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Key to Neuropeptide Y1 Receptor-Ligand Interactions


By: Carol A. Rouzer, VICB Communications
Published:  May 15, 2018

 

New crystal structures of the Y1 receptor complexed with two antagonists provide important information about ligand binding. 

 

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system. Just 36 amino acids long, NPY plays a regulatory role in such diverse processes as food intake, energy metabolism, anxiety and stress, pain perception, blood pressure, and even seizures. However, its ability to strongly stimulate food intake and fat storage has garnered particular attention in recent years, based on the hypothesis that inhibition of NPY signaling might be an effective treatment for obesity. NPY acts by binding to one of four G protein-coupled receptors, Y1R, Y2R, Y4R, and Y5R. Its effects on food intake are mediated primarily by Y1R-dependent signaling. Thus, drug discovery efforts have produced a number of Y1R antagonists, including the highly Y1R-selective UR-MK299 and BMS-193885, which has exhibited anorectic activity in animal models (Figure 1). Realizing that further progress in targeting Y1R would be greatly facilitated by structural information regarding receptor-ligand interactions, Vanderbilt Institute of Chemical Biology member Jens Meiler and his graduate student Brian Bender teamed up with investigators from the Chinese Academy of Sciences, Leipzig University, and the University of Regensburg to obtain crystal structures of Y1R complexed with UR-MK299 and BMS-193885 and to use those structures to develop a computational model of the Y1R-NPY complex [Z. Yang, S. Han, M. Keller, A. Keiser, B. Bender, et al., (2018) Nature, 556, 520].

 

 

 

FIGURE 1. Structures of UR-MK299 and BMS-193885

 

 

 

As is characteristic of G protein-coupled receptors, Y1R comprises seven transmembrane helices. The crystal structure data revealed that the overall structure of Y1R is similar in the two antagonist complexes (Figure 2). In both cases, helix VI adopts an "inward" conformation, characteristic of an inactive receptor as would be expected from binding of an antagonist.

 

 

 

FIGURE 2. (a) Cartoon representation of the structure of Y1R complexed with UR-MK299. The protein is in brown, and UR-MK299 is shown in spheres with carbons in yellow. (b) Cartoon representation of the structure of Y1R complexed with BMS-193885. The protein is in green, and BMS-193885 is shown in spheres with carbons in pink. Figure reproduced with permission from Macmillan Publishers, Ltd and Springer Nature from Z. Yang, S. Han, M. Keller, A. Keiser, B. Bender, et al., (2018) Nature, 556, 520. Copyright 2018.

 


The crystal structure data also revealed that UR-MK299 binds in a pocket formed by helices III through VII, with its diphenymethyl moiety surrounded by F282, F286, and F302 (Figure 3). UR-MK299 is a member of the argininamide class of Y1R antagonists, and mutation studies showed that hydrophobic interactions with F302 and F286 are necessary for potency of this inhibitor class. The hydroxyphenyl group of UR-MK299 lies between helices III and VI, making hydrophobic contact with five residues. Of these, Q120 is thought to also interact with the C-terminus of NPY and to be required for receptor activation. A Q120N mutation does not affect the potency of argininamide Y1R antagonists, but a Q120H mutation substantially increases potency, possibly due to the ability of histidine to establish π-stacking interactions with the antagonists. W276 is another key residue in this region. It serves as a "toggle" switch to convert the receptor from the inactive to active conformation. The loss of potency of UR-MK299 and similar antagonists when W276 is mutated to alanine suggests that hydrophobic interactions with this residue are important for binding. N283 forms hydrogen bonds with the α-nitrogen and carbonyl oxygen next to the hydroxybenzylamine moiety of UR-MK299. D297 forms a salt bridge to the guanidinyl moiety of the antagonist. These polar interactions are critical for UR-MK299 binding, as indicated by a marked loss of potency when they are eliminated by mutation of these residues. As noted above, UR-MK299 is highly selective for Y1R. The only residues in the binding pocket that differ between Y1R and other Y receptors are F173, Q219, N283, and F286. It is likely that these residues play a role in antagonist selectivity.

 

 

 

FIGURE 3. (a) Close-up of the ligand binding pocket containing UR-MK299. The protein is shown as gray ribbon with residues involved in ligand binding depicted as brown sticks. UR-MK299 is shown as sticks with carbons in yellow. (b) Schematic representation of the interactions between residues of Y1R and atoms of UR-MK299. Figure reproduced with permission from Macmillan Publishers, Ltd and Springer Nature from Z. Yang, S. Han, M. Keller, A. Keiser, B. Bender, et al., (2018) Nature, 556, 520. Copyright 2018.

 

 

The binding pocket for BMS-193885 is similar to that for UR-MK299 (Figures 2 and 4). The antagonist's dihydropyridine moiety lies between helices II, V, and VI, and forms a hydrogen bond with T280. Consistently, a T280A mutation reduces the potency of BMS-193885. The antagonist also makes hydrophobic contacts with I124, and mutation of this residue to alanine results in a loss of potency. The urea group of BMS-193885 forms hydrogen bonds with D287, while the piperidine and methoxyphenyl groups lie in close proximity to F282, F286, and F302.

 

 

 

FIGURE 4. (a) Close-up of the ligand binding pocket containing BMS-193885. The protein is shown as gray ribbon with residues involved in ligand binding depicted as green sticks. BMS-193885 is shown as sticks with carbons in pink. (b) Schematic representation of the interactions between residues of Y1R and atoms of BMS-193885. Figure reproduced with permission from Macmillan Publishers, Ltd and Springer Nature from Z. Yang, S. Han, M. Keller, A. Keiser, B. Bender, et al., (2018) Nature, 556, 520. Copyright 2018.

 

 

Previous work had suggested that it is the C-terminus of NPY that interacts with the binding pocket of Y1R to activate receptor signaling. The two residues at this terminus are arginine and tyrosinamide, which bear a structural resemblance to the hydroxyphenyl and guanidinyl groups of UR-MK299. This led the researchers to hypothesize that the crystal structure of the Y1R-UR-MK299 complex should serve as a good basis upon which to build a molecular model of the Y1R-NPY complex. Using these data, along with data from solid-state NMR and complementary mutagenesis studies as a guide, they docked NPY into the active site of Y1R and computationally refined the structure to produce a model of the complex (Figure 5). The model predicts that the C-terminal tetrapeptide of NPY inserts into the binding pocket of Y1R, while the N-terminus lies close to the second extracellular loop, and the central α-helix is positioned along extracellular loops 1 and 3. As predicted, the binding pose of R35 at the C-terminus of NPY is similar to that of the argininamide of UR-MK299 (Figure 6a). R35 of NPY forms a salt bridge with D287 of Y1R. Notably, the only G protein-coupled receptors that bear an aspartate or glutamate residue in a position analogous to that of D287 are those that bind peptides containing a C-terminal arginine-phenylalanine-amide motif. It is interesting that the model predicts that the C-terminal tyrosinamide of NYP does not occupy the same location as the hydroxyphenyl group of UR-MK299. Rather than pointing toward Q219 on helix V, it is directed towards and forms a hydrogen bond with Q120 in helix III. This is consistent with mutation data that demonstrate the importance of Q120 in ligand binding. The peptide also makes hydrophobic contacts with Y100, W106, I293, and N299. The functionality of these contacts was confirmed by mutation data.

 

 

FIGURE 5. Predicted binding pose of NPY (cyan) in the binding pocket of Y1R (tan) as obtained by computational modeling. Figure reproduced with permission from Macmillan Publishers, Ltd and Springer Nature from Z. Yang, S. Han, M. Keller, A. Keiser, B. Bender, et al., (2018) Nature, 556, 520. Copyright 2018.

 

 

Although it does not interact directly with the active site of Y1R, the N-terminus of NPY is required for binding. Deletion of the two N-terminal residues of NPY markedly reduces binding affinity; however these residues can be mutated to alanine with only minor effects. This suggests that the primary interactions are between the receptor and the backbone of NPY in this region. The model of the Y1R-NPY complex predicts that the N-terminus of NPY forms contacts with residues between T180 and F199 in the second extracellular loop (Figure 6b). Data from photocrosslinking and mutagenesis studies support the position predicted by the model.

 

 

 

FIGURE 6. (a) Close-up of the ligand binding pocket of Y1R containing the C-terminus of NPY. The receptor is shown as tan ribbon with residues involved in ligand binding depicted as brown sticks. NPY is in teal with residues involved in binding depicted as dark blue sticks. (b) Close-up of the interaction of Y1R and the N-terminus of NPY. The receptor is shown as tan ribbon with residues involved in ligand binding depicted as brown sticks. NPY is in teal with residues involved in binding depicted as dark blue sticks. Figure reproduced with permission from Macmillan Publishers, Ltd and Springer Nature from Z. Yang, S. Han, M. Keller, A. Keiser, B. Bender, et al., (2018) Nature, 556, 520. Copyright 2018.

 

 

The availability of crystal structure data revealing the key interactions between Y1R and two representative antagonists provides invaluable information for structure-based drug design that will ultimately enable the evaluation of Y1R as a target for the treatment of obesity.  Although a complete understanding of how NPY interacts with and activates Y1R must await direct crystal structure data acquired from a Y1R-NPY complex, the model created in these studies will serve to generate new hypotheses that will drive future research on NPY-dependent Y1R signaling.

 

 

View Nature article: Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

 

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