A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli

Egloff, Pascal; Deluigi, M.; Heine, Philipp; Balada, Stefanie B.; Plückthun, Andreas

doi:10.1016/j.pep.2014.10.006

Cited by 19 publications

(18 citation statements)

References 32 publications

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“…Expression and the purification of a signaling-competent, thermo-stabilized variant of rat neurotensin receptor 1 (termed HTGH4) were performed as described previously 21,22 with some modifications. Briefly, the full-length fusion protein consisting of maltose-binding protein (MBP), followed by a His tag, a 3C protease recognition site, NTR1, a second 3C protease recognition site, thioredoxin (TrxA), and a His tag at the C terminus was purified by Ni 2+ affinity chromatography.…”

Section: Methodsmentioning

confidence: 99%

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Covalently circularized nanodiscs for studying membrane proteins and viral entry

et al. 2016

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We engineered covalently circularized nanodiscs (cNDs) with enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of NMR spectra for both VDAC-1, a beta-barrel membrane protein, and the G protein-coupled receptor NTR1, an alpha-helical membrane protein. In addition, we utilized cNDs to visualize how simple, non-enveloped viruses translocate their genomes across membranes to initiate infection.

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Section: Methodsmentioning

confidence: 99%

“…Heterotrimeric G protein (α i1 β 1 γ 1 ) was expressed in Sf9 cells using a single baculovirus encoding all three subunits as previously described 22 and purified following a published procedure 23 .…”

Section: Methodsmentioning

confidence: 99%

Covalently circularized nanodiscs for studying membrane proteins and viral entry

et al. 2016

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“…These chemical shift changes cluster specifically to the Ras-like domain with little effect on the helical domain (gray and orange coloring in Gαi1 Interacts with an Activated GPCR in Phospholipid Nanodiscs. We next studied the interaction between uniformly 2 H, 15 N-labeled Gαi1Δ31 with a thermostabilized (25,26), signaling-competent (27) variant of rat neurotensin receptor subtype 1 [HTGH4 L167R (28)], which was purified from E. coli as described previously (27,29) and then incorporated in phospholipid nanodiscs (30,31 15 N-labeled Gαi1Δ31 in the apo, GDP-, or GMP-PNP-bound forms. We recorded NMR experiments with Gαi1Δ31 samples alone and after the addition of empty nanodiscs assembled with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids and membrane scaffold protein 1D1 (MSP1D1) and finally in complex with nanodiscs containing neurotensin-activated HTGH4 (Fig.…”

Section: Gαi1 Displays Ligand-dependent Changes As Probed With Nmrmentioning

confidence: 99%

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Goricanec

Stehle

Egloff

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDPbound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.H eterotrimeric G proteins are localized at the inner leaflet of the plasma membrane where they convey signals from cellsurface receptors to intracellular effectors (1). Heterotrimeric G proteins consist of two functional units, an α subunit (Gα) and a tightly associated βγ complex. The Gα subunit harbors the guanine nucleotide-binding site. In the inactive GDP-bound state, the Gα subunit is associated with the βγ complex. Exchange of GDP for GTP on the Gα subunit, triggered by interaction with the agonist-bound G-protein-coupled receptor (GPCR), results in a conformational change leading to GDP release and ultimately to GTP binding and subunit dissociation. The complexity of the mechanism by which a GPCR activates the Gα subunit based on available crystal structures has been discussed recently (2, 3). Both the Gα subunit and the βγ subunit interact with downstream effectors and regulate their activity. The intrinsic GTP hydrolysis of the Gα subunit returns the protein to the GDP-bound state, thereby increasing its affinity for the Gβγ subunit, and the subunits reassociate (Fig. 1A), ready for interaction with the agonist-bound GPCR. Throughout this cycle, the Gα subunit is engaged in specific interactions with the GPCR and/or the βγ subunit that stabilize the flexible parts of the protein, e.g., its switch regions. Only the GTP-bound form is stable enough to mediate downstream signaling.Crystallographic (4-9), biochemical (10), and biophysical (11-13) studies have elucidated details of the conformational states of the Gα subunit during the GTPase cycle. The Gα subunit has two structural domains, a nucleotide-binding domain (the Ras-like domain) and a helical domain (the α-H domain) that partially occludes the bound nucleotide (Fig. 1A). Because of this steric considerati...

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“…Expression and purification of a thermostable variant of rat NTR1 (TM86V-L167R DIC3B) was performed as described previously with some modifications 30,43 . This NTR1 variant consists of residues G50-G390, contains a deletion of E273-T290 in intracellular loop (ICL) 3, and has ten stabilizing mutations.…”

Section: Preparation Of Ntr1 In Cndsmentioning

confidence: 99%

“…Cells were then lysed and solubilized by sonication in buffer containing 100 mM HEPES (pH 8.0), 20% glycerol, 400 mM NaCl, 2.5 mM MgCl2, 0.6/0.12% CHAPS/cholesterol, 1.7% n-decyl-β-D-maltopyranoside (DM), 100 mg lysozyme, one tablet of protease inhibitor, and 250 U benzonase. Cell lysate was centrifuged, and the supernatant was mixed with pD-NT resin43 pre-equilibrated with 25 mM HEPES (pH 8.0), 10% glycerol, 600 mM NaCl and 0.5% DM at 4 °C for 1 hour. The flow-through from the pD-NT resin was then discarded, and the resin was washed with 25 mM HEPES (pH 7.0), 10% glycerol, 150 mM NaCl, 2 mM DTT and 0.3% DM.…”

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Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

Zhang

Gui

Wang

et al. 2020

Preprint

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G protein coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals 1,2 . Although several structures have been solved for GPCR-G protein complexes [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] , structural studies of the complex in a physiological lipid membrane environment are lacking. Additionally, most previous studies required additional antibodies/nanobodies and/or engineered G proteins for complex stabilization.In the absence of a native complex structure, the underlying mechanism of G protein activation leading to GDP/GTP exchange remains unclear. Here, we report cryo-EM structures of lipid bilayer-bound complexes of neurotensin, neurotensin receptor 1, and Gai1b1g1 protein in two conformational states, resolved to 4.1 and 4.2 Å resolution. The structures were determined without any stabilizing antibodies/nanobodies, and thus provide a native-like platform for understanding the structural basis of GPCR-G protein complex formation. Our structures reveal an extended network of protein-protein interactions at the GPCR-G protein interface compared to in detergent micelles, defining roles for the lipid membrane in modulating the structure and dynamics of complex formation, and providing a molecular explanation for the stronger interaction between GPCR and G protein in lipid bilayers. We propose a detailed allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling under near native conditions. G protein coupled receptors (GPCRs) sense extracellular stimuli including odorants, hormones, neurotransmitters, and photons. A stimulus leads to a shift in the conformational equilibrium of the GPCR towards a state which favors binding of the intracellular signal transducer, GDP-bound heterotrimeric Gabg protein 18 . Binding causes perturbation of the GDP binding pocket, leading to replacement of GDP by GTP and the dissociation of the Ga and Gbg subunits from each other and the GPCR 19 . The released Ga and Gbg subunits remain anchored to the membrane through lipid modifications but diffuse and interact with downstream effectors to stimulate signaling cascades 18 .Recent advances in X-ray crystallography and cryo-EM have allowed the determination of several GPCR-G protein complex structures to near-atomic resolution [3][4][5][6][7][8][11][12][13][14][15][16][17]20 . However, due to difficulties in preparing stable GPCR-G protein complexes in detergent micelles, a range of stabilization techniques had to be employed that compromise the function of the complexes. These include: i) antibodies or nanobodies that prevent GTP binding to Ga; ii) engineered dominantnegative Ga (DNGa) subunits that do not exhibit GDP/GTP exchange activity; or iii) engineered mini-G proteins that lack the a-helical domain (AHD) of Ga. Furthermore, all the previous structural studies reconstituted GPCR-G protein complexes in detergent micelles, which fail to replicate the properties of the nati...

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A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli

Cited by 19 publications

References 32 publications

Covalently circularized nanodiscs for studying membrane proteins and viral entry

Covalently circularized nanodiscs for studying membrane proteins and viral entry

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs

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