The human NPY receptors including four subtypes, namely Y 1, Y 2, Y 4, and Y 5 receptors, are widely distributed in central and peripheral nervous systems, as well as a variety of tissues and cell types. In response to three endogenous peptide ligands, NPY, peptide YY, and pancreatic polypeptide, NPY receptors play important roles in a variety of physiological processes, including food intake, angiogenesis, bone formation, and regulation of circadian rhythm and mood disorder. Therefore, NPY receptors have been proposed as important drug targets for the treatment of obesity, anxiety, cancer, and cardiovascular diseases. However, drugs that target NPY receptors are not currently available, partly due to the poor understanding of receptor–ligand interactions. Previous studies using mutagenesis, computational modeling, nuclear magnetic resonance, and various functional assays offered insights into ligand-binding modes of NPY receptors. In addition, crystal structures of Y 1 receptor (Y 1 R) bound to two structurally diverse antagonists were recently determined, providing molecular details of ligand recognition and selectivity of a NPY receptor. However, more structural information is essential to fully understand the molecular basis of ligand recognition and subtype specificity for the complex multiligand/multireceptor system of the NPY-Y receptor family. Y 2 R has attracted considerable interest as a drug target for its role in food intake and bone formation. A number of Y 2 R agonists and antagonists have shown therapeutic potential in the treatment of obesity and anxiety, but their clinical application has been limited by low potency and selectivity and poor blood–brain-barrier permeability. JNJ-31020028 is a potent, selective, brain penetrant small-molecule antagonist of Y 2 R, and has been suggested as a potential treatment for the negative affective states following alcohol withdrawal.
In this work, we report the crystal structure of Y 2 R in complex with JNJ-31020028 at 2.8 Å resolution. Together with extensive functional studies, our results provide key insights into the structural basis of Y 2 R ligand-binding mode and NPY receptor subtype specificity.
To obtain diffraction-quality crystals of Y 2 R–JNJ-31020028, an engineered Y 2 R construct was designed by truncating 28 amino acids (S354-V381) at C terminus and introducing two mutations H149 3.51 Y and S280 6.47 C (superscripts indicate nomenclature according to Ballesteros–Weinstein numbering system) to improve protein yield, homogeneity, and stability. Crystallization was further facilitated by fusing a modified T4 lysozyme (T4L) at the N terminus of the receptor and replacing six residues (S251-N256) in the third intracellular loop (ICL3) with a modified flavodoxin. Functional assays indicate that these modifications have little effect on binding and antagonistic activity of JNJ-31020028 and receptor signaling. The Y 2 R–JNJ-31020028 complex was obtained by copurifying the modified Y 2 R with JNJ-31020028. The complex structure was determined at 2.8 Å resolution. The N-terminal T4L fusion protein was not traced due to poor electron densities. The ligand JNJ-31020028 used in protein purification is a racemic mixture of R-isomer and S-isomer (molar ratio = 1:1), which have similar Y 2 R affinity. Strong and unambiguous electron densities are present for JNJ-31020028 in the Y 2 R structure with the S-isomer fitting better compared to the R-isomer. The following structural analysis is focused on the binding mode of the S-isomer.
The Y 2 R–JNJ-31020028 structure exhibits a canonical seven-transmembrane helical bundle (helices I–VII) architecture of G protein-coupled receptors (GPCRs). The second extracellular loop (ECL2) of the receptor adopts a β-hairpin conformation, which is a common structural feature shared by class A peptide GPCRs. This β-hairpin structure, together with the conserved disulfide bond connecting helix III and ECL2, stabilizes the conformation of the extracellular part of Y 2 R. Y 2 R is structurally similar to Y 1 R (PDB code: 5ZBQ), with a C α root-mean-square deviation of 0.8 Å within the helical bundle. Compared to the structures of inactive Y 1 R and active neurotensin receptor 1 (NTSR1) (PDB code: 6OS9), the extracellular tips of helices II and VI in the JNJ-31020028-bound Y 2 R structure move outward by 3.6 and 2.0 Å, respectively. This movement may be partially due to the ligand binding as JNJ-31020028 would form spatial clashes with these two helices if they were in similar conformations to those in Y 1 R and NTSR1. On the intracellular side, helix VI of Y 2 R adopts an inward conformation similar to that observed in the inactive Y 1 R structure but not in the active NTSR1 structure, suggesting an inactive conformational state of the Y 2 R–JNJ-31020028 structure.
a Side view of the Y 2 R–JNJ-31020028 structure. Y 2 R is shown in light blue cartoon representation. JNJ-31020028 (carbon in yellow, nitrogen in blue, oxygen in red, fluorine in cyan) is shown in sphere representation. The disulfide bond is shown as orange sticks. b, c Structural comparison of Y 2 R with inactive Y 1 R (PDB code: 5ZBQ) and active NTSR1 (PDB code: 6OS9). The helical bundles of Y 2 R, Y 1 R, and NTSR1 are colored light blue, light cyan, and pink, respectively. JNJ-31020028 is shown as sticks. b Extracellular view. Red ar
