20 Research Frontiers 2022 Research Frontiers 2022 Life Science Apelin receptor (APJR) belongs to the class A G-protein coupled receptor (GPCR) group, which is closely related to cardiac development, cardiovascular homeostasis and cardiac contraction [1,2]. The activation of the APJR signaling pathway can alleviate or treat many cardiovascular-related diseases such as heart failure and hypertension (Fig. 1). Many pharmaceutical companies, such as AMGEN, BMS and Sanofi, are trying to develop effective APJR agonist ligands; however, no drug has been successfully marketed until now. To develop new agonists of APJR, especially small-molecule drugs, understanding the molecular mechanism of the small-molecule binding mode is of critical importance. To investigate the molecular mechanism of a small- molecule ligand, we chose a potent small-molecule agonist, (1S,2R)-N-(4-(2,6-dimethoxyphenyl)-5-(6- methylpyridin-2-yl)-4H-1,2,4-triazol-3-yl)-1-hydroxy- 1-(5-methylpyrimidin-2-yl) propane-2-sulfonamide (termed cmpd644), which is similar to a clinical- stage drug candidate developed for treating heart failure (US patent WO2016187308A1, Amgen Inc.). After several rounds of optimization, we obtained cmpd644-bound APJR structures in a complex with its downstream heterotrimeric G a i G bg by cryo-EM methods [3]. Interestingly, we observed two types of APJR-Gi coupling stoichiometry from one cryo-EM dataset. Dimeric APJR and monomeric APJR adopt 2:1 and 1:1 (receptor: G protein) stoichiometric ratios, respectively (Fig. 2). This provides the first direct structural evidence of the coexistence of homodimers and monomers in the ligand-bound and G protein- coupled APJR signaling complexes. To understand the small-molecule ligand binding mode for APJR in greater detail, we also solved the co-crystal structure of APJR in a complex with cmpd644 at a high resolution (2.7 Å) in the absence of Gi protein by X-ray crystallography. To obtain a stable APJR protein for structural determination, we first back-mutated W261K to the wild type employing the previous construct used for obtaining the APJR- peptide co-crystal structure [4]. Then, we generated and screened multiple constructs and finally introduced three new mutations (i.e., E174C, M217C, and I250C) based on the wild-type APJR sequence to further stabilize the receptor, and conducted crystallization trials by the LCP method. Finally, with the help of beamline scientists at SPring-8 BL41XU , we obtained high-quality X-ray diffraction data sufficient for solving the structure. T h i s i s t h e f i r s t r e p o r t e d s m a l l - m o l e c u l e - bound APJR co-crystal structure and we named it xtal APJ cmpd644 (Fig. 3(a)). Alignment with our previously solved peptide-bound AMG3054-APJR co-crystal structure revealed an almost identical receptor conformation (r.m.s.d. 0.529Å) [4]. We are curious about the distinction between peptide and small- molecule binding modes. Upon close examination of the binding pocket, we found that cmpd644 only occupied “site 1” of the pocket and inserted itself deeper when aligning with the C-terminal portion of the peptide ligand, which is different from the two- site binding mode for the peptide ligand (Fig. 3(b)). Moreover, the dimethoxyphenyl group of cmpd644 simulates the phenyl ring of F17 in AMG3054, interacting with W85 2.60 , I109 3.32 , F110 3.33 , and F291 7.35 (Fig. 3(c)). There is an extended subpocket formed by the interaction between the methylpyridine ring in Crystal structure of the small-molecule-bound apelin receptor Fig. 1. Simplified illustration of APJR functional pathway. Peptide and small-molecule ligands can activate APJR signaling, which leads to cardiac development, cardiovascular homeostasis and cardiac contraction [1,2]. This figure was created at BioRender.com. Peptide ligands Small molecule ligands APJR Extracellular space Cell membrane Intracellular space Cardiac development Cardiovascular homeostasis Cardiac contraciton 21 Research Frontiers 2022 Research Frontiers 2022 cmpd644 and the surrounding hydrophobic residues of F78 2.53 , F110 3.33 , Y264 6.51 , and Y299 7.43 (Fig. 3(c)). The interacting residues W85 2.60 , F110 3.33 , K268 6.55 , Y271 6.58 , M288 7.32 , and F291 7.35 are consistent with the reported functional assay for the small-molecule AM- 8123, which shares a similar scaffold with cmpd644 [5]. We report the first co-crystal structure of APJR in a complex with the drug-candidate small-molecule compound, providing a precise molecular template for further pharmacology studies on tre a ti n g cardiovascular disease. Additionally, together with the G-protein-bound cryo-EM structures in both 2:1 and 1:1 stoichiometric ratios, we provide a different, exciting view of the class A GPCR signaling mechanism. References [1] B. F. O'Dowd et al. : Gene 136 (1993) 355. [2] S. C. Chng et al. : Dev. Cell 27 (2013) 672. [3] Y. Yue, L. Liu, L.-J. Wu, Y. Wu, L. Wang, F. Li, J. Liu, G.-W. Han, B. Chen, X. Lin, R. L. Brouillette, É. Breault, J.-M. Longpré, S. Shi, H. Lei, P. Sarret, R. C. Stevens, M. A. Hanson and F. Xu: Nat. Struct. Mol. Biol. 29 (2022) 688. [4] Y. Ma et al. : Structure 25 (2017) 858. [5] B. Ason et al. : JCI Insight 5 (2020) e132898. Yang Yue a and Fei Xu a,b, * a iHuman Institute, ShanghaiTech University, China b School of Life Science and Technology, ShanghaiTech University, China *Email: xufei@shanghaitech.edu.cn Fig. 2. Cryo-EM structures of the dimeric APJR-Gi (a) and monomeric APJR-Gi (b) complexes in the presence of cmpd644. Fig. 3. (a) Overall structure of xtal APJ cmpd644 . APJR is colored green and cmpd644 pink. (b) Comparison of xtal APJ cmpd644 with AMG3054-APJR co-crystal structure (PDB ID: 5VBL, AMG3054 is colored yellow and APJR gray). Site 1 is circled with dashed lines. (c) Binding pocket of cmpd644 in xtal APJ cmpd644 . Interacting residues of APJR are shown as sticks. The subpocket is circled with dashed lines. G G γ γ G G γ γ (a) (b) Dimeric AP JR Cmpd644 Cmpd644 Cmpd644 G G α α i G G α α i G G β β G G β β Monomeric AP JR TM4 TM2 TM5 TM3 TM6 TM1 TM7 Site1 Subpocket AMG3054 Cmpd644 Y271 6.58 M288 7.32 K268 6.55 F291 7.35 Y264 6.51 F110 3.33 W85 2.60 Y299 7.43 F78 2.53 (a) (b) (c)
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