Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

Abstract

Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. 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 mammals, namely 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 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, tumor1 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 two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors.

Carbaboranylation of Truncated C‐Terminal Neuropeptide Y Analogue Leads to Full hY1 Receptor Agonism

“…This underlines that there is a specific binding pocket for bulky substituents in hY 1 R that especially triggers arrestin recruitment. This binding pocket may be located around Phe 199 of the extracellular loop 2, as illustrated in Figure B, thereby mimicking the NPY N terminus around Tyr 1 (Figure A) . Internalisation studies with the use of fluorescence microscopy nicely complemented the results of the arrestin recruitment studies with one exception: lauroyl was able to induce internalisation despite a pronounced loss in potency in the BRET assay with activity only at the highest tested concentration.…”

“…Moreover, truncated versions may help to elucidate the role of the individual elements of the peptide structure in mediating receptor signalling. It was comparatively easy to identify such agonists for the related hY 2 R, but this has remained challenging for the hY 1 R. From a structure‐based model of NPY binding to this receptor, it becomes clear that binding of Arg 33 , Arg 35 , and Tyr 36 of the NPY C terminus to transmembrane helices 6 and 7 and the interaction of the flexible N‐terminal stretch with the extracellular loop 2 of hY 1 R are crucial . Truncation of the N terminus inevitably results in competitive binding and signalling of the antagonists of NPY, whereas truncation of the C terminus yields peptides that no longer bind at all.…”

“…It was comparatively easy to identify such agonists for the related hY 2 R, [16] butt his has remained challenging for the hY 1 R. From a structure-based model of NPY binding to this receptor,i tb ecomes clear that binding of Arg 33 ,A rg 35 ,a nd Tyr 36 of the NPY Cterminus to transmembrane helices 6a nd 7a nd the interaction of the flexible N-terminal stretch with the extracellular loop 2o fh Y 1 Ra re crucial. [5] Truncation of the Nterminus inevitably results in competitive binding and signalling of the antagonists of NPY,w hereas truncation of the Cterminus yields peptides that no longerb ind at all. The development of antagonists for hY 1 Rh as been ongoing for quite some time and has been reviewed; [17] however,s hort agonistsw ere not identified until 2009, when the first truncated NPY-derived peptide agonist was published.…”

“…Proposed binding site of Cpa-sNPY.A)Proposed binding mode of NPY to hY 1 Ra sp ublished [5]. N-terminal Tyr 1 of NPY in cyan, hY1 Ri nteraction with NPY Nterminus in dark blue, hY 1 Ri nteraction points with NPY Cterminus in lime green, and regiono fs NPY in NPY in pale green.B )Mimicking of NPY Nterminus by carbaboranec oupled to Lys(red) in sNPY.…”

Targeting of peptide‐binding receptors on cancer cells with peptide‐drug conjugates

“…[ 116 ] Furthermore, two amino acid substitutions in the N‐terminal and the very C‐terminal part of NPY led to the generation of the hY 1 R‐preferring, full‐length variant [F 7 ,P 34 ]‐NPY (Figure 4B) with nanomolar potency at the hY 1 R and highly reduced affinity for the hY 2 R. [ 117 ] Recently, the crystal structure of the hY 1 R in complex with two different antagonists was solved. [ 118 ] Use of this structural data, in combination with molecular docking, NMR, crosslinking, and functional studies, allowed for a more detailed characterization of the NPY binding site at the hY 1 R as previously known. This scientific breakthrough might enable further optimization of receptor‐selective ligands in the future.…”