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The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

ABSTRACT: 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.

SUBMITTER: Krug U

PROVIDER: S-EPMC7736470 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Krug Ulrike U Gloge Anika A Schmidt Peter P Becker-Baldus Johanna J Bernhard Frank F Kaiser Anette A Montag Cindy C Gauglitz Marcel M Vishnivetskiy Sergey A SA Gurevich Vsevolod V VV Beck-Sickinger Annette G AG Glaubitz Clemens C Huster Daniel D

Angewandte Chemie (International ed. in English) 20200930 52

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 conf ...[more]

PMID: 32790043

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Project description:G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

Dataset Information

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Publications

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

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