Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations

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Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of α i1 or α sL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β 5 . A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gα i1 βγ combinations containing γ 1 and γ 11 . Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.heterotrimeric G proteins | G-protein-coupled receptor | membrane protein | protein complex | protein-protein interaction

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

Hillenbrand

Schori

Schöppe

et al. 2015

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“…In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10 8 different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16,17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli.…”

Section: Significancementioning

confidence: 99%

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Egloff

Hillenbrand

Klenk

et al. 2014

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202

316

Crystallography has advanced our understanding of G proteincoupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type-like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein-coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein-coupled receptor signaling, receptor maturation, and desensitization. membrane proteins | protein stability | protein engineering | detergents N eurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson's disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1-4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein-coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin-and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G proteinindependent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracel...

“…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

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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...