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 13C‐labeled. NMR‐signal assignment was achieved by dynamic‐nuclear‐polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved “cysteine lock”‐Trp11623.50 was identified.

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.

NMR spectroscopy … …s heds light on receptor activation:M olecular switches are conserved motifs of Gprotein-coupled receptors that undergo structural changes during activation. Tr yptophan residues are often part of these molecular switches and thus provide insights in the activation mechanism of the molecule.I nt heir Research Article on page 23854, D. Huster and co-workers use NMR spectroscopy to monitor structural alterations of the neuropeptide Y 1 receptor with atomic resolution observable by changes in the chemical shifts of six native tryptophan residues.

Dynamische 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.