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Structural insight into apelin receptor-G protein stoichiometry

Nature Structural & Molecular Biology volume 29, pages 688–697 (2022)Cite this article

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Abstract

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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Fig. 1: Cryo-EM structures of the dimAPJRcmpd644–Gi and monAPJRcmpd644–Gi complexes.
Fig. 2: Structural comparison between dimAPJRcmpd644–Gi and monAPJRcmpd644–Gi complexes and dimer interface.
Fig. 3: ELA-32 activated structures of APJR–Gi complex and functional characterization of ‘dimer-switch’ mutant.
Fig. 4: Activation mechanism of APJR.
Fig. 5: Molecular basis for small-molecule and ELA-32 binding.

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Data availability

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.

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Acknowledgements

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.

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Author notes

  1. These authors contributed equally: Yang Yue, Lier Liu, Li-Jie Wu.

Authors and Affiliations

  1. iHuman Institute, ShanghaiTech University, Shanghai, China

    Yang Yue, Lier Liu, Li-Jie Wu, Yiran Wu, Ling Wang, Fei Li, Junlin Liu, Bo Chen, Xi Lin, Raymond C. Stevens & Fei Xu

  2. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Lier Liu, Raymond C. Stevens & Fei Xu

  3. University of Chinese Academy of Sciences, Beijing, China

    Lier Liu & Fei Xu

  4. Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA, USA

    Gye-Won Han

  5. Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institute of Pharmacology at Sherbrooke, University of Sherbrooke, Sherbrooke, Quebec, Canada

    Rebecca L. Brouillette, Émile Breault, Jean-Michel Longpré & Philippe Sarret

  6. Structure Therapeutics, South San Francisco, CA, USA

    Songting Shi, Hui Lei, Raymond C. Stevens & Michael A. Hanson

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Contributions

Y.Y. and L.L. performed cloning, protein purification, cryo-EM sample preparation, data collection and structure analysis. Y.Y. carried out crystallography and functional assay. L.-J.W. performed cryo-EM data processing, model building and refinement and 3D viability analysis. Y.W. assisted with the computational analysis. G.-W.H. was responsible for crystal structure refinement and quality control. L.W., F.L. and X.L. carried out the functional assay. J.L. assisted with protein expression. B.C. assisted with the cryo-EM data collection. R.L.B., E.B., J.-M.L. and P.S. assisted with the biosensor functional assay. S.S. and H.L. instructed the cmpd644 synthesis and characterization. R.C.S. instructed the manuscript preparation. M.A.H. processed the crystallographic data and analyzed the structure. F.X. conceived the project, designed and supervised experiments. All authors contributed to data interpretation and the writing of the manuscript. Y.Y., L.L., L.-J.W., M.A.H. and F.X. wrote the manuscript.

Corresponding author

Correspondence to Fei Xu.

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Competing interests

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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Nature Structural & Molecular Biology thanks Arun Shukla and Asuka Inoue for their contribution to the peer review of this work. Primary Handling Editor: Florian Ullrich, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

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.

Source data

Extended Data Fig. 2 Cryo-EM density map and refined structures of dimAPJRcmpd644-Gi complex.

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 v2.11.1.4.

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.

Source data

Extended Data Fig. 6 Cryo-EM density map and refined structures of WT-APJRELA-Gi.

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.

Extended Data Fig. 7 Biochemical and functional characterization of dimer interface mutations.

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.

Source data

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.

Source data

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.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Fig. 1.

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Supplementary Video 1

3D variability analysis of dimAPJRcmpd644–Gi_view1.

Supplementary Video 2

3D variability analysis of dimAPJRcmpd644–Gi_view2.

Supplementary Video 3

3D variability analysis of monAPJRcmpd644–Gi_view2.

Source data

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 1

Representative micrograph.

Source Data Extended Data Fig. 5

Unprocessed gels.

Source Data Extended Data Fig. 5

Representative micrograph.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed gels.

Source Data Extended Data Fig. 8

Unprocessed gels.

Source Data Extended Data Fig. 8

Representative micrograph.

Source Data Extended Data Fig. 10

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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