Inside Cover: The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes (Angew. Chem. Int. Ed. 52/2020)

Krug, Ulrike; Gloge, Anika; Schmidt, Péter; Becker‐Baldus, Johanna; Bernhard, Frank; Kaiser, Anette; Montag, Cindy; Gauglitz, Marcel; Vishnivetskiy, Sergey A.; Gurevich, Vsevolod V.; Beck‐Sickinger, Annette G.; Glaubitz, Clemens; Huster, Daniel

doi:10.1002/anie.202014454

Cited by 3 publications

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“…The activation of a GPCR is accompanied by characteristic changes in the energy landscape of these proteins [ 54 ], resulting in dynamic alterations. In addition to the characteristic changes observed upon activation and G-protein or arrestin binding [ 20 , 21 , 22 , 23 , 24 ], the equilibrium dynamics of a GPCR is subject to changes [ 35 , 44 , 45 ]. Here, we probed how the fluctuations of Y1R reconstituted into DMPC membranes would change upon agonist binding and subsequent interaction with arrestin.…”

Section: Resultsmentioning

confidence: 99%

“…Under the latter conditions, backbone order parameters amounted to surprisingly low values between 0.57 and 0.67 depending on the host membrane, corresponding to remarkable backbone motional amplitudes of the C–H bond vectors of 47° to 40°. In a recent study, the site-specific order parameters of all Trp residues, mostly residing in α-helical secondary structures of the Y2R in DMPC membranes, were measured site-specifically using CP excitation with a 700 µs contact time [ 24 ]. This study reported Trp order parameters between 0.71 to 0.85 in the apo state.…”

Section: Discussionmentioning

confidence: 99%

“…Whereas these structures provide a static picture of the different conformations of the receptor in individual states, the dynamics of the structural transitions between such states can be studied in detail by spectroscopic tools [ 19 ], in particular by NMR spectroscopy [ 9 ]. Using NMR spectroscopy in a solution [ 20 , 21 , 22 , 23 ] and in the solid state [ 24 , 25 ], the dynamics of the conformational transitions in GPCRs have been characterized in atomistic detail. Receptor activation is characterized by a seesaw-like swing of transmembrane Helices 6 (TM6) and 7 (TM7), by which the extra- and intracellular ends of the helices are moved in the opposite direction [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

“…Using uniform 13 C-labeled Y2R that did not provide site resolution, low average order parameters between 0.55 and 0.67 were determined for the backbone in different liquid crystalline membranes [ 35 , 44 ]. More specifically, site-specific Cα-Hα order parameters for the six Trp residues in Y2R in DMPC membranes prepared by cell-free synthesis ranged from 0.71 to 0.85 in the apo state [ 24 ]. The U- 13 C-labeled human growth secretagogue receptor 1a (GHSR) showed similarly high dynamics in membranes, with order parameters between 0.56 and 0.69 [ 45 ].…”

Section: Introductionmentioning

confidence: 99%

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The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

et al. 2020

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

et al. 2020

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“…In turn, desensitization processes on GPCRs have not yet been studied in as much detail as have activation processes. It will therefore be important to extend the NMR studies towards receptor desensitization to address, for example, biased agonism [ 52 , 53 , 54 ].…”

Section: Discussionmentioning

confidence: 99%

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

et al. 2020

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In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized β1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with β1 adrenergic receptor (β1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tβ1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.

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Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

et al. 2020

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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.

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Inside Cover: The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes (Angew. Chem. Int. Ed. 52/2020)

Cited by 3 publications

References 0 publications

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

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