Search

The μ-opioid receptor (μOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the μOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi.One-Sentence SummaryLigand-dependent conformational dynamics of the μ-opioid receptor determine downstream signaling efficacy.

There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites. Here, we describe a selection methodology to enrich for functionally modulatory antibodies using a yeast-displayed library of synthetic camelid antibody fragments called “nanobodies.” Using this platform, we discovered multiple nanobodies that act as antagonists of the angiotensin II type 1 receptor (AT1R). Following angiotensin II infusion in mice, we found that an affinity matured nanobody antagonist has comparable antihypertensive activity to the angiotensin receptor blocker (ARB) losartan. The unique pharmacology and restricted biodistribution of nanobody antagonists may provide a path for treating hypertensive disorders when small-molecule drugs targeting the AT1R are contraindicated, for example, in pregnancy.

The β(2) adrenergic receptor (β(2)AR) 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 β(2)AR 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 β(2)AR. 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 β(2)AR 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.

Purpose: To evaluate whether perfluorocarbon (PFO) liquids can exacerbate condensation on intraocular lenses (IOL).Methods: Two separate experiments were carried out. In the first experiment, a two-segment chamber was constructed out of glass and acrylic panels to serve as an in-vitro eye model. The chamber was placed on a non-activated cooling plate with two IOLs of the same material, one in each segment. 2.0 ml of PFO and/or water for the control were placed in the respective receptacle. The cooling plate was then activated to the desired temperatures. Condensation on the lenses was visually assessed via high-definition ultra-zoom camera by trained observers for three temperatures and three IOL materials.In the second experiment, Fourier transform infrared (FTIR) spectroscopy wasemployed to determine the composition of the droplets forming after condensation.Results: The presence of PFO liquid in a closed chamber exacerbates the intensity and likelihood of condensation on all intraocular material types. Condensation of PFO on surfaces in the presence of water was confirmed with FTIR spectroscopy by the isolation of specific absorption bands. Furthermore, material type also affects the characteristics of condensation, with silicone lenses inducing the fastest rate and intensity of condensation.Conclusions: Our study shows that the presence of perfluoro-n-octane is a significant factor in the formation of condensation on the posterior surface of IOLs when performing vitrectomy in a pseudophakic patient with posterior capsulotomy.

Signaling of the prototypical G protein–coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G(t)) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the “finger loop” of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G(t) α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G(t) α-subunit binding and both ArrFL-1–binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR–G protein/-arrestin signaling.

To evaluate the diagnostic performance of diastolic function parameters derived from long-axis (LAX) planimetry compared with short-axis (SAX) volumetry in cardiac magnetic resonance imaging. Cine steady-state free precession (SSFP) datasets of 15 healthy participants (8 young and 7 middle aged) and 25 patients with echocardiographically proven diastolic dysfunction (9 mild, 9 moderate, and 7 severe) were retrospectively included. Volume-time curves for assessing left ventricular (LV) function were obtained by manually contouring the LV endocardial borders in SAX and LAX datasets. The time needed for contouring was recorded for each dataset. The following LV parameters were determined: end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), myocardial mass (MM), time to peak filling rate (TPFR), normalized peak filling rate (nPFR), and the ratio of early to late peak filling rate (E/A ratio). A Wilcoxon signed-rank test was used to compare subgroups based on age and severity of diastolic dysfunction for statistical differences. Intraclass correlation coefficients were used to assess intermethod and interobserver reliability. Accuracy for the diagnosis of diastolic dysfunction was highest for E/A (mild diastolic dysfunction) and nPFR (any stage of diastolic dysfunction) derived from LAX datasets (E/A: area under the curve (AUC) = 0.97, sensitivity of 68 % and specificity of 100 %; nPFR: AUC = 0.84, sensitivity of 84 % and specificity of 80 %). Diastolic parameters showed a moderate to good intraclass correlation between both methods. The mean differences in EDV, ESV, EF, and MM were 5.3 ml, 1.9 ml, 3.5 %, and 11 g, respectively (each p 0.001). Significantly less time was needed to derive volume-time curves from LAX images (median 14:45 min, interquartile range 14:15-15:53 min versus median 29:25 min, interquartile range 28:12-32:22 min; p = 0.001). The interobserver reliability was generally good to excellent. Diastolic function parameters derived from left ventricular LAX planimetry have high diagnostic performance and can be obtained in significantly less time compared with SAX volumetry. These findings may pave the way for routine use of LAX planimetry in the clinical diagnosis of diastolic dysfunction. · Diastolic function parameters derived from long-axis datasets have high diagnostic performance.. · Generation of volume-time curves using long-axis datasets requires significantly less time.. · This time savings may allow use of cardiac MRI for the diagnosis of diastolic dysfunction in the clinical routine..· Schaafs LA, Wyschkon S, Elgeti M et al. Diagnosis of Left Ventricular Diastolic Dysfunction Using Cardiac Magnetic Resonance Imaging: Comparison of Volume-Time Curves Derived from Long- and Short-Axis Cine Steady-State Free Precession Datasets. Fortschr Röntgenstr 2020; 192: 764 - 775.ZIEL: Vergleich der diagnostischen Genauigkeit von diastolischen Funktionsparametern in der kardialen MRT, die mittels Langachsen (LAX)-Planimetrie bzw. Kurzachsen (SAX) -Volumetrie an Patienten mit diastolischer Dysfunktion erhoben wurden. Cine-steady-state-free-precession (SSFP)-Datensätze von 15 gesunden Teilnehmern (8 junge Teilnehmer, 7 Teilnehmer mittleren Alters) sowie 25 Patienten mit echokardiografisch gesicherter diastolischer Dysfunktion (9 mild, 9 moderat und 7 schwer) wurden retrospektiv ausgewertet. Volumen-Zeit-Kurven zur Bewertung der linksventrikulären Funktion wurden nach manuellem Einzeichnen der Endo- bzw. Epikardkontur auf SAX- und LAX-Datensätzen berechnet. Die für jeden Datensatz benötigte Zeit wurde gemessen. Im Anschluss wurden enddiastolisches (EDV) und endsystolisches Volumen (ESV), Ejektionsfraktion (EF), myokardiale Masse (MM), „time-to-peak-fillin-rate“ (TPFR), normalisierte „peak-filling-rate“ (nPFR) und E/A-Verhältnis (E/A) für den linken Ventrikel bestimmt. Unterschiede der Messergebnisse wurden mittels Wilcoxon-Vorzeichen-Rang-Test geprüft. Die Reliabilität zwischen den Auswertern und zwischen beiden Methoden wurde mittels Intra-Klassen-Korrelation gemessen. Die höchste diagnostische Genauigkeit wurde mit E/A (Diagnose einer milden diastolischen Dysfunktion) und nPFR (Diagnose einer diastolischen Dysfunktion unabhängig vom Stadium), jeweils generiert aus LAX-Datensätzen, erreicht (E/A: Fläche unter der Kurve = 0,97, 68 % Sensitivität und 100 % Spezifität; nPFR: Fläche unter der Kurve = 0,84, 84 % Sensitivität und 80 % Spezifität). Die diastolischen Funktionsparameter wiesen im Vergleich eine moderate bis gute Intra-Klassen-Korrelation zwischen beiden Methoden auf. Die mittlere Differenz für EDV, ESV, EF und MM betrug 5,3 ml, 1,9 ml, 3,5 % beziehungsweise 11 g (p 0,001). Die für die Erstellung von Volumen-Zeit-Kurven benötigte Zeit war signifikant kürzer bei der Verwendung von LAX-Datensätzen (Median 14:45 min, Interquartilsabstand 14:15–15:53 min versus median 29:25 min, Interquartilsabstand 28:12–32:22 min; p = 0,001). Die Reliabilität zwischen beiden Auswertern war gut bis exzellent. Die Planimetrie des linken Ventrikels auf Langachsen ergab diastolische Funktionsparameter mit einer hohen diagnostischen Genauigkeit und kann im Vergleich zur Volumetrie in deutlich kürzerer Zeit durchgeführt werden. Diese Erkenntnisse könnten den Einsatz der MRT zur Routinediagnostik der diastolischen Dysfunktion ermöglichen. · Mittels Langachsen-Planimetrie berechnete diastolische Funktionsparameter weisen eine hohe diagnostische Aussagekraft auf.. · Die Erstellung von Volumen-Zeit-Kurven durch Auswertung von Langachsen-Datensätzen benötigt deutlich weniger Zeit.. · Diese Zeitersparnis könnte den Einsatz der MRT zur Routinediagnostik der diastolischen Dysfunktion ermöglichen..

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.

“Biased” G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or β-arrestins. Here we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between ten pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an “open” conformation with an accessible transducer-binding site. β-Arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

Time-resolved Fourier transform infrared (FTIR) and UV/Vis difference spectra of light induced deactivation of the photoreceptor rhodopsin were simultaneously recorded on the same sample. The data were analyzed by a special designed combination of singular value decomposition and Global analysis to verify our recently published model of receptor deactivation. This mathematical approach enables us to obtain the pure difference spectra of the conversions between the species involved. We found two different species, which could be identified as key intermediates of the model. The new data allow deeper insights into the structural changes that come along with the formation of the deactivated state. By means of this method we can now separate and assign even strongly overlapping and highly coupled bands of the infrared difference spectra.

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.

In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the ‘finger loop’ region (ArrFL1–4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins., G-protein-coupled receptors (GPCRs) transmit signals through intracellular heterotrimeric G proteins and arrestins. Here, Szczepek et al. present the structure of a common binding interface for Gα and arrestin on rhodopsin to shed light on key interactions that mediate transduction of specific signals through a single GPCR.

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (Gαβγ, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active β2-adrenoceptor (β2AR*) in complex with C-terminal peptides derived from the key interaction site of Gα (GαCT) as surrogate of Gαβγ. We find that GiαCT and GsαCT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed β2AR*. The slim GiαCT stabilizes a β2AR* conformation, not accessible to the bulkier GsαCT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of β2AR* toward Gi or Gs.

Upon activation by agonists, G-protein coupled receptors (GPCRs) in their active R∗ form transmit extracellular signals into the cell by catalysing GDP/GTP exchange in heterotrimeric G-proteins (Gαsγ, G). The GPCR rhodopsin activates Gt to transmit the light signal into retinal rod cells. After photon capture, the rhodopsin activity jumps by more than one billion fold implying both high fidelity and speed of active rhodopsin (RhR∗)/Gt coupling. We employed all-atom molecular dynamics (MD) simulations to study the conformational diversity of membrane embedded rhodopsin and extend the static picture provided by the available crystal structures. An intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (ICL3) is identified. The MD data show how each protein state is split into several conformational substates. Only a single conformational substate with largely helical ICL3 is selectively stabilized by the Gtα C-terminus (GtαCT), as in the crystal structures. A mechanism for the fast and precise signal transfer from rhodopsin to Gt is proposed, which assumes a stepwise and mutual reduction of their conformational space. The analysis was extended to the interaction of GsαCT and GiαCT with activated β2-adrenoceptor (β2AR∗) to address the issue of receptor promiscuity. We find that a short region of GsαCT stabilizes the open β2AR∗ conformation provided by the β2AR∗/Gsαsγ crystal structure. In the crystal structures of RhR∗ with GtαCT, a close homologue of GiαCT, RhR∗ exhibits a more closed active conformation. With the slim GiαCT peptide we find that β2AR∗ adopts a similarly closed conformation as RhR∗ with GtαCT. This analysis elucidates how G-protein coupling specificity relies on the fold and primary structure of the Gα C-terminus.Scheerer et al. (2008), Crystal structure of opsin in its G-protein-interacting conformation, NATUREElgeti et al. (2013) Precision vs Flexibility in GPCR signaling, JACS

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric G protein transducin (Gt) to transmit the light signal into retinal rod cells. The rhodopsin activity is virtually zero in the dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity and speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy and supporting all-atom molecular dynamics (MD) simulations to study the conformational diversity of rhodopsin in membrane environment and extend the static picture provided by the available crystal structures. The FTIR results show how the equilibria of inactive and active protein states of the receptor (so-called metarhodopsin states) are regulated by the highly conserved E(D)RY and Yx7K(R) motives. The MD data identify an intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (CL3) and show how each protein state is split into conformational substates. The C-termini of the Gtγ- and Gtα-subunits (GαCT and GγCT), prepared as synthetic peptides, are likely to bind sequentially and at different sites of the active receptor. The peptides have different effects on the receptor conformation. While GγCT stabilizes the active states but preserves CL3 flexibility, GαCT selectively stabilizes a single conformational substate with largely helical CL3, as it is found in crystal structures. Based on these results we propose a mechanism for the fast and precise signal transfer from rhodopsin to Gt, which assumes a stepwise and mutual reduction of their conformational space. The mechanism relies on conserved amino acids and may therefore underlie GPCR/G protein coupling in general.

Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III "retinal storage" product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK approximately 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu(134)/Arg(135) cluster.