J P
authors: RJ
authors: Dawson
authors: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland.
authors: Dmitry B
authors: DB
authors: Veprintsev
authors: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland.
authors: Gebhard F X
authors: GF
authors: Schertler
mesh_terms: D000818:Animals; D002417:Cattle; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D012243:Rhodopsin; D015293:Transducin