The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling.
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The coordinates and cryo-EM maps of the dimAPJRcmpd644–Gi, monAPJRcmpd644–Gi, dimAPJRELA–Gi, monAPJRELA–Gi and ELA-APJRF101A–Gi have been deposited to PDB (EMDB) under accession codes 7W0L (EMD-32243), 7W0M (EMD-32244), 7W0N (EMD-32245), 7W0O (EMD-32246) and 7W0P (EMD-32247), respectively. The coordinate and structure of xtalAPJR–cmpd644 complex has been deposited to PDB under accession code 7SUS. Source data are provided with this paper.
Sleno, R. & Hébert, T. E. Shaky ground—the nature of metastable GPCR signalling complexes. Neuropharmacology 152, 4–14 (2019).
Milligan, G., Ward, R. J. & Marsango, S. GPCR homo-oligomerization. Curr. Opin. Cell Biol. 57, 40–47 (2019).
Pétrin, D. & Hébert, T. E. The functional size of GPCRs—monomers, dimers or tetramers? Subcell Biochem. 63, 67–81 (2012).
Dupré, D. J. & Hébert, T. E. Biosynthesis and trafficking of seven transmembrane receptor signalling complexes. Cell Signal 18, 1549–1559 (2006).
Lopez-Gimenez, J. F., Canals, M., Pediani, J. D. & Milligan, G. The alpha1b-adrenoceptor exists as a higher-order oligomer: effective oligomerization is required for receptor maturation, surface delivery, and function. Mol. Pharmacol. 71, 1015–1029 (2007).
Salahpour, A. et al. Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting. J. Biol. Chem. 279, 33390–33397 (2004).
Papasergi-Scott, M. M. et al. Structures of metabotropic GABA(B) receptor. Nature 584, 310–314 (2020).
Scholler, P. et al. Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation. Nat. Commun. 8, 1967 (2017).
Zhu, S. et al. Structure of a human synaptic GABA(A) receptor. Nature 559, 67–72 (2018).
Dijkman, P. M. et al. Dynamic tuneable G protein-coupled receptor monomer-dimer populations. Nat. Commun. 9, 1710 (2018).
Ferré, S. et al. G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives. Pharmacol Rev 66, 413–434 (2014).
Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl Acad. Sci. USA 97, 3684–3689 (2000).
Calebiro, D. et al. Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc. Natl Acad. Sci. USA 110, 743–748 (2013).
Möller, J. et al. Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors. Nat. Chem. Biol. 16, 946–954 (2020).
Fiorentini, C., Busi, C., Spano, P. & Missale, C. Dimerization of dopamine D1 and D3 receptors in the regulation of striatal function. Curr. Opin. Pharmacol. 10, 87–92 (2010).
Trettel, F. et al. Ligand-independent CXCR2 dimerization. J. Biol. Chem. 278, 40980–40988 (2003).
Işbilir, A. et al. Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists. Proc. Natl Acad. Sci. USA 117, 29144–29154 (2020).
Herrick-Davis, K., Grinde, E., Harrigan, T. J. & Mazurkiewicz, J. E. Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein. J. Biol. Chem. 280, 40144–40151 (2005).
Zhao, D. Y. et al. Cryo-EM structure of the native rhodopsin dimer in nanodiscs. J. Biol. Chem. 294, 14215–14230 (2019).
Liang, Y. et al. Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes. J. Biol. Chem. 278, 21655–21662 (2003).
Huang, J., Chen, S., Zhang, J. J. & Huang, X. Y. Crystal structure of oligomeric β1-adrenergic G protein-coupled receptors in ligand-free basal state. Nat. Struct. Mol. Biol. 20, 419–425 (2013).
Manglik, A. et al. Crystal structure of the µ-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012).
Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010).
Siddiquee, K., Hampton, J., McAnally, D., May, L. & Smith, L. The apelin receptor inhibits the angiotensin II type 1 receptor via allosteric trans-inhibition. Br. J. Pharmacol. 168, 1104–1117 (2013).
Cai, X., Bai, B., Zhang, R., Wang, C. & Chen, J. Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling. Sci. Rep. 7, 40335 (2017).
Chun, H. J. et al. Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis. J. Clin. Invest. 118, 3343–3354 (2008).
Li, Y. et al. Heterodimerization of human apelin and kappa opioid receptors: roles in signal transduction. Cell Signal 24, 991–1001 (2012).
Ji, B. et al. Roles for heterodimerization of APJ and B2R in promoting cell proliferation via ERK1/2-eNOS signaling pathway. Cell Signal 73, 109671 (2020).
O’Dowd, B. F. et al. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene 136, 355–360 (1993).
Edinger, A. L. et al. An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol. 72, 7934–7940 (1998).
Chng, S. C., Ho, L., Tian, J. & Reversade, B. Elabela: a hormone essential for heart development signals via the apelin receptor. Dev. Cell 27, 672–680 (2013).
Cox, C. M., D’Agostino, S. L., Miller, M. K., Heimark, R. L. & Krieg, P. A. Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo. Dev. Biol. 296, 177–189 (2006).
Kasai, A. et al. Retardation of retinal vascular development in apelin-deficient mice. Arterioscler. Thromb. Vasc. Biol. 28, 1717–1722 (2008).
Wang, Z. et al. Elabela-apelin receptor signaling pathway is functional in mammalian systems. Sci. Rep. 5, 8170 (2015).
Marsault, E. et al. The apelinergic system: a perspective on challenges and opportunities in cardiovascular and metabolic disorders. Ann. N. Y. Acad. Sci. 1455, 12–33 (2019).
Scimia, M. C. et al. APJ acts as a dual receptor in cardiac hypertrophy. Nature 488, 394–398 (2012).
Ma, Y. et al. Structural basis for apelin control of the human apelin receptor. Structure 25, 858–866 (2017).
Pauli, A. et al. Toddler: an embryonic signal that promotes cell movement via Apelin receptors. Science 343, 1248636 (2014).
Perjés, Á. et al. Characterization of apela, a novel endogenous ligand of apelin receptor, in the adult heart. Basic Res. Cardiol. 111, 2 (2016).
Dagamajalu, S. et al. The network map of Elabela signaling pathway in physiological and pathological conditions. J. Cell Commun. Signal. 16, 145–154 (2021).
Sato, T. et al. ELABELA-APJ axis protects from pressure overload heart failure and angiotensin II-induced cardiac damage. Cardiovasc. Res. 113, 760–769 (2017).
Deng, C., Chen, H., Yang, N., Feng, Y. & Hsueh, A. J. Apela regulates fluid homeostasis by binding to the APJ receptor to activate Gi signaling. J. Biol. Chem. 290, 18261–18268 (2015).
Ho, L. et al. ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice. Science 357, 707–713 (2017).
Chapman, F. A. et al. The therapeutic potential of apelin in kidney disease. Nat. Rev. Nephrol. 17, 840–853 (2021).
Chen, H. et al. ELABELA and an ELABELA fragment protect against AKI. J. Am. Soc. Nephrol. 28, 2694–2707 (2017).
Liet, B., Nys, N. & Siegfried, G. Elabela/toddler: new peptide with a promising future in cancer diagnostic and therapy. Biochim. Biophys. Acta, Mol. Cell. Res. 1868, 119065 (2021).
Chen, N. et al. Triazole agonists of the APJ receptor. Patent WO2016187308A1 (2016).
Ason, B. et al. Cardiovascular response to small-molecule APJ activation. JCI Insight 5, e132898 (2020).
Maeda, S. et al. Development of an antibody fragment that stabilizes GPCR/G-protein complexes. Nat. Commun. 9, 3712 (2018).
Shen, C. et al. Structural basis of GABAB receptor–Gi protein coupling. Nature 594, 594–598 (2021).
Lin, S. et al. Structures of G(i)-bound metabotropic glutamate receptors mGlu2 and mGlu4. Nature 594, 583–588 (2021).
Velazhahan, V. et al. Structure of the class D GPCR Ste2 dimer coupled to two G proteins. Nature 589, 148–153 (2021).
Ballesteros, J. A. & Weinstein, H. In Methods in Neurosciences Vol. 25 (ed Sealfon, S. C.) 366–428 (Academic Press, 1995).
Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Reggio, P. H. Computational methods in drug design: modeling G protein-coupled receptor monomers, dimers, and oligomers. AAAPS. J. 8, E322–E336 (2006).
Terrillon, S. et al. Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol. Endocrinol. 17, 677–691 (2003).
Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 (2015).
Barnea, G. et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl Acad. Sci. USA 105, 64–69 (2008).
Ma, Y. et al. Structure-guided discovery of a single-domain antibody agonist against human apelin receptor. Sci. Adv. 6, eaax7379 (2020).
Zhang, H. et al. Structural basis for selectivity and diversity in angiotensin II receptors. Nature 544, 327–332 (2017).
Scott, I. C. et al. The G protein-coupled receptor agtrl1b regulates early development of myocardial progenitors. Dev. Cell 12, 403–413 (2007).
Murza, A. et al. Discovery and structure–activity relationship of a bioactive fragment of ELABELA that modulates vascular and cardiac functions. J. Med. Chem. 59, 2962–2972 (2016).
Read, C. et al. International Union of Basic and Clinical Pharmacology. CVII. Structure and pharmacology of the apelin receptor with a recommendation that ELABELA/toddler is a second endogenous peptide ligand. Pharmacol. Rev. 71, 467–502 (2019).
Trân, K. et al. Structure–activity relationship and bioactivity of short analogues of ELABELA as agonists of the apelin receptor. J. Med. Chem. 64, 602–615 (2021).
Felce, J. H., Davis, S. J. & Klenerman, D. Single-molecule analysis of G protein-coupled receptor stoichiometry: approaches and limitations. Trends Pharmacol. Sci. 39, 96–108 (2018).
Murata, K. & Wolf, M. Cryo-electron microscopy for structural analysis of dynamic biological macromolecules. Biochim. Biophys. Acta, Gen. Subj. 1862, 324–334 (2018).
Lau, C., Hunter, M. J., Stewart, A., Perozo, E. & Vandenberg, J. I. Never at rest: insights into the conformational dynamics of ion channels from cryo-electron microscopy. J. Physiol. 596, 1107–1119 (2018).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Velazhahan, V. et al. Structure of the class D GPCR Ste2 dimer coupled to two G proteins. Nature 589, 148–153 (2020).
Koehl, A. et al. Structural insights into the activation of metabotropic glutamate receptors. Nature 566, 79–84 (2019).
Mao, C. et al. Cryo-EM structures of inactive and active GABA(B) receptor. Cell Res. 30, 564–573 (2020).
Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012).
Hua, T. et al. Activation and signaling mechanism revealed by cannabinoid receptor-G(i) complex structures. Cell 180, 655–665 (2020).
Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).
Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).
Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D. Biol. Crystallogr. 66, 133–144 (2010).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D. Biol. Crystallogr. 53, 240–255 (1997).
Smart, K. M., Blake, C., Staines, A., Thacker, M. & Doody, C. Mechanisms-based classifications of musculoskeletal pain: part 1 of 3: symptoms and signs of central sensitisation in patients with low back (± leg) pain. Man. Ther. 17, 336–344 (2012).
Ritchie, T. K. et al. Chapter 11—reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).
Olsen, R. H. J. et al. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat. Chem. Biol. 16, 841–849 (2020).
This work was supported by the Ministry of Science and Technology of China (grant no. 2018YFA0507000 to F.X.), the National Natural Science Foundation of China (grant nos. 32071194, 32111530085 and 81861128023 to F.X.), the Science and Technology Commission of Shanghai Municipality (grant no. 19XD1422800 to F.X.) and the Canadian Institutes of Health Research (grant no. FDN-148413 to P.S.). We thank Q. Tan, Q. Shi, L. Zhang, N. Chen, W. Xiao and F. Zhou for protein cloning, expression and assay support; Q. Sun, Y. Liu, Y. Wang, D. Liu and Z. Zhang at the Bio-EM facility at ShanghaiTech University, and the Instrumental Analysis Center at Shanghai Jiaotong University for technical support on cryo-EM data collection. The X-ray diffraction data were collected at BL41XU at SPring-8 with JASRI proposal no. 2019B2704.
S.S., H.L., R.C.S. and M.A.H. are current or former full-time employees and/or founders of Structure Therapeutics. The rest of the authors declare no competing interests.
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Extended Data Fig. 1 Cryo-EM sample preparation, data collection and processing for APJRcmpd644-Gi-scFv16.
a, Representative cryo-EM image from 7,337 micrographs of the APJRcmpd644-Gi-scFv16 complex. Scale bar, 50 nm. b, Representative 2D averages showing distinct secondary structural features from different views of the complex. c, Cryo-EM data processing workflow. The data was all processed by CryoSPARC and images of density maps were created in UCSF Chimera. The final 3D density maps are colored according to the local resolution. Gold-standard FSC curves from Phenix indicate overall nominal resolutions of 3.57 Å and 3.71 Å using the FSC = 0.143 criterion for the dimAPJRcmpd644-Gi map (light blue curve, left) and monAPJRcmpd644-Gi map (dark blue curve, right), respectively. d, Elution profile and gel image of APJR (comprising residues 1–350), Gαi, Gβ, Gγ and scFv16 after SEC purification. The collected fraction for cryo-EM sample were marked between dashed lines.
a, The cryo-EM density map and models of transmembrane helices in the ProtA of dimAPJRcmpd644-Gi complex. b, The Cryo-EM density map and models of transmembrane helices in the ProtB of dimAPJRcmpd644-Gi complex. c, Cryo-EM maps for the cmpd644 in the structure of dimAPJRcmpd644-Gi and monAPJRcmpd644-Gi complexes, respectively.
Extended Data Fig. 3 Receptor-G protein interface comparison between dimAPJRcmpd644-Gi and monAPJRcmpd644-Gi complexes.
a–c, Interactions between dimeric APJR ProtA (rainbow colored surface) and Gαi protein (blue cartoon). Interface residues are shown as sticks. d–f, Interactions between monomeric APJR (rainbow colored surface) and Gαi protein (yellow cartoon). Interface residues are shown as sticks. g, Sequence alignment of APJR-Gαi interface (residues on the receptor within 5 Å to the G protein) with other class A GPCR-Gi structures using Jalview v126.96.36.199.
Extended Data Fig. 4 Snake plot of the amino acid sequence of APJR and sequence analysis of dimer interface (F23.52G3.21T3.22F3.23F3.24) with all 289 class A GPCRs.
a, Residues that form the dimer interface are shown in green, residues that interact with cmpd644 are shown in red and residues that interact with the G protein are shown in blue or circled in red in dimer or monomer, respectively. b, Calculation of covered dimer interface area in APJR, mGlu5 (PDB ID: 6N51), GABAB (PDB ID: 6W2Y), CaSR (PDB ID: 7DTV), and class D GPCR Ste2 receptor (PDB ID: 7AD3). c, Sequence alignment of F23.52G3.21T3.22F3.23F3.24 within all 289 class A GPCRs, revealing that F23.52, G3.21 are highly conserved (45% and 62%, respectively), F3.23 and F3.24 are intermediately conserved (13% and 10%, respectively), and T3.22 is the least conserved (6.5%). The proportion of the aromatic group at each position of this sequence string (from position 23.52 to 3.24) is 45.7%, 1.3%, 9.7%, 19.7%, and 14.5%, respectively.
Extended Data Fig. 5 Cryo-EM sample preparation, data collection and processing for APJRELA-Gi-scFv16.
a, Representative cryo-EM image from 9,316 micrographs of the APJRELA-Gi-scFv16 complex. Scale bar, 50 nm. b, Representative 2D averages showing distinct secondary structural features from different views of the APJRELA-Gi-scFv16 complex. c, Cryo-EM data processing workflow. The data was all processed by CryoSPARC and images of density maps were created in UCSF Chimera. The final 3D density maps are colored according to the local resolution. Gold-standard FSC curves from Phenix indicate overall nominal resolutions of 4.21 Å and 3.78 Å using the FSC = 0.143 criterion for the dimAPJRELA-Gi map (left), monAPJRELA-Gi map (right), respectively. d, Elution profile and gel image of APJR (comprising residues 1–350), Gαi, Gβ, Gγ and scFv16 after SEC purification. The collected fraction for cryo-EM sample were marked between dashed lines.
a, The overall cryo-EM density map and atomic models in the dimAPJRELA-Gi and monAPJRELA-Gi complexes. b, The Cryo-EM density map and atomic models for ELA-32 in dimAPJRELA-Gi and monAPJRELA-Gi complexes, the dimer interface and TM7-H8 in dimAPJRELA-Gi complex. c, The cryo-EM density map and models of transmembrane helices in monAPJRELA-Gi complex.
a, Relative surface expression levels of mutants were monitored by FACS staining assay and normalized to the expression levels of WT-APJR. Data were presented as mean ± S.E.M of three biologically independent experiments. b, SEC profile of APJR mutants expressed in the absence of Gi protein. Single mutation F1013.24A disrupted the dimer formation (arrow showed F1013.24A shift towards the monomeric GPCR control (FLAG-BRIL-fused GPR52)). c, SEC profile of APJR mutants-Gi co-expression (arrow showed peak-shift of F1013.24A-Gi compared to WT-Gi). d, SEC profile and SDS-PAGE gel image of APJRWT-Gi or APJRF101A-Gi protein complex in the nanodisc system. Black arrow indicates peak shift from dimer to monomer species. e, Influence of dimer interface mutations on the downstream signaling measured by cAMP response in the presence of cmpd644. f, Influence of ‘dimer-switch’ F101A mutation on the downstream signaling measured by BRET2 bio-sensor assay for both ELA-32 and cmpd644 (normalized response relative to %WT Emax). Basal activity of F101A mutant relative to WT was denoted. For e and f, data were presented as mean ± S.E.M of three biologically independent experiments.
Extended Data Fig. 8 Cryo-EM structure of the ELA-APJRF101A–Gi complex and analysis of activation motifs.
a, Representative cryo-EM image of from 9,096 micrographs the ELA-APJRF101A-Gi complex. Scale bar, 50 nm. b, Representative 2D averages showing distinct secondary structural features from different views of the ELA-APJRF101A-Gi complex. c, Cryo-EM data processing workflow. The data was all processed by CryoSPARC and images of density maps were created in UCSF Chimera. The final 3D density maps are colored according to the local resolution. Gold-standard FSC curves from Phenix indicate overall nominal resolutions of 3.16 Å using the FSC = 0.143 criterion. d, Elution profile of APJRF101A (comprising residues 1-350), Gαi, Gβ, Gγ and scFv16 after SEC purification. The collected fraction for cryo-EM sample were marked between dashed lines. The gel image of APJRF101A-Gi complex in the presence of ELA-32 (labeled in red color) compared with APJRWT-Gi complexes in the presence of cmpd644 and ELA-32 respectively. The three part of gel images were from different gels and split with white boarder. e, Atomic model of ELA-APJRF101A-Gi complex and global fitting of the structure into the cryo-EM density map (scFv16 is omitted for clarity). f, Superposition of ELA-monAPJRF101A-Gi with ELA-monAPJRWT-Gi complex structures. g, Conformational rearrangements in the activation-related key motifs. PIF and ‘toggle switch’ residues in the active−state ProtAcmpd644 (blue) show side−chain movement compared to the inactive-state APJR (pink), left. Conformational rearrangement related to the Na+ pocket. D752.50 forms polar interaction with N461.50 in the active-state structure (ProtAcmpd644, blue). Dashed lines represent polar interaction, middle. Conformational changes in DRY and NPxxY motif, right. The hydrogen bond is depicted as a dashed line. Related residues are presented as sticks. h, Comparison of helix 8 (H8) in ProtBcmpd644 from dimAPJRcmpd644-Gi with that in AT2R (PDB ID: 5UNF). The H8 showed inverted orientation in both structures.
Extended Data Fig. 9 Ligand binding mode comparison in different cmpd644-bound APJR structures reported in this study.
a, The crystal packing, SEC profile and crystal images from 112 crystals of xtalAPJRcmpd644. b, Structural alignment of cytoplasmic portion of TM5/6/7 in the xtalAPJRcmpd644 structure (gray) suggested inactive conformation when compared to inactive-state AMG3054-APJR co-crystal structure (PDB ID: 5VBL, green) and active-state ProtA in dimAPJRcmpd644–Gi complex (blue). c, Comparison of binding pockets between cmpd644 (yellow) in ProtA (blue) and AMG3054 (purple) in APJR co-crystal structure (green). Related residues are shown in sticks. Dashed lines circled the dimethoxyphenyl group of cmpd644 that mimics the phenyl ring of F17 in AMG3054. d, Extended interactions of cmpd644 with xtalAPJR in the subpocket. Interacting residues of APJR are shown as sticks in pink. e, Structural comparison of the residues in the ligand binding pocket (left) or underneath (right). Conformational changes of key residues in the active-state ProtA (blue) from that of inactive-state xtalAPJR (gray) are indicated. f, Orthogonal views of superimposed cmpd644 in ProtA (yellow), monAPJR (green) and xtalAPJRcmpd644 structures (orange), respectively. xtalAPJR is shown as surface in gray. g, Cmpd644 binding pocket superposition between ProtA (blue) and monAPJR (purple). Cmpd644 are shown as yellow and cyan, respectively. h, Superposition of cmpd644 and comparison of cmpd644 binding pockets between ProtA (blue) and ProtB (gray). Cmpd644 are shown as yellow and pink, respectively. Related residues are presented as sticks and dashed lines represent hydrogen bonds.
Extended Data Fig. 10 Peptide sequence alignment and functional assessment of mutations in the ELA-32 binding pocket.
a, Multiple sequence alignment of ELA-32, apelin-17 and AMG3054. Numbering for the peptide is colored in brown. The 1-14 residues of ELA-32 are omitted. b, cAMP functional assay to measure ELA-32 potency on the mutant APJR in comparison to the WT. c, Ligand potency analysis for mutations in the ELA-32 binding pocket for ELA-32 and Apelin-13, respectively. Data are presented as mean ± S.E.M of three biologically independent experiments.
Supplementary Tables 1–3 and Fig. 1.
3D variability analysis of dimAPJRcmpd644–Gi_view1.
3D variability analysis of dimAPJRcmpd644–Gi_view2.
3D variability analysis of monAPJRcmpd644–Gi_view2.
Statistical source data.
Statistical source data.
Statistical source data.
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Yue, Y., Liu, L., Wu, LJ. et al. Structural insight into apelin receptor-G protein stoichiometry. Nat Struct Mol Biol 29, 688–697 (2022). https://doi.org/10.1038/s41594-022-00797-5
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