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17 pages, 3347 KiB

Open AccessArticle

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

by Philip Rößler, Daniel Mayer, Ching-Ju Tsai, Dmitry B. Veprintsev, Gebhard F. X. Schertler and Alvar D. Gossert

Molecules 2020, 25(24), 5984; https://doi.org/10.3390/molecules25245984 - 17 Dec 2020

Cited by 10 | Viewed by 5297

Abstract

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-G_s and nanobody 60 (Nb60), which [...] Read more.

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-G_s 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-G_s 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. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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22 pages, 4825 KiB

Open AccessArticle

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

by Alexander Vogel, Mathias Bosse, Marcel Gauglitz, Sarah Wistuba, Peter Schmidt, Anette Kaiser, Vsevolod V. Gurevich, Annette G. Beck-Sickinger, Peter W. Hildebrand and Daniel Huster

Molecules 2020, 25(23), 5489; https://doi.org/10.3390/molecules25235489 - 24 Nov 2020

Cited by 9 | Viewed by 3926

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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21 pages, 4388 KiB

Open AccessArticle

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

by Jeannette M. Laugwitz, Haleh H. Haeri, Anette Kaiser, Ulrike Krug, Dariush Hinderberger, Annette G. Beck-Sickinger and Peter Schmidt

Molecules 2020, 25(18), 4143; https://doi.org/10.3390/molecules25184143 - 10 Sep 2020

Cited by 5 | Viewed by 3263

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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Review

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24 pages, 9909 KiB

Open AccessReview

The Structural Basis of Peptide Binding at Class A G Protein-Coupled Receptors

by Oanh Vu, Brian Joseph Bender, Lisa Pankewitz, Daniel Huster, Annette G. Beck-Sickinger and Jens Meiler

Molecules 2022, 27(1), 210; https://doi.org/10.3390/molecules27010210 - 30 Dec 2021

Cited by 12 | Viewed by 3881

Abstract

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides [...] Read more.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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19 pages, 3949 KiB

Open AccessReview

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies

by Yann Waltenspühl, Janosch Ehrenmann, Christoph Klenk and Andreas Plückthun

Molecules 2021, 26(5), 1465; https://doi.org/10.3390/molecules26051465 - 8 Mar 2021

Cited by 5 | Viewed by 3842

Abstract

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and [...] Read more.

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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39 pages, 5759 KiB

Open AccessReview

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

by Andrew J. Y. Jones, Florian Gabriel, Aditi Tandale and Daniel Nietlispach

Molecules 2020, 25(20), 4729; https://doi.org/10.3390/molecules25204729 - 15 Oct 2020

Cited by 38 | Viewed by 8169

Abstract

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in [...] Read more.

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in order to reproduce the various effects that lipids and membranes exert on the biological function for these allosteric receptors, in vitro studies of GPCRs need to be conducted under conditions that adequately approximate the native lipid bilayer environment. In the first part of this review, we assess some of the more general effects that a membrane environment exerts on lipid bilayer-embedded proteins such as GPCRs. This is then followed by the consideration of more specific effects, including stoichiometric interactions with specific lipid subtypes. In the final section, we survey a range of different membrane mimetics that are currently used for in vitro studies, with a focus on NMR applications. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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18 pages, 931 KiB

Open AccessReview

Structure-Function Analyses of Human Bitter Taste Receptors—Where Do We Stand?

by Maik Behrens and Florian Ziegler

Molecules 2020, 25(19), 4423; https://doi.org/10.3390/molecules25194423 - 26 Sep 2020

Cited by 19 | Viewed by 5544

Abstract

The finding that bitter taste receptors are expressed in numerous tissues outside the oral cavity and fulfill important roles in metabolic regulation, innate immunity and respiratory control, have made these receptors important targets for drug discovery. Efficient drug discovery depends heavily on detailed [...] Read more.

The finding that bitter taste receptors are expressed in numerous tissues outside the oral cavity and fulfill important roles in metabolic regulation, innate immunity and respiratory control, have made these receptors important targets for drug discovery. Efficient drug discovery depends heavily on detailed knowledge on structure-function-relationships of the target receptors. Unfortunately, experimental structures of bitter taste receptors are still lacking, and hence, the field relies mostly on structures obtained by molecular modeling combined with functional experiments and point mutageneses. The present article summarizes the current knowledge on the structure–function relationships of human bitter taste receptors. Although these receptors are difficult to express in heterologous systems and their homology with other G protein-coupled receptors is very low, detailed information are available at least for some of these receptors. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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33 pages, 3039 KiB

Open AccessReview

A Paradigm for Peptide Hormone-GPCR Analyses

by Fred Naider and Jeffrey M. Becker

Molecules 2020, 25(18), 4272; https://doi.org/10.3390/molecules25184272 - 18 Sep 2020

Cited by 6 | Viewed by 3734

Abstract

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored [...] Read more.

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure. Full article

(This article belongs to the Special Issue New Breakthroughs in GPCR Structure, Function and Their Impact on Drug Discovery)

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