Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology1,2. The NPY–Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumour1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.
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Zhang, L., Bijker, M. S. & Herzog, H. The neuropeptide Y system: pathophysiological and therapeutic implications in obesity and cancer. Pharmacol. Ther. 131, 91–113 (2011).
Morales-Medina, J. C., Dumont, Y. & Quirion, R. A possible role of neuropeptide Y in depression and stress. Brain Res. 1314, 194–205 (2010).
Michel, M. C. et al. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol. Rev. 50, 143–150 (1998).
Yulyaningsih, E., Zhang, L., Herzog, H. & Sainsbury, A. NPY receptors as potential targets for anti-obesity drug development. Br. J. Pharmacol. 163, 1170–1202 (2011).
Sousa, D. M., Herzog, H. & Lamghari, M. NPY signalling pathway in bone homeostasis: Y1 receptor as a potential drug target. Curr. Drug Targets 10, 9–19 (2009).
Antal-Zimanyi, I. et al. Pharmacological characterization and appetite suppressive properties of BMS-193885, a novel and selective neuropeptide Y1 receptor antagonist. Eur. J. Pharmacol. 590, 224–232 (2008).
Leibowitz, S. F., Sladek, C., Spencer, L. & Tempel, D. Neuropeptide Y, epinephrine and norepinephrine in the paraventricular nucleus: stimulation of feeding and the release of corticosterone, vasopressin and glucose. Brain Res. Bull. 21, 905–912 (1988).
MacNeil, D. J. NPY Y1 and Y5 receptor selective antagonists as anti-obesity drugs. Curr. Top. Med. Chem. 7, 1721–1733 (2007).
Reubi, J. C., Gugger, M., Waser, B. & Schaer, J. C. Y. Y1-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets. Cancer Res. 61, 4636–4641 (2001).
Keller, M. et al. Nω-carbamoylation of the argininamide moiety: an avenue to insurmountable NPY Y1 receptor antagonists and a radiolabeled selective high-affinity molecular tool ([3H]UR-MK299) with extended residence time. J. Med. Chem. 58, 8834–8849 (2015).
White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012).
Yin, J. et al. Structure and ligand-binding mechanism of the human OX1 and OX2 orexin receptors. Nat. Struct. Mol. Biol. 23, 293–299 (2016).
Yin, J., Mobarec, J. C., Kolb, P. & Rosenbaum, D. M. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant. Nature 519, 247–250 (2015).
Shihoya, W. et al. Activation mechanism of endothelin ETB receptor by endothelin-1. Nature 537, 363–368 (2016).
Ballesteros, J. A. & Weinstein, H. in Methods in Neurosciences Vol. 25 (ed. S. Sealfon) 366–428 (Elsevier, Amsterdam, 1995).
Kaiser, A. et al. Unwinding of the C-terminal residues of neuropeptide Y is critical for Y2 receptor binding and activation. Angew. Chem. Int. Edn Engl. 54, 7446–7449 (2015).
Venkatakrishnan, A. J. et al. Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).
Standfuss, J. et al. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656–660 (2011).
Sautel, M. et al. Neuropeptide Y and the nonpeptide antagonist BIBP 3226 share an overlapping binding site at the human Y1 receptor. Mol. Pharmacol. 50, 285–292 (1996).
Keller, M. et al. Guanidine–acylguanidine bioisosteric approach in the design of radioligands: synthesis of a tritium-labeled N G-propionylargininamide ([3H]-UR-MK114) as a highly potent and selective neuropeptide Y Y1 receptor antagonist. J. Med. Chem. 51, 8168–8172 (2008).
Sjödin, P. et al. Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study. Biochem. J. 393, 161–169 (2006).
Poindexter, G. S. et al. Dihydropyridine neuropeptide Y Y1 receptor antagonists. Bioorg. Med. Chem. Lett. 12, 379–382 (2002).
Poindexter, G. S. et al. Dihydropyridine neuropeptide Y Y1 receptor antagonists 2: bioisosteric urea replacements. Bioorg. Med. Chem. 12, 507–521 (2004).
Pedragosa-Badia, X., Stichel, J. & Beck-Sickinger, A. G. Neuropeptide Y receptors: how to get subtype selectivity. Front. Endocrinol. (Lausanne) 4, 5 (2013).
Bender, B. J. et al. Protocols for molecular modeling with Rosetta3 and RosettaScripts. Biochemistry 55, 4748–4763 (2016).
Merten, N. et al. Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands. J. Biol. Chem. 282, 7543–7551 (2007).
Xu, B. et al. Mutagenesis and computational modeling of human G-protein-coupled receptor Y2 for neuropeptide Y and peptide YY. Biochemistry 52, 7987–7998 (2013).
Lindner, D., Walther, C., Tennemann, A. & Beck-Sickinger, A. G. Functional role of the extracellular N-terminal domain of neuropeptide Y subfamily receptors in membrane integration and agonist-stimulated internalization. Cell. Signal. 21, 61–68 (2009).
Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).
Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007).
Roth, C. B., Hanson, M. A. & Stevens, R. C. Stabilization of the human β2-adrenergic receptor TM4–TM3–TM5 helix interface by mutagenesis of Glu1223.41, a critical residue in GPCR structure. J. Mol. Biol. 376, 1305–1319 (2008).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. 67, 355–367 (2011).
Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D. 68, 368–380 (2012).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. 66, 486–501 (2010).
Keller, M. et al. Mimicking of arginine by functionalized N ω-carbamoylated arginine as a new broadly applicable approach to labeled bioactive peptides: high affinity angiotensin, neuropeptide Y, neuropeptide FF, and neurotensin receptor ligands as examples. J. Med. Chem. 59, 1925–1945 (2016).
Yung-Chi, C. & Prusoff, W. H. Relationship between the inhibition constant (K I) and the concentration of inhibitor which causes 50 per cent inhibition (I 50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).
Burkert, K. et al. A deep hydrophobic binding cavity is the main interaction for different Y2R antagonists. ChemMedChem. 12, 75–85 (2017).
Els, S., Beck-Sickinger, A. G. & Chollet, C. Ghrelin receptor: high constitutive activity and methods for developing inverse agonists. Methods Enzymol. 485, 103–121 (2010).
Kostenis, E. Is Gα16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? Trends Pharmacol. Sci. 22, 560–564 (2001).
Pedragosa-Badia, X. et al. Pancreatic polypeptide is recognized by two hydrophobic domains of the human Y4 receptor binding pocket. J. Biol. Chem. 289, 5846–5859 (2014).
Hoffmann, S., Rist, B., Videnov, G., Jung, G. & Beck-Sickinger, A. G. Structure-affinity studies of C-terminally modified analogs of neuropeptide Y led to a novel class of peptidic Y1 receptor antagonist. Regul. Pept. 65, 61–70 (1996).
Gerald, C. et al. A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382, 168–171 (1996).
Schmidt, P. et al. A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment. Biophys. Chem. 150, 29–36 (2010).
Casiraghi, M. et al. Functional modulation of a G protein-coupled receptor conformational landscape in a lipid bilayer. J. Am. Chem. Soc. 138, 11170–11175 (2016).
Hohwy, M., Rienstra, C. M., Jaroniec, C. P. & Griffin, R. G. Fivefold symmetric homonuclear dipolar recoupling in rotating solids: application to double quantum spectroscopy. J. Chem. Phys. 110, 7983–7992 (1999).
Raveh, B., London, N., Zimmerman, L. & Schueler-Furman, O. Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors. PLoS ONE 6, e18934 (2011).
Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013).
Schwarz, D. et al. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat. Protocols 2, 2945–2957 (2007).
Bosse, M. et al. Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding. Biochemistry 50, 9817–9825 (2011).
Wilkins, M. R. et al. Detailed peptide characterization using PEPTIDEMASS—a World-Wide-Web-accessible tool. Electrophoresis 18, 403–408 (1997).
We are grateful to T. Zellmann for his contribution to the modelling in the early state of the project and for the technical support of R. Reppich-Sacher (mass spectrometry) and K. Löbner (cell culture). We thank H. A. Scheidt for support with solid-state NMR measurements and M. Beer-Krön, D. Fritsch, S. Bollwein and B. Wenzl for expert help in performing radioligand-binding experiments. The synchrotron radiation experiments were performed at the BL41XU of SPring-8 with approval of the Japan Synchrotron Radiation Research Institute (proposal no. 2015B2026, 2015B2027, 2016A2517, 2016A2518, 2016B2517 and 2016B2518). We thank the beamline staff members K. Hasegawa, H. Okumura, N. Mizuno, T. Kawamura and H. Murakami of the BL41XU for help with X-ray data collection. This work was supported by CAS Strategic Priority Research Programs XDB08020000 (B.W.) and XDB08030102 (R.Z.), the Key Research Program of Frontier Sciences, CAS, Grant no. QYZDB-SSW-SMC024 (B.W.) and QYZDB-SSW-SMC054 (Q.Z.), the National Science Foundation of China grants 31570739 (B.W.), 81525024 (Q.Z.), 3170040264 (Z.Y.) and 31470792 (S.Y.), Program of Shanghai Academic/Technology Research Leader no. 18XD1404800 (Z.Y., Q.Z.), the European Community, the Free State of Saxony (SAB 100148835 to D.H. and 100881433 to A.G.B.-S.) and the Deutsche Forschungsgemeinschaft (DFG) (Be1264-16, SFB 1052/A3, research grant KE 1857/1-1 and Graduate Training Program GRK 1910). Work in the Meiler laboratory is supported by the NIH (R01 GM080403, R01 DK097376, R01 HL122010) and NSF (CHE 1305874).
Nature thanks N. Holliday and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Crystal packing and structural features of Y1R and chemical structures of Y1R ligands.
a, b, Crystal packing of Y1R–UR-MK299 (a) and Y1R–BMS-193885 (b) complexes. Y1R is shown in cartoon representation and coloured brown and green in the Y1R–UR-MK299 and Y1R–BMS-193885 complexes, respectively. The T4L fusion is shown in grey cartoon representation. UR-MK299 and BMS-193885 are displayed as yellow and pink spheres, respectively. c, Cutaway view of the UR-MK299-binding pocket in Y1R. The receptor is shown in brown cartoon and surface representations. The ligand is shown as yellow sticks. d, Comparison of Y1R in the Y1R–UR-MK299 crystal structure (brown) and the Y1R–NPY model (green). Side chains of Q1203.32 and W2766.48 are shown as sticks. R35–Y36 of NPY is displayed as cyan sticks. The hydrogen bond between Q1203.32 and Y36 of NPY is shown as a green dashed line. e–j, Chemical structures of the argininamide Y1R antagonists BIBP3226 (e), UR-HU404 (f), UR-MK299 (g), BIBO3304 (h), UR-MK289 (i) and UR-MK136 (j). k, Chemical structure of BMS-193885. l, Scaffold of NPY C-terminal residues R35 and Y36. Key differences between R35–Y36 of NPY and UR-MK299 are chirality of the arginine derivative and alteration of bond connectivity leading to the hydroxyphenyl group.
Extended Data Fig. 2 Expression of wild-type and mutant Y1 receptors in transiently transfected COS-7 cells.
a, Live-cell fluorescence microscopy verifies all Y1R variants to be properly folded and exported to the cell membrane like the wild-type receptor. Nuclei stained with Hoechst33342. Scale bars, 10 μm. Pictures are representative of two independent experiments with similar results. b, The total expression level was determined by fluorescence reading and expression was confirmed to be similar to the wild type. Transfection of only 50% or 25% of the DNA amount (with total DNA amount held constant by empty vector), led to a proportional decrease of fluorescence, and thus, expression level. Data represent mean ± s.e.m. of three to five independent experiments performed in technical triplicate (see Source Data for sample size of each mutant). c, Estimation of the receptor reserve in functional inositol phosphate accumulation assays. Transfection of half of the vector encoding the receptor (with a constant total DNA amount including chimeric G protein, see a) still produces maximum signal, while further reduction results in signal loss at comparable potency. Thus, there is only a small receptor reserve in the functional readout, allowing potency alteration to be directly related to compromised ligand binding. Data represent mean ± s.e.m. of three independent experiments performed in technical duplicate. cNPY, concentration of NPY. Source data
Colours represent the similarity of residues: red background, identical; red text, strongly similar. Key residues in the UR-MK299-binding pocket, which are conserved or variable among receptors, are indicated by red or black arrows, respectively. The alignment was generated using UniProt (http://www.uniprot.org/align/) and the graphic was prepared on the ESPript 3.0 server (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).
Extended Data Fig. 4 Pharmacological characterization of refolded Y1R and NMR studies of Y1R-bound NPY.
a, Binding of Atto 520-labelled NPY (50 nM) to increasing amounts of bicelles containing Y1R or empty bicelles. Data reflect fluorescence enhancement upon binding. An inflection point at EC50 = 52 nM was determined. Two independent experiments were performed in technical duplicate with similar results. Data shown are from a representative experiment. a.u., arbitrary units. c(Y1R), concentration of Y1R. b, Typical 13C MAS single-quantum (SQ)/double-quantum (DQ) correlation spectrum of NPY in the presence of Y1R reconstituted into large bicelles at −30 °C. NMR spectra were acquired from one to three independent preparations for each labelled amino acid with similar results (see d). Data shown are from a representative experiment. c, Table showing 13C-NMR chemical shifts of assigned amino acids of NPY bound to Y1R (referenced to tetramethylsilane) as acquired in solid-state NMR experiments. d, 13C-chemical-shift index of NPY bound to Y1R in large DMPC/DHPC-c7 bicelles (q > 20) compared with docked models. Plotted in black is the measured chemical shift difference (Cα − Cβ) for each individual residue of NPY minus the chemical shift difference of the same amino acid type in random-coil conformation. Individual data points from one to three independent experiments for each labelled amino acid are shown. Typical experimental error when determining chemical shifts under these conditions are ± 1 p.p.m. Chemical shifts were back-calculated for the top docking solutions and filtered against the experimental data to generate a final ensemble of docked poses. Their average chemical-shift index and associated s.d. from the top ten docked poses are shown in red.
a, Mass spectra of photo-crosslinked Y1R with [Bpa1, K4[(Ahx)2-biotin]]NPY. Exemplary MALDI–TOF mass spectra of photo-crosslinked samples enzymatically digested by rLys-C and Glu-C. Potential Y1R fragments are labelled. Two independent experiments were performed with similar results. N, N terminus of Y1R (blue); E, ECL2 (red). b, Respective regions of NPY N terminus at Y1R. Amino acid sequence of Y1R with a C-terminal His-tag. The two detected regions within Y1R (N terminus (blue), ECL2 (red)) after crosslinking with [Bpa1,K4[(Ahx)2-biotin]]NPY are emphasized in boxes. The different sizes of the boxes represent different detected fragments (Extended Data Table 5). Experiments were repeated twice independently with similar results, and only fragments that were observed in both experiments are listed here and in Extended Data Table 5. c, Binding of Atto 520-labelled NPY (50 nM) to increasing amounts of cell-free produced Y1R in Brij-58. Data reflect fluorescence enhancement upon binding. An EC50 value of 69 nM was determined. Data shown are mean ± s.e.m. from six independent experiments performed in technical triplicate. c(Y1R), concentration of Y1R. Source data
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Yang, Z., Han, S., Keller, M. et al. Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor. Nature 556, 520–524 (2018). https://doi.org/10.1038/s41586-018-0046-x
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