The interaction of the finger loop sequence of arrestin-3 with the neuropeptide Y1 and Y2 G protein-coupled receptors was studied. In cell assays, the finger loop part of arrestin was shown to be essential for Y2R internalization, but had no relevance for the interaction with Y1R. Here, we combined experimental data from nanoBRET, EPR, and NMR spectroscopy studies using a small library of NMR- and EPR-labeled finger loop peptides. Computer models of arrestin interacting with both receptors were built using AlphaFold2 and filtered by experimental constraints. Overall, Y1 and Y2 receptors show different binding modalities with the interacting finger loop region. In the interaction with Y1 receptor, the finger loop features a tail conformation critical for binding but also optional interactions. The Y2 receptor showed fewer contacts with the finger loop and two different conformations were found, which allow for higher intrinsic flexibility of this arrestin-3 segment.

Previous efforts in delineating molecular mechanisms of G protein-coupled receptor (GPCR) activation have focused on transmembrane regions and ligand-receptor contacts of the extracellular loops. The role of the highly flexible N-termini of rhodopsin-like GPCRs have not been well characterized to date. We hypothesize that transient contacts between the peptide ligand and the intrinsically disordered N-terminus (NT) of the neuropeptide Y (NPY) receptor Y2 (Y2R) will affect receptor signaling. We employ cross-linking mass spectrometry to capture ligand-receptor contacts including transient binding modes. A photo-reactive NPY analogue allows mapping the interaction between NPY and Y2R NT resulting in a total number of 40 cross-links. The cross-links provide distance constraints for deriving structural models of the interaction. Molecular dynamics simulations highlight the structural flexibility and rapid interconversion of ligand-receptor contacts. Mutagenesis of Y2R and functional characterization suggest that the cross-linking hotspots in the NT electrostatically control its conformational ensemble. The NT engages in transient contacts to the peptide and prolongs ligand residence time, which is required for efficient interaction of Y2R with arrestin-3, but not Gi. We delineate structure-function relationships for the intrinsically disordered Y2R NT and propose a functional role for transient binding modes involving the NT of a peptide-binding receptor.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor–cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

Many peptide-activated rhodopsin-like GPCRs share a β-hairpin folding motif in the extracellular loop 2 (ECL2), which interacts with the peptide ligand while at the same time being connected to transmembrane helix 3 (TM3) via a highly conserved disulfide bond. Currently, it remains unknown whether the coupling of the specifically shaped ECL2 to TM3 influences the activation of peptide-activated GPCRs. We investigated this possibility in a selection of peptide GPCRs with known structures. Most of the receptors with cysteine to alanine mutations folded like the respective wild-type and resided in the cell membrane, challenging pure folding stabilization by the disulfide bridge. G-protein signaling of the disulfide mutants was retained to a greater extent in secretin-like GPCRs than in rhodopsin-like GPCRs, while recruitment of arrestin was completely abolished in both groups, which may be linked to alterations in ligand residence time. We found a correlation between receptor activity of the neuropeptide Y2 receptor and alterations in ECL2 dynamics using engineered disulfide bridges or site-directed spin labeling and EPR spectroscopy. These data highlight the functional importance of the TM3-ECL2 link for the activation of specific signaling pathways in peptide-activated GPCRs, which might have implications for future drug discovery.

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 μs (with one exception, where the trajectory length was 10 μs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.

AbstractDynamische Strukturübergänge innerhalb des Sieben‐Transmembran‐Bündels stellen den Mechanismus dar, durch den G‐Protein‐gekoppelte Rezeptoren ein extrazelluläres chemisches Signal in eine intrazelluläre biologische Antwort umwandeln. In dieser Arbeit wurde die Konformationsdynamik des Neuropeptid‐Y‐Rezeptors Typ 2 (Y2R) während des Aktivierungsprozesses untersucht. Es wurden der apo‐Zustand, der Zustand des gebundenen vollen Agonisten und der Arrestin‐gebundene Zustand durch zellfreie Expression, funktionelle Faltung und Rekonstitution in Lipidmembranen präpariert. Um die konformationellen Übergänge zwischen den Zuständen zu untersuchen, wurden alle sechs Tryptophane des Y2R 13C‐markiert. Die NMR‐Signale wurden zugeordnet und die funktionalen Zustände des Rezeptors charakterisiert. Die Y2R‐Aktivierung wird durch molekulare Schalter wie das hoch konservierte Trp2816.48 und Trp3277.55 vermittelt. Trp11623.50 bildet mit einer Disulfidbrücke ein konformationell konserviertes cysteine‐lock‐Motiv.

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were(13)C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring(13)C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp281(6.48)of the highly conserved SWLP motif and Trp327(7.55)adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp116(23.50)was identified.

NMR spectroscopy sheds light on receptor activation: Molecular switches are conserved motifs of G protein-coupled receptors that undergo structural changes during activation. Tryptophan residues are often part of these molecular switches and thus provide insights in the activation mechanism of the molecule. In their Research Article on page 23854, D. Huster and co-workers use NMR spectroscopy to monitor structural alterations of the neuropeptide Y1 receptor with atomic resolution observable by changes in the chemical shifts of six native tryptophan residues.

NMR-Spektroskopie beleuchtet die Rezeptoraktivierung: Molekulare Schalter sind konservierte Motive von G-Protein-gekoppelten Rezeptoren, die während der Aktivierung ihre Struktur ändern. Tryptophanreste sind häufig Teil dieser molekularen Schalter und können Einblicke in die Rezeptoraktivierung liefern. Daniel Huster et al. nutzen in ihrem Forschungsartikel auf S. 24062 NMR-Spektroskopie, um Strukturänderungen des Neuropeptid-Y1-Rezeptors mit atomarer Auflösung zu beschreiben, indem Änderungen der chemischen Verschiebung von sechs nativen Tryptophanen analysiert werden.

Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for 13 C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged 1 H- 13 C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly 13 C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions.

AbstractPeptidtherapeutika und ihre Rolle in der Behandlung und Diagnostik von Tumorerkrankungen; G‐Protein‐gekoppelte Rezeptoren – ihre Struktur und Funktionsweise; Mini‐Organe aus Stammzellen; Kopplung elektrochemischer und biologischer Systeme für Synthesen und zur Stromerzeugung.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology1,2. The NPY–Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumour1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors. Crystal structures of the neuropeptide Y1 receptor in complex with two distinct antagonists combined with NMR, molecular docking and mutagenesis studies inform a proposed model for receptor–agonist binding.

Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli . refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15 N and 13 C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15 N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15 N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1 H- 13 C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

assays, namelymobilization of intracellular calcium in the Fura-2/AM and in an aequorin-based assay with mitochondrially targeted photoprotein. Herewe present the results of investigations on the humanNPYY2 andY4 receptors. Native pNPY, K4hPP as well as newNPY Y4R ligands developed in our laboratory were studied on CHO cells, stably expressing the receptor of interest, the chimeric G protein qi5 and apoaequorin (Ziemek et al., 2006, 2007). Both peptides showed higher potency when studied with the label-free technique [EC50 (pNPY)= 3.80 nM, EC50 (K4hPP)= 2.95 nM] compared to the Fura-2/AM [EC50 (pNPY)= 4.57 nM, EC50 (K4hPP)= 11.22 nM] and the aequorin-based assay [EC50 (pNPY)= 14.45 nM, EC50 (K4hPP)= 75.86 nM]. Interestingly, the agonist-induced signals were completely suppressed after pretreatmentwith the selective Gαq/11 inhibitor UBO-QIC in the conventional assays, whereas the DMR signal was just partially reduced (approximately by 20%). Inversely, pertussis toxin almost completely prevented the DMR signal, but only partially inhibited the calcium response, suggesting additional cellular reactions to contribute to the holistic readout.