The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes

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.202006075

Cited by 27 publications

(33 citation statements)

References 68 publications

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“…This suggests that the receptor assumes an overall more rigid conformation in complex with arrestin. Our recent study on the Y2R also confirms that the Y2R in complex with Arr3-3A predominantly assumes a single conformation, concluded from the distinct chemical shifts observed for five out of six Trp residues in the molecule [24].…”

Section: Discussionsupporting

confidence: 79%

“…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%

“…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: Molecular Dynamics Of Y1r In the Presence Of The Agonist Andmentioning

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: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Molecular Dynamics Of Y1r In the Presence Of The Agonist Andmentioning

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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Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

et al. 2021

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Dedicated to Horst Kunz on the occasion of his 80th birthday.Fluorescence microscopy imaging enables receptor proteins to be investigated within their biological context. A key challenge is to site-specifically incorporate reporter moieties into proteins without interfering with biological functions or cellular networks. Small peptide tags offer the opportunity to combine inducible labeling with small tag sizes that avoid receptor perturbation. Herein, we review the current state of live-cell labeling of peptide-tagged cell-surface proteins. Considering their importance as targets in medicinal chemistry, we focus on membrane receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We discuss peptide tags that i) are subject to enzyme-mediated modification reactions, ii) guide the complementation of reporter proteins, iii) form coiled-coil complexes, and iv) interact with metal complexes. Given our own contributions in the field, we place emphasis on peptide-templated labeling chemistry.

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E. coli “Stablelabel” S30 lysate for optimized cell-free NMR sample preparation

et al. 2023

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Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative “Stablelabel” containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in “Stablelabel” lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.

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The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes

Cited by 27 publications

References 68 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

Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

E. coli “Stablelabel” S30 lysate for optimized cell-free NMR sample preparation

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