Time-resolved cryo-EM of G-protein activation by a GPCR.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.

Viewing rare conformations of the ß2 adrenergic receptor with pressure-resolved DEER spectroscopy.

The ß2 adrenergic receptor (ß2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The ß2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free ß2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in ß2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Structural equilibrium underlying ligand-dependent activation of ß2-adrenoreceptor.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins mediating cellular signals in response to extracellular stimuli. Although three-dimensional structures showcase snapshots that can be sampled in the process and nuclear magnetic resonance detects conformational equilibria, the mechanism by which agonist-activated GPCRs interact with various effectors remains elusive. Here, we used paramagnetic nuclear magnetic resonance for leucine amide resonances to visualize the structure of ß2-adrenoreceptor in the full agonist-bound state, without thermostabilizing mutations abolishing its activity. The structure exhibited a unique orientation of the intracellular half of the transmembrane helix 6, forming a cluster of G-protein-interacting residues. Furthermore, analyses of efficacy-dependent chemical shifts of the residues near the pivotal PIF microswitch identified an equilibrium among three conformations, including one responsible for the varied signal level in each ligand-bound state. Together, these results provide a structural basis for the dynamic activation of GPCRs and shed light on GPCR-mediated signal transduction.

Investigation on temperature-induce conformational change of immobilized ß2 adrenergic receptor.

The ß2 adrenergic receptor (ß2-AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. Purified ß2-AR was immobilized on macroporous silica gel to obtain liquid chromatographic stationary phase. The resulting phase was packed into a stainless steel column (4.6 × 50 mm, 7 µm) and used for on-line chromatographic system. When column oven temperature increased from 20.0 °C to 40.0 °C, uncomplete separate chromatographic peaks of ephedrine and pseudoephedrine as receptor conformational probe were gradually merged into one peak, meanwhile retention time and resolution of the probes were reduced correspondingly, which suggested that temperature could regulate protein conformation. Temperature-induced conformational change of immobilized ß2-AR, especially changes at higher temperatures, indicated that constructed receptor chromatography could simulate fever disease state of human body and clarify receptor conformation change at pathological condition. At the same time this study could also provide new ideas for screening active components in pathological conditions.

Conformational changes in the G protein Gs induced by the ß2 adrenergic receptor.

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human ß(2) adrenergic receptor (ß(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the ß(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the ß(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the ß-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and ß-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

Helix 3 acts as a conformational hinge in Class A GPCR activation: An analysis of interhelical interaction energies in crystal structures.

A collection of crystal structures of rhodopsin, ß2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the activation process of some glutamic and aspartic acidic residues and, as a consequence, the influence of variations on local pH is highlighted. Structural hypotheses that arose from the analysis of rhodopsin, ß2-adrenergic and adenosine A2A receptors were tested on the active and inactive M2 muscarinic acetylcholine receptor structures and further discussed in the context of the new mechanistic insights provided by the recently determined active and inactive crystal structures of the µ-opioid receptor. Overall, the structural and energetic analyses of the interhelical interactions present in this collection of Class A GPCRs suggests the existence of a common general activation mechanism featuring a chemical space useful for drug discovery exploration.

Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied ß(2)-adrenoceptor (ß(2)AR) in complex with the heterotrimeric G protein Gs (Gαsßγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

Concerted interconversion between ionic lock substates of the beta(2) adrenergic receptor revealed by microsecond timescale molecular dynamics.

The recently solved crystallographic structures for the A(2A) adenosine receptor and the beta(1) and beta(2) adrenergic receptors have shown important differences between members of the class-A G-protein-coupled receptors and their archetypal model, rhodopsin, such as the apparent breaking of the ionic lock that stabilizes the inactive structure. Here, we characterize a 1.02 mus all-atom simulation of an apo-beta(2) adrenergic receptor that is missing the third intracellular loop to better understand the inactive structure. Although we find that the structure is remarkably rigid, there is a rapid influx of water into the core of the protein, as well as a slight expansion of the molecule relative to the crystal structure. In contrast to the x-ray crystal structures, the ionic lock rapidly reforms, although we see an activation-precursor-like event wherein the ionic lock opens for approximately 200 ns, accompanied by movements in the transmembrane helices associated with activation. When the lock reforms, we see the structure return to its inactive conformation. We also find that the ionic lock exists in three states: closed (or locked), semi-open with a bridging water molecule, and open. The interconversion of these states involves the concerted motion of the entire protein. We characterize these states and the concerted motion underlying their interconversion. These findings may help elucidate the connection between key local events and the associated global structural changes during activation.

Nanoscale organization of beta2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells.

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize beta(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(2)AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the beta(2)AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of beta(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

Structure of an endosomal signaling GPCR-G protein-ß-arrestin megacomplex.

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by ß-arrestin (ß-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-ß-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine ß-arr to the core and phosphorylated tail, respectively, of a single active human chimeric ß2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (ß2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and ß-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

[Increase in beta-adrenergic receptors in rabbits in long-term local administration of beta-blockers]. / Zunahme von beta-adrenergen Rezeptoren bei Kaninchen unter lokaler Langzeitapplikation von beta-Blockern.

Chronic application of beta-blockers often induces tachyphylaxia of unknown origin. After long-term topical pretreatment of pindolol and timolol in rabbit eyes (up to 12 weeks, twice a day) beta 2-adrenergic receptors were localized and quantified with autoradiographic methods. Frozen sections of albino rabbit eyes were labelled with [125I] cyanopindolol. Quantification revealed a significant increase in the density of beta 2-receptors after premedication with pindolol in the ciliary body and the corneal epithelium. This increase was detectable after 2 weeks of premedication and reached its maximum after 4 weeks. After premedication with timolol a significant increase of beta 2-receptors in the epithelium of the ciliary body and the corneal epithelium was visible. Following pindolol over an equivalent time-course, to that of timolol administrations, an increasing number of beta 2-receptor sites was also observed. No significant changes were visible for either kind of premedication investigated (pindolol/timolol) in the corneal endothelium, the lens or the choroid. Chronic application of topically instilled beta-blocking agents leads to a significant increase in receptor density in ocular structures that are involved in aqueous humor production. This up-regulation of beta 2-adrenergic receptors might explain the intraocular events at receptor level in relation to the phenomenon of tachyphylaxis.